TW202525856A - Bifunctional proteins and uses thereof - Google Patents

Abstract

Provided herein are compositions and methods of treating muscular dystrophies by administering an effective amount of a composition comprising a bifunctional protein comprising a first binding moiety that binds to muscle-specific molecules and a second functional domain that activates Notch signaling, thereby spatiotemporally activating Notch signaling in muscle tissues to boost satellite cell renewal in situ, and thus enhancing muscle repair/regeneration to improve muscle function as a treatment of various muscular dystrophies and sarcopenia. Also provided are engineered DLL4 extracellular domains and protein constructs comprising thereof.

Description

Bifunctional proteins and their uses

The present invention relates to bifunctional protein constructs comprising a first binding moiety that specifically binds to a muscle-specific molecule and a second binding moiety that specifically binds to and activates the Notch receptor. The present invention also relates to methods for preparing these bifunctional protein constructs and their use in treating muscle-related diseases, such as muscular dystrophy and sarcopenia. The present invention also provides engineered DLL4 extracellular domains (ECDs) and protein constructs comprising the same.

Muscular dystrophy (MD) is a diverse group of muscle diseases with numerous subtypes, characterized by varying degrees of muscle weakness and degeneration. MD is typically associated with defects in one or more genes encoding proteins critical for muscle integrity or function. Degenerative MD is typically caused by defects in structural proteins directly or indirectly associated with the dystrophin-related glycoprotein complex (DGC), which is essential for maintaining muscle cell membrane integrity.

A direct link between MD and Notch signaling in humans was established in limb-girdle muscular dystrophy (LGMD R21), which is caused by double mutations in the protein O-glucosyltransferase 1 (POGLUT1) gene, impaired Notch signaling, and satellite cell loss (Servián-Morilla et al., 2016, EMBO Mol Med. 8(11):1289-1309; Servián-Morilla et al., 2020, Acta Neuropathol. 139(3):565-582).

All references, including patent applications, patent publications, and Genbank accession numbers, cited herein are incorporated by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

In one aspect, the present application provides a bifunctional protein construct comprising a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to a notch receptor and activates the notch receptor.

In some embodiments of the bifunctional protein constructs described above, the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or within the basal lamina. In some embodiments, the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlecan, and M-calcification protein (CDH15). In some embodiments, the target antigen is a component of the dystrophin-related glycoprotein complex (DGC). In some embodiments, the target antigen is selected from the group consisting of α-dystroglycan (α-DG), β-DG, laminin-211, perlein, collagen, α-sarcosin, β-sarcosin, γ-sarcosin, δ-sarcosin, ε-sarcosin, ζ-sarcosin, biglycan, sarcospan, and matrix glycans on α-DG. In some embodiments, the target antigen is laminin subunit α-2 (LAMA2), CDH15, α-DG, or matrix glycans on α-DG.

In some embodiments of the bifunctional protein constructs described above, the first binding moiety comprises an antibody portion that specifically binds to a muscle-specific molecule. In some embodiments, the antibody portion is selected from the group consisting of a full-length antibody, Fab, Fab', F(ab') ₂ , scFv, and sdAb.

In some embodiments of the bifunctional protein constructs described above, the antibody portion specifically binds to LAMA2 (anti-LAMA2 antibody portion). In some embodiments, the anti-LAMA2 antibody portion comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: (iii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (iv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (v) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (vi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (vii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (viii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (ix) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. (i) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (x) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xiii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xiv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (xvi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xvii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xviii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. (xxi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 64; (xiii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66. (xxii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xxiii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xxiv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (xxv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xxvi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xxvii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. (xxviii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xxix) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xxx) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65. (xxxi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xxxii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xxxiii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xxxiv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xxxv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xxxvi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. (xxxvii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xxxviii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xxxix) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xl) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xli) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xlii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xliv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xlv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. (xlvii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xlviii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xlviv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67. 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xlix) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (l) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (li) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (i) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (ii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (iii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (iv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: (iv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (lvi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (lvii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (iviii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (lix) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (lx) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (i) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (ii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (iii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (i) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; (i) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (ii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (iii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (lxvii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (lxviii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxix) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 6 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxx) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxxi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxxii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: (lxxiv) HC-CDR1, HC-CDR2 and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2 and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 65; (lxxv) HC-CDR1, HC-CDR2 and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2 and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 65; (lxxv) HC-CDR1, HC-CDR2 and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2 and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 66. (lxxvi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (lxxvii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (lxxvii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxxviii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 6 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxxix) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxxx) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxxxi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: (lxxxiii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (lxxxiv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 65, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66. 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (lxxxv) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (lxxxvi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxxxvii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 68, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 69; (lxxxviii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxxxix) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xc) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 72; (xci) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73; (xcii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65; (xciii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 65, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66; (xciv) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67; (xcv) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; (xcvi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68; 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69; (xcvii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70; (xcviii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71; (xcix) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. (c) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72; or (d) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-LAMA2 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments of any of the bifunctional protein constructs described above, the anti-LAMA2 antibody portion is an anti-LAMA2 scFv. In some embodiments, the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-LAMA2 antibody portion is an anti-LAMA2 Fab. In some embodiments, the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 52 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 53.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the anti-LAMA2 antibody portion comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of any of the bifunctional protein constructs described above, the anti-LAMA2 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 67; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 68; (v) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 69; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (vii) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 71. NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (viii) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 72; (ix) a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 73; (x) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (xi) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (xii) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 67; (xiii) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: (xv) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (xvi) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (xvii) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 72; (xviii) a VH comprising the amino acid sequence of SEQ ID NO: 55 and a VL comprising the amino acid sequence of SEQ ID NO: 73; (xix) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: (xx) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (xx) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (xxi) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 67; (xxii) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 68; (xxiii) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 69; (xxiv) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (xxv) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (xxvi) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 72. NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 72; (xxvii) a VH comprising the amino acid sequence of SEQ ID NO: 56 and a VL comprising the amino acid sequence of SEQ ID NO: 73; (xxviii) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (xxix) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (xxx) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 67; (xxxi) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 68; (xxxii) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 69. NO: 69; (xxxiii) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (xxxiv) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (xxxv) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 72; (xxxvi) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 73; (xxxvii) a VH comprising the amino acid sequence of SEQ ID NO: 58 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (xxxviii) a VH comprising the amino acid sequence of SEQ ID NO: 58 and a VL comprising the amino acid sequence of SEQ ID NO: (xli) a VH comprising the amino acid sequence of SEQ ID NO: 58 and a VL comprising the amino acid sequence of SEQ ID NO: 69; (xlii) a VH comprising the amino acid sequence of SEQ ID NO: 58 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (xliii) a VH comprising the amino acid sequence of SEQ ID NO: 58 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (xliv) a VH comprising the amino acid sequence of SEQ ID NO: 58 and a VL comprising the amino acid sequence of SEQ ID NO: (xlv) a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (xlvii) a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (xlviii) a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 67; (xlix) a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 68; (l) a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: (iv) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (lvi) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (lvii) a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 67; (lviii) a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 68; (lviv) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 69; (lv) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 63; (lviv) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 64; (lviii) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65 (1x) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (1x) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (1xiii) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 72; (1xiii) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 73. NO: 73; (lxiv) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (lxv) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (lxvi) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 67; (lxvii) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxviii) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxix) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: (lxx) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (lxx) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxxi) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 72; (lxxii) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 73; (lxxiii) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (lxxiv) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (lxxv) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: (lxxix) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxxx) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 72; (lxxxi) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: (lxxxv) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 68; (lxxxvi) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxxxvii) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 69; (lxxxviii) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (lxxxix) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 72; (xc) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 73; (xci) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 65; (xcii) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (xciii) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: (xcv) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 68; (xcv) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 69; (xcvi) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (xcvii) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 71; (xcviii) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: 72; or (xcix) a VH comprising the amino acid sequence of SEQ ID NO: 64 and a VL comprising the amino acid sequence of SEQ ID NO: VL of the amino acid sequence of 73.

In some embodiments of any of the bifunctional protein constructs described above, the anti-LAMA2 antibody portion is an anti-LAMA2 scFv. In some embodiments, the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145.

In some embodiments of any of the bifunctional protein constructs described above, the anti-LAMA2 antibody portion is an anti-LAMA2 Fab. In some embodiments, the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166.

In some embodiments of the bifunctional protein constructs described above, the antibody portion specifically binds to matrix glycans on α-DG (anti-matrix glycan antibody portion). In some embodiments, the anti-matrix antibody portion comprises: (i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79; (ii) HC-CDR1, HC-CDR2, and HC-CDR3 of the VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of the VL comprising the amino acid sequence of SEQ ID NO: 95; (iii) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: (iv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; (v) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (vi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 99; (i) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (vii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (viii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; (ix) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 98; (x) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (xi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (xii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 96, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: (xiii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (xiv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (xv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 94, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96. (xvi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (xvi) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; or (xvii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the anti-matrix antibody portion comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93 and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94-98.

In some embodiments of any of the bifunctional protein constructs described above, the anti-matrix glycan antibody portion comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 89 and a VL comprising the amino acid sequence of SEQ ID NO: 94; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 95; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 96; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 97; (v) a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 98; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 91 and a VL comprising the amino acid sequence of SEQ ID NO: (vii) a VH comprising the amino acid sequence of SEQ ID NO: 91 and a VL comprising the amino acid sequence of SEQ ID NO: 96; (viii) a VH comprising the amino acid sequence of SEQ ID NO: 91 and a VL comprising the amino acid sequence of SEQ ID NO: 97; (ix) a VH comprising the amino acid sequence of SEQ ID NO: 91 and a VL comprising the amino acid sequence of SEQ ID NO: 98; (x) a VH comprising the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ ID NO: 95; (xi) a VH comprising the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ ID NO: 96; (xii) a VH comprising the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ ID NO: 97; (xiii) a VH comprising the amino acid sequence of SEQ ID NO: 9 (xxvi) a VH comprising the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid sequence of SEQ ID NO: 97; or (xxvii) a VH comprising the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid sequence of SEQ ID NO: 98.

In some embodiments of any of the bifunctional protein constructs described above, the anti-matrix glycan antibody portion is an anti-matrix glycan scFv. In some embodiments, the anti-matrix glycan scFv comprises the amino acid sequence of SEQ ID NO: 99.

In some embodiments of any of the bifunctional protein constructs described above, the anti-matrix glycan antibody portion is an anti-matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 100 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 101.

In some embodiments of the bifunctional protein constructs described above, the antibody portion specifically binds to CDH15 (anti-CDH15 antibody portion). In some embodiments, the anti-CDH15 antibody portion comprises: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 102, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 103, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 105, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.

In some embodiments of the bifunctional protein construct according to any one of the above descriptions, the anti-CDH15 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 108 and a VL comprising the amino acid sequence of SEQ ID NO: 109.

In some embodiments of any of the bifunctional protein constructs described above, the anti-CDH15 antibody portion is an anti-CDH15 scFv. In some embodiments, the anti-CDH15 scFv comprises the amino acid sequence of SEQ ID NO: 110.

In some embodiments of any of the bifunctional protein constructs described above, the anti-CDH15 antibody portion is an anti-CDH15 Fab. In some embodiments, the anti-CDH15 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 111 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 112.

In some embodiments of any of the bifunctional protein constructs described above, the first binding moiety comprises a non-antibody portion that specifically binds to a muscle-specific molecule. In some embodiments, the non-antibody portion comprises a protein domain selected from the group consisting of the laminin G-like domain (LG domain) of laminin, the LG domain of agrin, the LG domain of nidogen, the LG domain of perlein, the laminin coiled-coil binding domain of agrin, and the laminin gamma binding domain of nidogen. In some embodiments, the non-antibody portion comprises the LG domain of laminin. In some embodiments, the non-antibody portion comprises the LG domain of laminin subunit alpha-1 (LAMA1), LAMA2, laminin subunit alpha-3 (LAMA3), laminin subunit alpha-4 (LAMA4), or laminin subunit alpha-5 (LAMA5). In some embodiments, the non-antibody portion comprises the amino acid sequence of any one of SEQ ID NOs: 113-117.

In some embodiments of any of the bifunctional protein constructs described above, the non-antibody portion comprises the LG domain of LAMA2. In some embodiments, the non-antibody portion comprises the LG4-5 domain of LAMA2 (LAMA2 LG4-5). In some embodiments, the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 118 or 119.

In some embodiments of any of the bifunctional protein constructs described above, the non-antibody portion comprises the LG domain of perlein. In some embodiments, the non-antibody portion comprises the amino acid sequence of SEQ ID NO: 120.

In some embodiments of any of the bifunctional protein constructs described above, the non-antibody portion comprises the LG domain of aggregin. In some embodiments, the non-antibody portion comprises the amino acid sequence of SEQ ID NO: 121.

In some embodiments of any of the bifunctional protein constructs described above, the non-antibody portion comprises a laminin coiled-coil binding domain of an aggrecan protein. In some embodiments, the laminin coiled-coil binding domain of an aggrecan protein comprises the amino acid sequence of SEQ ID NO: 122.

In some embodiments of any of the bifunctional protein constructs described above, the non-antibody portion comprises the laminin gamma binding domain of nidogenin. In some embodiments, the laminin gamma binding domain of nidogenin comprises the amino acid sequence of SEQ ID NO: 123 or 124.

In some embodiments of the bifunctional protein construct according to any one of the above descriptions, the second binding moiety comprises the extracellular domain (ECD) of a Notch ligand or a variant thereof, the Notch ligand being selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2.

In some embodiments of any of the bifunctional protein constructs described above, the second binding moiety comprises DLL4 ECD or a variant thereof. In some embodiments, the second binding moiety comprises DLL4 ECD, and wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, wherein the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the MNNL domain, DSL domain, and EGF1-5 domain of DLL4, or a sequence having at least about 90% sequence identity to the sequence of the MNNL domain, DSL domain, and EGF1-5 domain of DLL4. In some embodiments, the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267.

In some embodiments of the bifunctional protein constructs described above, the second binding moiety comprises DLL1 ECD or a variant thereof. In some embodiments, the second binding moiety comprises DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130.

In some embodiments of the bifunctional protein constructs described above, the second binding moiety comprises DLL3 ECD or a variant thereof. In some embodiments, the second binding moiety comprises DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131.

In some embodiments of any of the bifunctional protein constructs described above, the second binding moiety comprises Jag1 ECD or a variant thereof. In some embodiments, the second binding moiety comprises Jag1 ECD, and wherein the Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, wherein the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises the MNNL domain, DSL domain, and EGF1-6 domain of Jag1, or a sequence having at least about 90% sequence identity to the sequence of the MNNL domain, DSL domain, and EGF1-6 domain of Jag1. In some embodiments, the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.

In some embodiments of any of the bifunctional protein constructs described above, the second binding moiety comprises Jag2 ECD or a variant thereof. In some embodiments, the second binding moiety comprises Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135.

In some embodiments of any of the bifunctional protein constructs described above, the second binding moiety is an anti-notch antibody moiety that activates the notch receptor. In some embodiments, the anti-notch agonist antibody moiety is selected from the group consisting of: full-length antibody, Fab, Fab', F(ab') ₂ , scFv, and sdAb.

In some embodiments of any of the bifunctional protein constructs described above, the first binding moiety is fused to the second binding moiety via an optional linker. In some embodiments, the first binding moiety is fused to the N-terminus of the second binding moiety. In some embodiments, the first binding moiety is fused to the C-terminus of the second binding moiety. In some embodiments, the bifunctional protein construct comprises: (i) a second binding moiety comprising the ECD of a Notch ligand or a variant thereof, the Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and (ii) a first binding moiety comprising: (a) an antibody moiety, wherein the antibody moiety is an scFv, Fab, or sdAb that specifically binds to a muscle-specific molecule; or (b) a non-antibody moiety, wherein the non-antibody moiety comprises a protein domain selected from the group consisting of the LG domain of laminin, the LG domain of agrin, the LG domain of nidogen, and the LG domain of perlein. In some embodiments, the first binding moiety comprises two or more non-antibody moieties linked in tandem.

In some embodiments of any of the bifunctional protein constructs described above, the first binding moiety comprises LAMA2 LG4-5, which comprises the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NO: 126-129 and 260-267. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of any one of SEQ ID NO: 136-139. In some embodiments, the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 142 or 143. In some embodiments, the second binding moiety comprises a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 144.

In some embodiments of any of the bifunctional protein constructs described above, the first binding moiety is an anti-LAMA2 scFv. In some embodiments, the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of any one of SEQ ID NOs: 146-154.

In some embodiments of any of the bifunctional protein constructs described above, the first binding moiety comprises an anti-matrix glycan scFv. In some embodiments, the anti-matrix glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of any one of SEQ ID NOs: 156-164.

In some embodiments of the bifunctional protein constructs described above, the first binding moiety comprises a Fab that specifically binds to a muscle-specific molecule. In some embodiments, the second binding moiety is fused to the N-terminus of the VL of the Fab via an optional linker.

In some embodiments of the bifunctional protein constructs described above, the first binding moiety comprises an anti-LAMA2 Fab. In some embodiments, the anti-LAMA2 Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a fusion polypeptide comprising a second binding moiety and the second anti-LAMA2 Fab polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 167-175.

In some embodiments of the bifunctional protein constructs described above, the first binding moiety comprises an anti-matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a fusion polypeptide comprising a second binding moiety and the second anti-matrix glycan Fab polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 178-186.

In some embodiments of the bifunctional protein constructs described above, the first binding moiety is fused to the second binding moiety via a carrier protein. In some embodiments, the carrier protein is selected from the group consisting of human serum albumin (HSA), an anti-HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the first binding moiety is fused to the N-terminus of the carrier protein via an optional first linker, and the second binding moiety is fused to the C-terminus of the carrier protein via an optional second linker. In some embodiments, the first binding moiety is fused to the C-terminus of the carrier protein via an optional first linker, and the second binding moiety is fused to the N-terminus of the carrier protein via an optional second linker. In some embodiments, the bifunctional protein construct comprises: (i) a second binding moiety comprising the ECD of a Notch ligand or a variant thereof, the Notch ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and (ii) a first binding moiety comprising: (a) an antibody moiety, wherein the antibody moiety is an scFv, Fab, or sdAb that specifically binds to a muscle-specific molecule; or (b) a non-antibody moiety, wherein the non-antibody moiety comprises a protein domain selected from the group consisting of the LG domain of laminin, the LG domain of agrin, the LG domain of nidogen, and the LG domain of perlein. In some embodiments, the first binding moiety comprises two or more non-antibody moieties linked in tandem.

In some embodiments of any of the bifunctional protein constructs described above, the bifunctional protein construct further comprises an Fc domain comprising a first subunit and a second subunit. In some embodiments, the Fc domain is derived from human IgG1, human IgG2, or human IgG4. In some embodiments, the Fc domain is derived from human IgG1 comprising the amino acid sequence of SEQ ID NO: 187. In some embodiments, each subunit of the Fc domain comprises a mutation that reduces or abolishes effector function. In some embodiments, each subunit of the Fc domain comprises the L234A/L235A mutation (EU numbering).

In some embodiments of the bifunctional protein constructs described above, the first Fc domain unit or the second Fc domain unit comprises H435R/Y436F mutations (EU numbering). In some embodiments, the Fc domain comprises a knob-into-hole mutation, and wherein: i) the first Fc domain unit comprises a knob mutation and the second Fc domain unit comprises a hole mutation; or ii) the second Fc domain unit comprises a knob mutation and the first Fc domain unit comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, the Fc domain comprises charge pair mutations, and wherein: i) the amino acid residues in the first subunit of the Fc domain are replaced with positively charged residues, and the amino acid residues in the second subunit of the Fc domain are replaced with negatively charged residues; or ii) the amino acid residues in the first subunit of the Fc domain are replaced with negatively charged residues, and the amino acid residues in the second subunit of the Fc domain are replaced with positively charged residues. In some embodiments, the Fc domain comprises: i) the amino acid residue at D399 (EU numbering) in the first unit of the Fc domain is substituted with a positively charged residue, and the amino acid residue at K409 (EU numbering) in the second unit of the Fc domain is substituted with a negatively charged residue; or ii) the amino acid residue at K409 (EU numbering) in the first unit of the Fc domain is substituted with a negatively charged residue, and the amino acid residue at D399 (EU numbering) in the second unit of the Fc domain is substituted with a positively charged residue.

In some embodiments of any of the bifunctional protein constructs described above, each subunit of the Fc domain comprises a mutation that shortens half-life. In some embodiments, each subunit of the Fc domain comprises an H435A mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the bifunctional protein construct comprises: i) a first unit that specifically binds to a second binding moiety of a first nick receptor, and ii) a second unit that specifically binds to a second binding moiety of a second nick receptor, wherein the first unit of the second binding moiety and the second unit of the second binding moiety each independently comprise an ECD of a nick ligand or a variant thereof, the nick ligand being selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprise a DLL4 ECD, and wherein the DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 125-129. In some embodiments, the first unit of the second binding moiety is identical to the second unit of the second binding moiety. In some embodiments, the first unit of the second binding moiety is different from the second unit of the second binding moiety. In some embodiments, the bifunctional protein construct comprises: i) a first unit of a first binding moiety that specifically binds to a first muscle-specific molecule, and ii) a second unit of a first binding moiety that specifically binds to a second muscle-specific molecule. In some embodiments, the first unit of the first binding moiety is identical to the second unit of the first binding moiety. In some embodiments, the first unit of the first binding moiety is different from the second unit of the first binding moiety. In some embodiments, the first unit of the first binding moiety comprises a first antibody portion that specifically binds to a first muscle-specific molecule, the second unit of the first binding moiety comprises a second antibody portion that specifically binds to a second muscle-specific molecule, and wherein the first antibody portion and the second antibody portion are each independently selected from the group consisting of: Fab, scFv, and sdAb.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first Fab (Fab1), and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit specifically binding to the second binding portion of a first nick receptor - optionally a linker - a first unit of an Fc domain - optionally a linker - VH1-(H1-CH1); ii) a second polypeptide comprising, from N' to C': a second unit specifically binding to the second binding portion of a second nick receptor - optionally a linker - The second unit of the Fc domain - optionally a linker - VH2-(H2-CH1); iii) a third polypeptide comprising, from N' to C', VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C', VL2-(L2-CL); wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the first antibody portion is a first Fab (Fab1), and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit specifically binding to the second binding portion of the first nicked receptor - optionally a linker - a first unit of the Fc domain - optionally a linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': a second unit specifically binding to the second binding portion of the second nicked receptor - optionally a linker - The invention further comprises the following: a first polypeptide comprising, from N' to C', a linker (if present), a second Fc domain unit (if present), a linker (if present), and a second Fc domain unit (if present); iii) a third polypeptide comprising, from N' to C', VH1-(H1-CH1); and iv) a fourth polypeptide comprising, from N' to C', VH2-(H2-CH1); wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

In some embodiments of any of the bifunctional protein constructs described above, the bifunctional protein construct comprises Fab1 and Fab2, both of which specifically bind to LAMA2. In some embodiments, the anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of any one of SEQ ID NOs: 189-192. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, and the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 193. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 194. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. In some embodiments, the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 195 or 196. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 165, and the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 197.

In some embodiments of any of the bifunctional protein constructs described above, the bifunctional protein construct comprises Fab1 and Fab2, both of which specifically bind to matrix glycans. In some embodiments, anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the third polypeptide and the fourth polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 176, and wherein: (i) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of any one of SEQ ID NOs: 198-201; (ii) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 202; (iii) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 203; (iv) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 204 or 205; or (v) the first polypeptide and the second polypeptide of the bifunctional protein construct each comprise the amino acid sequence of SEQ ID NO: 206.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first sdAb (sdAb1), and the second antibody portion is a second sdAb (sdAb2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit-optionally a linker-Fc domain that specifically binds to a first nicked receptor; and ii) a second polypeptide comprising, from N' to C', a second unit-optionally a linker-Fc domain that specifically binds to a second nicked receptor; and ii) a second polypeptide comprising, from N' to C', a second unit-optionally a linker-Fc domain that specifically binds to a second nicked receptor. In some embodiments, the first antibody portion is a first scFv (scFv1), and the second antibody portion is a second scFv (scFv2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit specifically binding to the second binding portion of a first nick receptor, optionally a linker, and an Fc domain; and ii) a second polypeptide comprising, from N' to C', a second unit specifically binding to the second binding portion of a second nick receptor, optionally a linker, and an Fc domain; and optionally a linker and scFv2.

In some embodiments of any of the bifunctional protein constructs described above, the bifunctional protein construct comprises scFv1 and scFv2, both of which specifically bind to LAMA2. In some embodiments, the anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprises the amino acid sequence of SEQ ID NO: 145 or 268. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NO: 207-222. In some embodiments, the first Fc domain subunit and the second Fc domain subunit each comprise the amino acid sequence of SEQ ID NO: 188. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise the amino acid sequence of any one of SEQ ID NOs: 223-226. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO: 227. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO: 228. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO: 229 or 230. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 231.

In some embodiments of the bifunctional protein constructs described above, the bifunctional protein construct comprises scFv1 and scFv2, both of which specifically bind to matrix glycans. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each of which comprises: (i) the amino acid sequence of any one of SEQ ID NOs: 232-235; (ii) the amino acid sequence of SEQ ID NO: 236; (iii) the amino acid sequence of SEQ ID NO: 237; (iv) the amino acid sequence of SEQ ID NOs: 238 or 239; or (v) the amino acid sequence of SEQ ID NO: 240.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first Fab (Fab1), and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': VH1-(H1-CH1)-optionally a linker-a first unit of an Fc domain-optionally a linker-a first unit of a second binding moiety that specifically binds to a first notched receptor; ii) a second polypeptide comprising, from N' to C': VH2- (H2-CH1)-optional linker-second unit of the Fc domain-optional linker-second unit that specifically binds to the second binding portion of the second nicked receptor; iii) a third polypeptide comprising, from N' to C': VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C': VL2-(L2-CL); wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first sdAb (sdAb1), and the second antibody portion is a second sdAb (sdAb2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': sdAb1 - optionally a linker - a first unit of an Fc domain - optionally a linker - a first unit that specifically binds to a second binding moiety of a first nicked receptor; and ii) a second polypeptide comprising, from N' to C': sdAb2 - optionally a linker - a second unit of an Fc domain - optionally a linker - a second unit that specifically binds to a second binding moiety of a second nicked receptor.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first scFv (scFv1), and the second antibody portion is a second scFv (scFv2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': scFv1 - optionally a linker - a first unit of an Fc domain - optionally a linker - a first unit that specifically binds to the second binding portion of a first nicked receptor; and ii) a second polypeptide comprising, from N' to C': scFv2 - optionally a linker - a second unit of an Fc domain - optionally a linker - a second unit that specifically binds to the second binding portion of a second nicked receptor.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first Fab (Fab1), and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first subunit of VH1-(H1-CH1)-optionally a linker-Fc domain; ii) a second polypeptide comprising, from N' to C', a second subunit of VH2-(H2-CH1)-optionally a linker-Fc domain; iii) a third A polypeptide comprising, from N' to C', VL1-(L1-CL)-optionally a linker-a first unit that specifically binds to the second binding portion of a first nicked receptor; and iv) a fourth polypeptide comprising, from N' to C', VL2-(L2-CL)-optionally a linker-a second unit that specifically binds to the second binding portion of a second nicked receptor; wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first Fab (Fab1), and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first subunit of VH1-(H1-CH1)-optionally a linker-Fc domain; ii) a second polypeptide comprising, from N' to C', a second subunit of VH2-(H2-CH1)-optionally a linker-Fc domain; iii) a third A polypeptide comprising, from N' to C', a first unit that specifically binds to the second binding portion of a first nick receptor - optionally a linker - VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C', a second unit that specifically binds to the second binding portion of a second nick receptor - optionally a linker - VL2-(L2-CL); wherein VL1-(L1-CL) forms Fab1 with VH1-(H1-CH1), and VH2-(H2-CH1) forms Fab2 with VL2-(L2-CL).

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first Fab (Fab1), and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding portion that specifically binds to a first nicked receptor - optionally a linker - VH1-(H1-CH1) - optionally a linker - a first unit of an Fc domain; ii) a second polypeptide comprising, from N' to C': a first unit of the second binding portion that specifically binds to a first nicked receptor - optionally a linker - VH1-(H1-CH1) - optionally a linker - a first unit of an Fc domain; the second unit of the second binding portion of the second notched receptor - optionally a linker - VH2-(H2-CH1) - optionally a linker - a second unit of the Fc domain; iii) a third polypeptide comprising, from N' to C', VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C', VL2-(L2-CL); wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the bifunctional protein construct further comprises: a third unit of the second binding moiety that specifically binds to a third nick receptor, and a fourth unit of the second binding moiety that specifically binds to a fourth nick receptor, wherein the third unit of the second binding moiety and the fourth unit of the second binding moiety each independently comprise an ECD of a nick ligand or a variant thereof, the nick ligand being selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2. In some embodiments, the third unit of the second binding moiety and the fourth unit of the second binding moiety each independently comprise an ECD of DLL4 or a variant thereof. In some embodiments, the third unit of the second binding moiety and/or the fourth unit of the second binding moiety comprises a DLL4 ECD, wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the third unit of the second binding moiety and/or the fourth unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the MNNL domain, DSL domain, and EGF1-5 domain of DLL4. In some embodiments, the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. In some embodiments, all four units of the second binding moiety are identical. In some embodiments, at least one of the four units of the second binding moiety is different from the other units.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the first antibody portion is a first Fab (Fab1), and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit specifically binding to the second binding portion of a first notch receptor - optionally a linker - VH1-(H1-CH1) - optionally a linker - a first subunit of an Fc domain; ii) a second polypeptide comprising, from N' to C': a second unit specifically binding to the second binding portion of a second notch receptor - optionally a linker - VH2 -(H2-CH1)-optionally a linker-a second unit of the Fc domain; iii) a third polypeptide comprising, from N' to C': a third unit that specifically binds to the second binding portion of a third nick receptor-optionally a linker-VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C': a fourth unit that specifically binds to the second binding portion of a fourth nick receptor-optionally a linker-VL2-(L2-CL); wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2.

In some embodiments of the bifunctional protein constructs described above, the first unit of the first binding moiety comprises a first non-antibody moiety that specifically binds to a first muscle-specific molecule, the second unit of the first binding moiety comprises a second non-antibody moiety that specifically binds to a second muscle-specific molecule, and wherein the first non-antibody moiety and the second non-antibody moiety are each independently selected from the group consisting of the LG domain of laminin, the LG domain of agrin, the LG domain of nidogen, and the LG domain of perlein. In some embodiments, the first unit of the first binding moiety comprises two or more first non-antibody moieties linked in series, and the second unit of the first binding moiety comprises two or more second non-antibody moieties linked in series. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises LAMA2 LG4-5, which comprises the amino acid sequence of SEQ ID NO: 118 or 119.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the bifunctional protein construct comprises: i) a first polypeptide comprising, from N′ to C′: a first unit of a first binding moiety, optionally a linker, a first unit of an Fc domain, optionally a linker, a first unit of a second binding moiety that specifically binds to a first nicked receptor; and ii) a second polypeptide comprising, from N′ to C′: a second unit of the first binding moiety, optionally a linker, a second unit of an Fc domain, optionally a linker, a second unit of a second binding moiety that specifically binds to a second nicked receptor.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the bifunctional protein construct comprises: i) a first polypeptide comprising, from N′ to C′, a first unit that specifically binds to the second binding moiety of a first nick receptor, optionally a linker, a first unit that binds to the Fc domain, optionally a linker, and a first unit of the first binding moiety; and ii) a second polypeptide comprising, from N′ to C′, a second unit that specifically binds to the second binding moiety of a second nick receptor, optionally a linker, and a second unit that binds to the Fc domain, optionally a linker, and a second unit of the first binding moiety.

In some embodiments of the bifunctional protein constructs described above, the first unit of the first binding moiety comprises two LAMA2 LG4-5 linked in tandem, and the second unit of the first binding moiety comprises two LAMA2 LG4-5 linked in tandem. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 241-244. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO: 245. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO: 246. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO: 247 or 248. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the bifunctional protein construct comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO: 249.

In some embodiments of the bifunctional protein constructs described above, i) the first binding moiety is fused to the N-terminus of the first Fc domain subunit via a first linker, and the second binding moiety is fused to the N-terminus of the second Fc domain subunit via a second linker, or ii) the first binding moiety is fused to the N-terminus of the second Fc domain subunit via a first linker, and the second binding moiety is fused to the N-terminus of the first Fc domain subunit via a second linker. In some embodiments, the first binding moiety is a Fab that specifically binds to a muscle-specific molecule, and the C-terminus of the CH1 of the Fab is fused to the N-terminus of the first Fc domain subunit or the second Fc domain subunit via a first linker, or In some embodiments, the second binding moiety comprises the ECD of a Notch ligand or a variant thereof, wherein the Notch ligand is selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the bifunctional protein construct comprises: i) a first unit that specifically binds to a second binding moiety of a first nick receptor, and ii) a second unit that specifically binds to a second binding moiety of a second nick receptor; wherein the first unit of the second binding moiety is fused to the N-terminus of the first unit of the Fc domain via an optional first linker, and the second unit of the second binding moiety is fused to the N-terminus of the second unit of the Fc domain via an optional second linker; and wherein the first binding moiety is fused to the C-terminus of the first unit of the Fc domain or the second unit of the Fc domain via an optional third linker. In some embodiments, the bifunctional protein construct comprises: i) a first unit that specifically binds to a first binding moiety of a first muscle-specific molecule, and ii) a second unit that specifically binds to a first binding moiety of a second muscle-specific molecule; wherein the first unit of the first binding moiety is fused to the C-terminus of the first unit of the Fc domain via an optional third linker, and the second unit of the first binding moiety is fused to the C-terminus of the second unit of the Fc domain via an optional fourth linker.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the second binding moiety is fused to the N-terminus of the first subunit of the Fc domain via a linker, if applicable; wherein the bifunctional protein construct comprises one or more subunits of the first binding moiety that specifically bind to one or more muscle-specific molecules; and wherein each of the one or more subunits of the first binding moiety is independently fused via a linker, if applicable, to one of: i) the C-terminus of the first subunit of the Fc domain; ii) the N-terminus of the second subunit of the Fc domain; and iii) the C-terminus of the second subunit of the Fc domain.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the second binding moiety is fused to the N-terminus of the second subunit of the Fc domain via a linker, if applicable; wherein the bifunctional protein construct comprises one or more subunits of the first binding moiety that specifically bind to one or more muscle-specific molecules; and wherein each of the one or more subunits of the first binding moiety is independently fused via a linker, if applicable, to one of: i) the N-terminus of the first subunit of the Fc domain; ii) the C-terminus of the first subunit of the Fc domain; and iii) the C-terminus of the second subunit of the Fc domain.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the bifunctional protein construct comprises: i) a first unit that specifically binds to a second binding moiety of a first nick receptor, and ii) a second unit that specifically binds to a second binding moiety of a second nick receptor; wherein the first unit of the second binding moiety is fused to the C-terminus of the first unit of the Fc domain via an optional first linker, and the second unit of the second binding moiety is fused to the C-terminus of the second unit of the Fc domain via an optional second linker; and wherein the first binding moiety is fused to the N-terminus of the first unit of the Fc domain or the second unit of the Fc domain via an optional third linker. In some embodiments, the bifunctional protein construct comprises: i) a first unit that specifically binds to a first binding moiety of a first muscle-specific molecule, and ii) a second unit that specifically binds to a first binding moiety of a second muscle-specific molecule; wherein the first unit of the first binding moiety is fused to the N-terminus of the first unit of the Fc domain via an optional third linker, and the second unit of the first binding moiety is fused to the N-terminus of the second unit of the Fc domain via an optional fourth linker.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the second binding moiety is fused to the C-terminus of the first subunit of the Fc domain via a linker, if applicable; wherein the bifunctional protein construct comprises one or more subunits of the first binding moiety that specifically bind to one or more muscle-specific molecules; and wherein each of the one or more subunits of the first binding moiety is independently fused via a linker, if applicable, to one of: i) the N-terminus of the first subunit of the Fc domain; ii) the N-terminus of the second subunit of the Fc domain; and iii) the C-terminus of the second subunit of the Fc domain.

In some embodiments of the bifunctional protein construct according to any of the above descriptions, the second binding moiety is fused to the C-terminus of the second subunit of the Fc domain via a linker, if applicable; wherein the bifunctional protein construct comprises one or more subunits of the first binding moiety that specifically bind to one or more muscle-specific molecules; and wherein each of the one or more subunits of the first binding moiety is independently fused via a linker, if applicable, to one of: i) the N-terminus of the first subunit of the Fc domain; ii) the C-terminus of the first subunit of the Fc domain; and iii) the N-terminus of the second subunit of the Fc domain.

In some embodiments of the bifunctional protein constructs according to any of the above descriptions, i) one or more units of the second binding moiety each independently comprises an ECD of a Notch ligand or a variant thereof, wherein the Notch ligand is selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and ii) one or more units of the first binding moiety each independently comprises an antibody portion that specifically binds to a muscle-specific molecule, wherein the antibody portion is independently selected from the group consisting of scFv, Fab, and sdAb. In some embodiments, i) the two units of the second binding moiety are identical; and/or ii) the two or more units of the first binding moiety are identical. In some embodiments, i) the two units of the second binding moiety are different; and/or ii) at least one of the two or more units of the first binding moiety is different from the other units. In some embodiments, the Fc domain comprises a knob-in-hole mutation and/or a charge pair mutation. In some embodiments, (i) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 348, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 255; (ii) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 255, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 348; (iii) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 256, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 257; (iv) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 257, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 256; (v) the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 258, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: or (vi) the first Fc domain subunit comprises the amino acid sequence of SEQ ID NO: 259, and the second Fc domain subunit comprises the amino acid sequence of SEQ ID NO: 258.

In another aspect, provided herein is one or more isolated nucleic acids encoding any of the bifunctional protein constructs described above.

In another aspect, provided herein are one or more vectors comprising one or more isolated nucleic acids described above. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

In another aspect, provided herein is a host cell expressing any of the bifunctional protein constructs described above, one or more isolated nucleic acids described above, or one or more vectors described above. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell or a HEK293 cell.

Also provided are pharmaceutical compositions comprising: i) any of the bifunctional protein constructs described above, one or more isolated nucleic acids described above, or one or more vectors described above; and ii) a pharmaceutically acceptable excipient.

In another aspect, provided herein is a method for producing a bifunctional protein construct, comprising: i) culturing a host cell comprising one or more isolated nucleic acids or one or more vectors described above, or a host cell described above, under conditions suitable for expression of the bifunctional protein construct; and ii) recovering the expressed bifunctional protein construct from the cultured host cell. In some embodiments, the method further comprises introducing the one or more isolated nucleic acids or the one or more vectors into the host cell.

In another aspect, provided herein is a method for treating a muscle-related disease in a subject, comprising administering to the subject an effective amount of any of the bifunctional protein constructs described above or the pharmaceutical composition described above. In some embodiments, the muscle-related disease is selected from the group consisting of Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing distal myopathy, myofibrillar myopathy, and muscular dystrophy. In some embodiments, the muscle-related disease is muscular dystrophy. In some embodiments, the muscular dystrophy comprises one or more of: Duchenne muscular dystrophy (DMD), Beck muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital myopathy, Charcot-Marie-Tooth (CMT) disease, and Emery-Dreifuss muscular dystrophy (EDMD). In some embodiments, the bifunctional protein construct or the pharmaceutical composition is administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the subject is a human.

In another aspect, the present invention provides an antibody construct comprising an antibody portion that specifically recognizes plasmoglycan on α-DG (anti-plasmoglycan antibody portion), wherein the anti-plasmoglycan antibody portion comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79; (ii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: (iii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (iv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; (v) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: (viii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (viii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (viii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: (i) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (ii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (iii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95; (xiii) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98; (xiv) HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 94. (xv) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96; (xvi) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97; or (xvii) HC-CDR1, HC-CDR2 and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2 and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: LC-CDR1, LC-CDR2 and LC-CDR3 of VL of the amino acid sequence of 98.

In some embodiments of the antibody constructs described above, the anti-matrix antibody portion comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93 and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94-98. In some embodiments, the anti-matrix antibody portion comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 89 and a VL comprising the amino acid sequence of SEQ ID NO: 94; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 95; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 96; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 97; (v) a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 98; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 91 and a VL comprising the amino acid sequence of SEQ ID NO: (x) a VH comprising the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ ID NO: 95; (xi) a VH comprising the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ ID NO: 96; (xii) a VH comprising the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ ID NO: 97; (xiii) a VH comprising the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid sequence of SEQ ID NO: 94; (xiv) a VH comprising the amino acid sequence of SEQ ID NO: 94 and a VL comprising the amino acid sequence of SEQ ID NO: 95; (xv) a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO: 96; (xv) a VH comprising the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid sequence of SEQ ID NO: 97; (xv) a VH comprising the amino acid sequence of SEQ ID NO: 94 and a VL comprising the amino acid sequence of SEQ ID NO: 97 (xvi) a VH comprising the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid sequence of SEQ ID NO: 97; or (xvii) a VH comprising the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid sequence of SEQ ID NO: 98.

In some embodiments of the antibody constructs described above, the anti-matrix glycan antibody portion is selected from the group consisting of a full-length antibody, a Fab, a Fab', a F(ab') ₂ , and a scFv. In some embodiments, the anti-matrix glycan antibody portion is an anti-matrix glycan scFv. In some embodiments, the anti-matrix glycan scFv comprises the amino acid sequence of SEQ ID NO: 99.

In some embodiments of the antibody constructs described above, the anti-matrix glycan antibody portion is an anti-matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 100 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 101.

In some embodiments of the antibody constructs described above, the anti-matrix glycan antibody portion is an anti-matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 100 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 101.

In some embodiments of the antibody constructs described above, the anti-matrix glycan antibody portion is a full-length anti-matrix glycan antibody. In some embodiments, the full-length anti-matrix glycan antibody comprises an Fc domain derived from human IgG1, human IgG2, or human IgG4. In some embodiments, the Fc domain is derived from human IgG1 comprising the amino acid sequence of SEQ ID NO: 187. In some embodiments, each subunit of the Fc domain comprises an L234A/L235A mutation (EU numbering). In some embodiments, each subunit of the Fc domain comprises an H435A mutation (EU numbering). In some embodiments, each subunit of the Fc domain comprises an L234A/L235A/P329G mutation (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277.

In some embodiments of the antibody constructs described above, the antibody construct further comprises a second binding moiety that specifically binds to a second target molecule. In some embodiments, the second binding moiety specifically binds to and activates a nicked receptor. In some embodiments, the second binding moiety comprises the ECD of a nicked ligand or a variant thereof, the nicked ligand selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2.

In another aspect, the present invention provides an engineered DLL4 extracellular domain (ECD), wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of T52N and T135N, and wherein the amino acid positions are relative to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, the engineered DLL4 ECD further comprises a mutation at one or more amino acid positions selected from the group consisting of G2, E14, R66, P80, F81, H168, Q220, N231, and N260. In some embodiments, the additional mutation is selected from the group consisting of G2S, E14H, R66S, R66T, P80L, F81L, H168Y, Q220H, N231D, and N260D. In some embodiments, the engineered DLL4 ECD comprises a mutation selected from the group consisting of: (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (v) G2S, T52N, F81L, T135N, and H168Y; (vi) G2S, T52N, F81L, R66S, T135N, and H168Y; (vii) In some embodiments, the engineered DLL4 ECD comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 261-264.

In another embodiment of any of the engineered DLL4 ECDs described above, the present invention provides a protein construct comprising the engineered DLL4 ECD. In some embodiments, the protein construct further comprises a binding moiety that specifically binds to a muscle-specific molecule.

It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form further embodiments of the invention.

Cross-Reference to Related Applications This patent application claims priority to U.S. Provisional Patent Application No. 63/639,356, filed April 26, 2024, and U.S. Provisional Patent Application No. 63/537,382, filed September 8, 2023, the contents of each of which are incorporated herein by reference in their entirety. Citation of Electronic Sequence Listing

The contents of the electronic sequence listing (340742000141SEQLIST.xml; size: 457,221 bytes; creation date: September 5, 2024) are incorporated herein by reference in their entirety.

This application provides bifunctional protein constructs that specifically target and deliver Notch-activated muscle tissue in a spatiotemporally controlled manner. The bifunctional protein constructs bind to muscle tissue via proteins on the myofibril membrane or in the extracellular matrix (basal lamina), thereby anchoring the bifunctional protein construct and the Notch-activating moiety (e.g., a Notch ligand or an agonist antibody that specifically recognizes Notch 1-4) contained therein, which is required for Notch signaling. Thus, the bifunctional protein constructs disclosed in this disclosure meet three requirements for specific Notch signaling in muscle: 1) targeting to muscle to ensure tissue specificity; 2) anchoring of the Notch ligand required for transactivation; and 3) timely control of Notch activation to renew satellite cells while allowing myoblast differentiation during muscle repair.

Therefore, in one aspect, the present application provides a bifunctional protein construct comprising a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to a notch receptor and activates the notch receptor.

In another aspect, a method for preparing a bifunctional protein construct described herein is provided. Also provided are isolated nucleic acids, vectors, and host cells encoding any of the bifunctional protein constructs. Also provided are methods for treating muscle-related diseases in an individual (e.g., a human) by administering to the individual an effective amount of a bifunctional protein construct described herein or a pharmaceutical composition thereof.

In another aspect, an engineered DLL4 extracellular domain (ECD) is provided that comprises a mutation selected from the group consisting of T52N and T135N, wherein the amino acid positions are relative to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. Protein constructs comprising the DLL4 ECD described herein are also provided. I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, exemplary methods and materials are described.

The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

"Antibody" may refer to an immunoglobulin molecule or fragment thereof (including a basic four-chain antibody unit) that is capable of specifically binding to a particular antigenic determinant of an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies of the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), antigen-binding fragments (such as Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ ), as well as single-chain antibodies (scFv), heavy-chain antibodies such as camel antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

A full-length antibody consists of two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. The variable regions of both chains typically contain three hypervariable loops, known as complementation-determining regions (CDRs). (Light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3.) The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or designated according to the rules of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are interposed between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified into several classes based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, a bifunctional antibody, Fab, Fab', F(ab') ₂ , an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized bifunctional antibody (dsbifunctional antibody), a single-chain Fv (scFv), an scFv dimer (a bivalent bifunctional antibody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single-domain antibody (sdAb) (e.g., a camelized single-domain antibody), a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise the structure of a complete antibody. The antigen-binding fragment can bind to the same antigen as the parent antibody or parent antibody fragment (e.g., parent scFv). In some embodiments, the antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This fragment consists of a dimer of a heavy chain variable region and a light chain variable region in tight, non-covalent association. These two domains fold to form six hypervariable loops (three loops each in the heavy and light chains), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even though a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can recognize and bind antigen, its affinity is lower than that of the entire binding site.

"Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising VH and VL antibody domains linked into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the required structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies , 113th ed., Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

The basal four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basal four-chain units and an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2-5 basal four-chain units that can aggregate to form multivalent assemblies with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to the H chain via a covalent disulfide bond, and the two H chains are linked to each other via one or more disulfide bonds, depending on the H chain isotype. Each H chain and L chain also possesses regularly spaced intrachain disulfide bridges. Each H chain possesses a variable domain (VH) at its N-terminus, followed by three constant domains (CH) of each of the α and γ chains, as well as four CH domains of the μ and ε isotypes. Each L chain possesses a variable domain (VL) at its N-terminus, followed by a constant domain at its opposite end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. The VH and VL pair together to form a single antigen-binding site. For information on the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. L chains from any vertebrate species can be assigned to one of two distinct types, designated kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of their heavy chain constant domains (CH), immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. Based on the relatively small differences in CH sequence and function, the gamma and alpha classes are further divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

The Fc fragment comprises the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector function of an antibody is determined by sequences within the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The heavy and light chain variable domains are referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site. Heavy-chain-only antibodies from species of the Camelidae family possess a single heavy-chain variable region, designated "VHH." Therefore, VHH is a specialized type of VH.

The term "variable" refers to the fact that the sequences of certain segments of the variable domains vary widely between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in the light chain variable domain and the heavy chain variable domain. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions connected by three HVRs, primarily in a β-sheet configuration. The HVRs form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The HVRs in each chain are held together by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 15th ed., National Institute of Health, Bethesda, Md. (1991)). The homeodomains are not directly involved in antibody-antigen binding but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or possible minor post-translational modifications (e.g., isomerization, amidation). Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used in accordance with the present application can be produced by a variety of techniques, including, for example, hybridoma methods (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., Monoclonal Antibodies and T - Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and techniques for producing human or human-like antibodies in animals that have part or all of a human immunoglobulin locus or gene encoding a human immunoglobulin sequence (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, a full-length four-chain antibody includes an antibody having heavy and light chains, including an Fc region. The constant domain can be a native sequence constant domain (e.g., a native human constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may possess one or more effector functions.

The term "bifunctional antibody" refers to small antibody fragments prepared by constructing an sFv fragment (see previous paragraph) between the VH and VL domains using a short linker (approximately 5-10 residues), achieving interchain, rather than intrachain, pairing of the V domains, thereby generating a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific bifunctional antibodies are heterodimers of two "crossover" sFv fragments, in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Bifunctional antibodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chains is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATTZFACTOR D® antibodies, in which the antigen-binding region of the antibody is derived from an antibody generated, for example, by immunizing macaques with the antigen of interest. As used herein, "humanized antibodies" are used as a subset of "chimeric antibodies."

As used herein, the term "CDR" or "complementary determining region" is intended to refer to the non-contiguous antigen combining sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described in: Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest"(1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol. , 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MP et al., Dev. Comp. Immunol. , 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol. , 309:657-670 (2001), wherein these definitions include overlaps or subgroups of amino acid residues when compared to one another. However, CDRs or variants thereof of antibodies or grafted antibodies referred to using any of the definitions are intended to be within the scope of the terms as defined and used herein. The amino acid residues encompassing the CDRs defined in each of the above-cited references are shown in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res. , 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res. , 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more of the technical solutions herein. Table 1: CDR Definition Kabat ¹ Chothia ² MacCallum ³ IMGT ⁴ Aho ⁵ VH CDR1 31-35 26-32 30-35 27-38 25-40 VH CDR2 50-65 53-55 47-58 56-65 58-77 VH CDR3 95-102 96-101 93-101 105-117 109-137 VL CDR1 24-34 26-32 30-36 27-38 25-40 VL CDR2 50-56 50-52 46-55 56-65 58-77 VL CDR3 89-97 91-96 89-96 105-117 109-137 ^1. Residue numbering follows the nomenclature of Kabat et al. ^2. Residue numbering follows the nomenclature of Chothia et al. ^3. Residue numbering follows the nomenclature of MacCallum et al. ^4. Residue numbering follows the nomenclature of Lefranc et al. ^5. Residue numbering follows the nomenclature of Honegger and Plückthun

The phrase "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refers to the numbering system of the aforementioned Kabat et al. for heavy chain variable domains or light chain variable domains used for antibody compilation. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of a FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). For a given antibody, the Kabat numbering of the residues can be determined by aligning the homologous regions of the antibody sequence with the "standard" Kabat numbering sequence.

Unless otherwise indicated herein, the numbering of residues in immunoglobulin heavy chains is as per the EU index in Kabat et al., supra, with minor modifications. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

"Framework" or "FR" residues are those variable domain residues other than the CDR residues as defined herein.

As used herein, "immunoassay" refers to any binding assay that uses antibodies capable of specifically binding to a target molecule to detect and quantify the target molecule.

"Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human antibodies. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable regions (HVRs) of the recipient are replaced with residues from the hypervariable regions of a non-human species (donor antibodies; such as mouse, rat, rabbit, or non-human primate, which possess the desired antibody specificity, affinity, and potency). In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in either the recipient or donor antibodies. These modifications are made to further improve antibody potency. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are FRs of human immunoglobulin sequences. A humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Suitable human acceptor antibodies can be selected from databases based on nucleotide and amino acid sequence homology to the donor antibody, such as the KABAT database, the Los Alamos database, the AbM database, and the Swiss Protein database. Human antibodies characterized by amino acid homology to the framework regions of the donor antibody can be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies capable of providing light chain constant regions or variable framework regions can be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains do not need to originate from the same acceptor antibody. The prior art describes several methods for generating such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951). For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human and/or produced using any of the human antibody production techniques disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). In addition, methods for preparing human monoclonal antibodies are described in Cole et al., Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals that have been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, such as immunized xenogeneic mice (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). For human antibodies generated by human B cell hybridoma technology, see, for example, Li et al., Proc . Natl . Acad . Sci . USA , 103:3557-3562 (2006).

The term "donor antibody" refers to an antibody (monoclonal and/or recombinant antibody) that contributes the amino acid sequence of a variable region, CDR, or other functional fragment or its analog to a first immunoglobulin partner to provide an altered immunoglobulin coding region and enables the resulting antibody to exhibit the antigenic specificity and neutralizing activity characteristic of the donor antibody.

The term "acceptor antibody" refers to an antibody (monoclonal and/or recombinant antibody) that is heterologous to the donor antibody and that contributes all (or any portion, but in some embodiments, all) of the amino acid sequence encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner. In certain embodiments, the acceptor antibody is a human antibody.

As used herein, the terms "attach," "attached," "fuse," or "fused" refer to joining or uniting two or more components by a bond, linkage, force, or tie so as to hold them together, encompassing direct or indirect linkages, such as where a first polypeptide is directly bound to a second polypeptide or material, and where one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc.) are positioned between the first polypeptide and the second polypeptide or material.

"Percent amino acid sequence identity (%)" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the compared polypeptide, after alignment of the sequences, taking into account any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the compared sequences. However, for the purposes of this article, % amino acid sequence identity values were generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

"Homology" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions times 100. For example, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. In general, a comparison is performed when two sequences are aligned for maximum homology.

The term "constant domain" refers to a portion of an immunoglobulin molecule that has a highly conserved amino acid sequence relative to the other portion of the immunoglobulin containing the antigen-binding site (i.e., the variable domain). The constant domain comprises the heavy chain _CH1 , _CH2 , and _CH3 domains (collectively referred to as _CH ) and the light chain CHL (or _CL ) domain.

The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their homeostatic domains.

The "CH1 domain" (also called the "C1" of the "H1" domain) typically extends from approximately amino acid 118 to approximately amino acid 215 (EU numbering system).

The "hinge region" is generally defined as the region of IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form the interheavy chain SS bond in the same position.

The "CH2 domain" (also called the "C2" domain) of a human IgG Fc domain typically extends from approximately amino acid 231 to approximately amino acid 340. The CH2 domain is unique in that it does not closely pair with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact native IgG molecule. It is hypothesized that the carbohydrates provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

The "CH3 domain" (also called the "C3" domain) comprises the stretch of residues C-terminal to the CH2 domain in the Fc domain (i.e., from approximately amino acid residue 341 to the C-terminus of the antibody sequence, typically at amino acid residue 446 or 447 in IgG).

The terms "Fc domain" or "fragment crystallizable region" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence and variant Fc domains. Although the boundaries of an immunoglobulin heavy chain Fc domain can vary, the human IgG heavy chain Fc domain is typically defined as the stretch from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc domain (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc domains for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc domain of an antibody. Preferred FcRs are native sequence human FcRs. Furthermore, preferred FcRs are receptors that bind to IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Daëron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.

As used herein, the term "antigenic determinant" refers to a specific atomic group or amino acid on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they may bind to the same antigenic determinant on the antigen.

As used herein, the terms "specifically bind,""specificallyrecognize," and "specific for" refer to a measurable and reproducible interaction, such as binding, between a target and an antibody or antibody portion that determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody or antibody portion that specifically recognizes a target (which may be an antigenic determinant) is one that binds to that target with greater affinity, avidity, ease, and/or duration than it binds to other targets. In some embodiments, the extent of binding of the antibody to an unrelated target is about 10% less than the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an ^antibody that specifically binds to a target has a dissociation constant ( _KD ) ^{of ≤10-5} M, ^≤10-6 M, ^≤10-7 M, ^≤10-8 M, ^≤10-9 M, ^≤10-10 M, ^{≤10-11 M} , ^≤10-12 M, ^{≤10-13 M} , or ^{≤10-14 M.} In some embodiments ^, an ^antibody specifically binds to ^an antigenic determinant on a protein that is conserved among proteins from different species. In some embodiments ^, specific binding may include exclusive binding, but this is ^not required. The binding specificity of an antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA assays, RIA assays, ECL assays, IRMA assays, EIA assays, BIACORE ^™ assays, and peptide scanning.

The term "specificity" refers to the selective recognition of a particular antigenic determinant on an antigen by an antigen-binding protein or antibody. For example, natural antibodies are monospecific. As used herein, the term "multispecific" means that an antigen-binding protein or antibody has two or more antigen-binding sites, at least two of which bind to different antigens or to different antigenic determinants on the same antigen. As used herein, "bispecific" means that an antigen-binding protein or antibody has two different antigen-binding specificities. As used herein, the term "monospecific" antibody means an antibody that has one or more binding sites, each of which binds to the same antigenic determinant on the same antigen.

"Effector cells" are white blood cells that express one or more FcRs and perform effector functions. In one embodiment, effector cells express at least FcγRIII and perform ADCC effector functions. Examples of human white blood cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be obtained from natural sources, such as by isolation from blood. Effector cells are generally lymphocytes associated with the effector phase and function to produce interleukins (helper T cells), kill cells that infect pathogens (cytotoxic T cells), or secrete antibodies (differentiated B cells).

"Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) bound to their cognate antigen. To assess complement activation, a CDC assay can be performed, such as that described in Gazzano-Santoro et al. , J. Immunol. Methods 202: 163 (1996). Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent No. 6,194,551 B1 and WO99/51642. The contents of these patent disclosures are specifically incorporated herein by reference. See also Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

"Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this application. Specific illustrative and exemplary examples for measuring binding affinity are described below.

As used herein, "on-rate,""rate of association,""associationrate," or " _kon " can also be measured using methods such as biofilm interferometry and surface plasmon resonance (SPR), as described above.

An "isolated" polypeptide is one that has been identified, separated, and/or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from all other components of its production environment. Contaminant components of its production environment (e.g., produced by recombinant or transfected cells) are materials that would typically interfere with the research, diagnostic, or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide is (1) purified to greater than 95% antibody by weight as determined by, for example, the Lowry method, and in some embodiments to greater than 99% by weight; (2) purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer; or (3) purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptides include antibodies in situ within recombinant cells, since at least one component of the polypeptide's natural environment will not be present. However, typically, isolated polypeptides will be prepared by at least one purification step.

An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment of production. Preferably, the isolated nucleic acid is free of association with all components associated with the environment of production. Isolated nucleic acid molecules encoding the polypeptides and antibodies described herein are in a form or configuration other than that in which they are found in nature. Thus, isolated nucleic acid molecules are distinct from nucleic acids naturally present in cells encoding the polypeptides and antibodies described herein. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a protein prior to participating in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, an enhancer need not be contiguous. Linking is achieved by ligation at appropriate restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to known practices.

"Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in the normal environment of a living organism is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural environment is "isolated." An isolated nucleic acid or protein can exist in a substantially pure form or in a non-natural environment, such as a host cell.

As used herein, the term "hybrioma" refers to cells derived from the fusion of a B lymphocyte with a fusion partner, such as a myeloma cell. Hybriomas can be selected and maintained indefinitely in cell culture and are capable of producing monoclonal antibodies. Hybriomas can also be considered hybrid cells.

The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and refer to polymers of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to a linear sequence of nucleotides that makes up a nucleic acid molecule or polynucleotide. An "isolated nucleic acid" refers to a segment or fragment of a nucleic acid that has been separated from the sequences that flank it in its naturally occurring state, i.e., a DNA fragment that has been removed from the sequences with which it is normally adjacent (i.e., the sequences with which it is adjacent in its naturally occurring genome). The term also applies to nucleic acids that have been substantially purified from other components with which they naturally accompany the nucleic acid (i.e., the RNA or DNA or proteins with which they naturally accompany the nucleic acid in cells). Thus, the term includes, for example, recombinant DNA that is incorporated into a vector; incorporated into an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or exists as a separate molecule (i.e., a cDNA or genomic or cDNA fragment generated by PCR or restriction enzyme digestion) independent of other sequences. It also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

As used herein, the term "complementary" refers to nucleic acids and refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form a specific hydrogen bond ("base pairing") with a residue in a second nucleic acid region (if the residue is thymine or uracil), which is antiparallel to the first region. Similarly, it is known that a cytosine residue in a first nucleic acid strand can base pair with a residue in a second nucleic acid strand (if the residue is guanine), which is antiparallel to the first strand. A first region and a second region are complementary if, when a first region of nucleic acid and a second region of the same or different nucleic acid are arranged in an antiparallel manner, at least one nucleotide residue in the first region is capable of pairing with a residue base in the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first portion and the second portion are arranged in an antiparallel manner, at least about 50% and/or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues in the first portion are capable of pairing with a nucleotide residue base in the second portion. In some embodiments, all nucleotide residues in the first portion are capable of pairing with a nucleotide residue base in the second portion.

As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. A vector may be a viral vector, a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be an adeno-associated virus (AAV) vector or a lentiviral vector. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector or a vector that integrates into the host genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

"Encode" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, that serves as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties that derive therefrom in biological processes. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand and the non-coding strand that serves as a template for transcription of a gene or cDNA may be said to encode the protein or other product of that gene or cDNA. The nucleotide sequence of the coding strand is identical to the mRNA sequence and is typically provided in a sequence listing.

The terms "polypeptide" and "peptide" are used interchangeably to refer to polymers of amino acid residues, without limitation to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues. Full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In addition, "polypeptide" includes modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative), as long as the polypeptide maintains the desired activity. Such modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein or errors caused by PCR amplification.

As used herein, "conjugated" refers to the covalent attachment of one molecule to a second molecule.

As used herein, the term "variant" refers to a nucleic acid sequence or peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence, respectively, but retains the basic biological properties of the reference molecule. Sequence variations in nucleic acid variants may not change the amino acid sequence of the peptide encoded by the reference amino acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Sequence variations in peptide variants are typically limited or conservative, such that the sequences of the reference peptide and the variant are generally very similar and identical in many regions. The amino acid sequence of the variant and the reference peptide may differ by one or more substitutions, additions, or deletions in any combination. Variants of nucleic acids or polypeptides may be naturally occurring, such as allelic variants, or they may be unknown naturally occurring variants. Non-naturally occurring variants of nucleic acids and peptides may be prepared by mutation-inducing techniques or by direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, or at least 85% identical to the reference sequence.

As used herein, the term "regulate" can mean any method of altering the level or activity of a substrate. Non-limiting examples of regulation of proteins include affecting expression (including transcription and/or translation), affecting folding, affecting degradation or protein turnover, and affecting protein localization. Non-limiting examples of regulation of enzymes further include affecting enzyme activity. A "regulator" is a molecule whose activity includes affecting the level or activity of a substrate. A regulator can be direct or indirect. A regulator can act to activate, inhibit, or otherwise regulate its substrate.

The term "control sequences" refers to DNA sequences required for expression of an operably linked coding sequence in a particular host organism. Examples of control sequences suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A "pharmaceutically acceptable carrier" refers to a nontoxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation aid, or vehicle commonly used with a therapeutic agent in this art to form a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is nontoxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the intended formulation.

The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and that does not contain other components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

A "sterile" formulation is sterile or essentially free of viable microorganisms and their spores.

A "reconstituted" formulation is one prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed therein. The reconstituted formulation is suitable for administration (e.g., subcutaneously) to a patient to be treated with the protein of interest, and in certain embodiments, may be a formulation suitable for parenteral or intravenous administration.

An "isotonic" formulation is one whose osmotic pressure is essentially the same as that of human blood. Isotonic formulations will generally have an osmotic pressure of about 250 to 350 mOsm. The term "hypotonic" describes a formulation having an osmotic pressure lower than that of human blood. Correspondingly, the term "hypertonic" is used to describe a formulation having an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using a vapor pressure or ice-cold osmometer. The formulations of the present application may be rendered hypertonic by the addition of salts and/or buffers.

As used herein, the term "transfection," "transformation," or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected," "transformed," or "transduced" cell is one that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include primary somatic cells and their progeny.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not have identical nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as the original transformed cell screened or selected for are included herein.

As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: relief of one or more symptoms caused by a disease, reduction in disease severity, stabilization of the disease (e.g., preventing or delaying disease progression), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease symptoms, providing disease remission (partial or complete), reduction in the dosage of one or more other agents required to treat the disease, delaying disease progression, enhancing or improving quality of life, increasing weight gain, and/or prolonging survival. The methods of this application encompass any one or more of these treatment modalities.

As used herein, the terms "effective amount" and "pharmaceutically effective amount" refer to an amount of an agent sufficient to provide a desired biological result. The result can be a reduction (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and/or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system.

The term "inhibition" or "inhibit" refers to the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the occurrence, extent, or likelihood of such characteristic. "Reduction" or "inhibition" means to reduce, decrease, or arrest the activity, function, and/or amount compared to the activity, function, and/or amount of a reference. In certain embodiments, "reduction" or "inhibition" means the ability to cause an overall reduction of 20% or more (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). In another embodiment, "reduction" or "inhibition" means the ability to cause an overall reduction of 50% or more. In yet another embodiment, "reduce" or "inhibit" or "neutralize" means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more.

As used herein, "inhibiting" a function or activity means reducing the function or activity when compared to an otherwise identical condition or parameter except for the condition or parameter of interest, or when compared to another condition. For example, an antibody that inhibits tumor growth reduces the tumor growth rate compared to the tumor growth rate in the absence of the antibody.

As used herein, the terms "patient," "subject," "individual," and similar terms are used interchangeably herein and refer to any animal, in some embodiments, a mammal, and in some embodiments, a human, that has a complement system, including humans in need of treatment for or susceptible to a condition or its sequelae. Individuals can include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, mice, and humans. In some embodiments, the individual is a human.

An "affinity matured" antibody is one that has one or more alterations in one or more CDRs that improve the antibody's affinity for the antigen compared to a parent antibody lacking the alteration(s). In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describe affinity maturation by shuffling VH and VL domains. Random mutation induction of CDRs and/or framework residues is described, for example, in: Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mal. Biol. 226:889-896 (1992).

It should be understood that the embodiments of the present application described herein include "consisting of" and/or "consisting essentially of" the embodiments.

Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

As used herein, reference to "not being" a certain value or parameter generally means and describes "except for a certain value or parameter." For example, reference to "not being" a certain value or parameter generally means that the method is used to treat a type of disease other than type X.

As used herein, the term "about X-Y" has the same meaning as "about X to about Y."

As used herein and in the appended claims, the singular forms "a,""an," and "the" include plural referents unless the context clearly dictates otherwise. II. Bifunctional Protein Constructs

In one aspect, the present application provides a bifunctional protein construct comprising a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to and activates a notch receptor. It should be understood that the term "bifunctional protein construct" as used herein refers to a protein construct that has binding capabilities for both a muscle-specific molecule and a notch receptor, and may encompass constructs with additional functions. For example, in some embodiments, the bifunctional protein construct is multispecific. In some embodiments, the bifunctional protein construct comprises a first binding moiety and a second binding moiety, wherein the first binding moiety comprises a means for binding to a muscle-specific molecule, and wherein the second binding moiety comprises a means for binding to a notch receptor and activates the notch receptor.

In some embodiments, the first binding moiety comprises an antibody portion, including but not limited to a full-length antibody, scFv, Fab, or sdAb. In some embodiments, the first binding moiety comprises a non-antibody portion. Exemplary first binding moieties include but are not limited to anti-LAMA2 antibody portions (e.g., any of the anti-human LAMA2 antibody portions described herein), anti-matrix glycan antibody portions (e.g., any of the anti-human ADG41 antibody portions described herein), and anti-CDH15 antibodies (e.g., any of the anti-human CDH15 antibody portions described herein). These antibody portions are described in more detail in the following sections. The components described herein for binding to muscle-specific molecules may be any of the antibody portions described herein and their functional equivalents.

In some embodiments, the first binding moiety comprises a non-antibody binding moiety that specifically binds to a muscle-specific molecule, such as a protein domain of an extracellular matrix (ECM) protein that can bind to stromal glycans (e.g., a laminin G-like domain (LG domain)). In some embodiments, the non-antibody moiety comprises a protein domain selected from the group consisting of the laminin LG domain, the agrin LG domain, the nidogen LG domain, and the perlein LG domain. In some embodiments, the first binding moiety comprises two or more non-antibody moieties linked in tandem. The components described herein for binding to muscle-specific molecules can be any of the non-antibody moieties described herein, and their functional equivalents.

The second binding moiety described herein specifically binds to and activates the nick receptor. In some embodiments, the second binding moiety comprises the extracellular domain (ECD) of a nick ligand selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises a DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130. In some embodiments, the second binding moiety comprises a DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 131. In some embodiments, the second binding moiety comprises a DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 125, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 125. In some embodiments, the second binding moiety comprises a variant DLL4 ECD comprising the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the second binding moiety comprises a Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 132, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 132. In some embodiments, the second binding moiety comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133 or 134, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 133 or 134. In some embodiments, the second binding moiety comprises a Jag2 ECD comprising the amino acid sequence of SEQ ID NO: 135, or a variant thereof comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 135. The means described herein for binding to (and optionally activating) a notch receptor can be any of the non-antibody moieties described herein and functional equivalents thereof. In some embodiments, the second binding moiety is an anti-notch antibody moiety that activates the notch receptor. The means described herein for binding to the notch receptor (and optionally activating the notch receptor) can be any of the anti-notch antibody moieties described herein and their functional equivalents.

The first binding moiety and the second binding moiety described herein can be fused (i.e., covalently linked) to each other directly or indirectly. In some embodiments, the first binding moiety is fused to the second binding moiety via a carrier protein (e.g., albumin, an anti-human serum albumin antibody, or a monomeric Fc domain). In some embodiments, the first binding moiety is fused to the second binding moiety via an optional peptide linker (e.g., any of the peptide linkers described under the "Linker" subsection), e.g., a peptide linker of no more than about 30 (e.g., no more than about 25, 20, or 15) amino acids in length. In some embodiments, the bifunctional protein constructs provided herein comprise an Fc domain comprising a first subunit and a second subunit.

The bifunctional protein constructs provided herein can be used with any of the various bispecific or multispecific antibody construct formats known in the art. Numerous formats have been developed to address the therapeutic opportunities offered by molecules with multiple binding specificities. Several methods have been described for preparing bispecific antibody constructs in which a specific antibody light chain or fragment is paired with a specific antibody heavy chain or fragment.

For example, International Patent Application No. PCT/EP2011/056388 (WO2011/131746) describes an in vitro method for generating heterodimeric proteins in which asymmetric mutations are introduced into the CH3 regions of two monospecific starting proteins to drive directed "Fab arm" or "half-molecule" exchange between two monospecific IgG4 or IgG4-like antibodies when incubated under reducing conditions.

Schaefer et al. (Roche Diagnostics GmbH) describe a method for assembling a human bivalent, bispecific IgG antibody from two heavy chains and two light chains derived from two existing antibodies without the use of an artificial linker (PNAS (2011) 108(27): 11187-11192 and US 2009/0232811). The method involves exchanging one or more heavy and light chain domains within the antigen-binding fragment (Fab) of a bispecific antibody (CrossMab). Based on knob-in-hole technology that enables heavy chain heterodimerization, the correct association of the light chain with its cognate heavy chain is achieved by exchanging the heavy and light chain domains within the antigen-binding fragment (Fab) of a bispecific antibody. This "crossover" preserves antigen-binding affinity but dissimilarizes the two arms, eliminating light chain mispairing. See WO2009/080251, WO2009/080252, WO2009/080253, and WO2009/080254, each of which is incorporated herein by reference in its entirety. 1. Direct fusion or fusion via a carrier protein

The first binding moiety and the second binding moiety can be directly or indirectly linked to each other (i.e., via a peptide linker or a carrier protein, such as a monomeric carrier protein). The first binding moiety can be fused to the second binding moiety via any of the linkers (e.g., SEQ ID NOs: 211, 212, 337, and 338), as appropriate. The first binding moiety and the second binding moiety can also be directly fused to each other. The first binding moiety can be fused to the N-terminus or C-terminus of the second binding moiety, such as to the N-terminus or C-terminus of any polypeptide of the second binding moiety.

In some embodiments, the first binding moiety and the second binding moiety are linked to each other via a carrier protein. In some embodiments, the carrier protein is selected from the group consisting of: human serum albumin (HSA), an anti-HSA antibody moiety, and a subunit of an Fc domain. Anti-HSA antibody moieties are known in the art, including, for example, Mandrup et al., Commun Biol ., 4(1):310 (2021) and Benjamin et al., Hybridoma , 6(2):183-90 (1987). In some embodiments, the first binding moiety is fused to the N-terminus of the carrier protein via a first linker, which is optionally present, and the second binding moiety is fused to the C-terminus of the carrier protein via a second linker, which is optionally present. In some embodiments, the first binding moiety is fused to the C-terminus of the carrier protein via an optionally present first linker, and the second binding moiety is fused to the N-terminus of the carrier protein via an optionally present second linker. In some embodiments, the carrier protein (e.g., HSA) extends the half-life of the bifunctional protein construct (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

In some embodiments, the first binding moiety comprises an scFv that specifically binds to a muscle-specific molecule, the scFv comprising a VH and a VL, wherein the scFv is fused to a second binding moiety that specifically binds to a notch receptor. The second binding moiety can be fused to the N-terminus or C-terminus of the scFv that specifically recognizes a muscle-specific molecule via an optional linker. In some embodiments, the optional linker between the second binding moiety and the scFv comprises a carrier protein, such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding moiety and the scFv is a peptide linker. In some embodiments, the bifunctional protein construct comprises, from N' to C': [VH - a first optional linker (e.g., a peptide linker) - VL], forming an scFv - a second optional linker (e.g., a peptide linker or a carrier protein) - a second binding moiety that specifically binds to a nicked receptor. In some embodiments, the bifunctional protein construct comprises, from N' to C': [VL - a first optional linker (e.g., a peptide linker) - VH], forming an scFv - a second optional linker (e.g., a peptide linker or a carrier protein) - a second binding moiety that specifically binds to a nicked receptor. In some embodiments, the bifunctional protein construct comprises, from N' to C', the following: a second binding moiety that specifically binds to a nick receptor - a first, optionally present linker (e.g., a peptide linker or a carrier protein) - [VH - a second, optionally present linker (e.g., a peptide linker) - VL], forming an scFv. In some embodiments, the bifunctional protein construct comprises, from N' to C', the following: a second binding moiety that specifically binds to a nick receptor - a first, optionally present linker (e.g., a peptide linker or a carrier protein) - [VL - a second, optionally present linker (e.g., a peptide linker) - VH], forming an scFv. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. In some embodiments, the first binding moiety comprises an anti-matrix glycan scFv (e.g., SEQ ID NO: 155). In some embodiments, the first binding moiety comprises an anti-LAMA2 scFv (e.g., SEQ ID NO: 145 or 268). In some embodiments, the second binding moiety comprises an ECD of a nicked ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (eg, two or three) scFvs optionally linked in tandem via a peptide linker (eg, any one of SEQ ID NOs: 211, 212, 337, and 338).

In some embodiments, the first binding moiety comprises a Fab comprising: a first polypeptide chain comprising VH and CH1 and a second polypeptide chain comprising VL and CL, wherein the Fab specifically binds to a muscle-specific molecule and is fused to a second binding moiety that specifically binds to a nick receptor via an optional linker. In some embodiments, the second binding moiety is fused to the N-terminus of the first polypeptide chain of the Fab. In some embodiments, the second binding moiety is fused to the C-terminus of the first polypeptide chain of the Fab. In some embodiments, the second binding moiety is fused to the N-terminus of the second polypeptide chain of the Fab. In some embodiments, the second binding moiety is fused to the C-terminus of the second polypeptide chain of the Fab. In some embodiments, the bifunctional protein construct comprises: i) a fusion polypeptide comprising, from N' to C', VH-CH1 - optionally, a linker (e.g., a peptide linker or a carrier protein) - a second binding moiety that specifically binds to a nicked receptor; and ii) a second polypeptide comprising, from N' to C', VL-CL; wherein VH-CH1 and VL-CL form a Fab that specifically binds to a muscle-specific molecule. In some embodiments, the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', VH-CH1; and ii) a fusion polypeptide comprising, from N' to C', VL-CL, optionally a linker (e.g., a peptide linker or a carrier protein), and a second binding moiety that specifically binds to a nicked receptor; wherein VH-CH1 and VL-CL form a Fab that specifically binds to a muscle-specific molecule. In some embodiments, the bifunctional protein construct comprises: i) a fusion polypeptide comprising, from N' to C': a second binding moiety that specifically binds to a nick receptor, optionally a linker (e.g., a peptide linker or a carrier protein); VH-CH1; and ii) a second polypeptide comprising, from N' to C': VL-CL; wherein VH-CH1 and VL-CL form a Fab that specifically binds to a muscle-specific molecule. In some embodiments, a bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', VH-CH1; and ii) a fusion polypeptide comprising, from N' to C', a second binding moiety that specifically binds to a nick receptor, optionally a linker (e.g., a peptide linker or a carrier protein), and VL-CL; wherein VH-CH1 and VL-CL form a Fab that specifically binds to a muscle-specific molecule. In some embodiments, the optional linker between the second binding moiety and the Fab comprises a carrier protein, such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding moiety and the Fab is a peptide linker. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. In some embodiments, the first binding moiety comprises an anti-matrix glycan Fab, such as an anti-matrix glycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the first binding moiety comprises an anti-LAMA2 Fab, such as an anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the second binding moiety comprises an ECD of a nicked ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (e.g., two or three) Fabs, optionally linked in tandem via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

In some embodiments, the first binding moiety comprises an sdAb that specifically binds to a muscle-specific molecule, comprising a VHH domain, wherein the sdAb is fused to a second binding moiety that specifically binds to a nick receptor. The second binding moiety can be fused to the N-terminus or C-terminus of the sdAb. In some embodiments, the bifunctional protein construct comprises, from N' to C': an sdAb that specifically binds to a muscle-specific molecule (e.g., a VHH) - an optional linker (e.g., a peptide linker or a carrier protein) - a second binding moiety that specifically binds to a nick receptor. In some embodiments, the bifunctional protein construct comprises, from N' to C', a second binding moiety that specifically binds to a nick receptor, an optional linker (e.g., a peptide linker or carrier protein), and an sdAb (e.g., a VHH) that specifically binds to a muscle-specific molecule. In some embodiments, the optional linker between the second binding moiety and the sdAb comprises a carrier protein, such as HSA, an anti-HSA antibody moiety, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding moiety and the sdAb is a peptide linker. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and basal glycans on α-DG. In some embodiments, the second binding moiety comprises an ECD of a nicked ligand selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (e.g., two or three) sdAbs, optionally linked in tandem via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

In some embodiments, the first binding moiety comprises a non-antibody portion that specifically binds to a muscle-specific molecule, wherein the non-antibody portion comprises an LG domain, for example, the LG domain selected from the group consisting of the LG domain of laminin, the LG domain of agrin, the LG domain of nidogen, and the LG domain of perlein. In some embodiments, the bifunctional protein construct comprises, from N' to C': a non-antibody portion that specifically binds to a muscle-specific molecule, an optional linker (e.g., a peptide linker or carrier protein), and a second binding moiety that specifically binds to a nick receptor. In some embodiments, the bifunctional protein construct comprises, from N' to C', a second binding moiety that specifically binds to a nick receptor, an optional linker (e.g., a peptide linker or a carrier protein), and a non-antibody portion that specifically binds to a muscle-specific molecule. In some embodiments, the optional linker between the second binding moiety and the non-antibody portion comprises a carrier protein, such as HSA, an anti-HSA antibody portion, and a subunit of an Fc domain. In some embodiments, the optional linker between the second binding moiety and the non-antibody portion is a peptide linker. In some embodiments, the first binding moiety comprises LAMA2 LG4-5, which comprises the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the second binding moiety comprises an ECD of a nicked ligand or a variant thereof selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the first binding moiety comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) non-antibody moieties (such as LG domains) optionally linked in tandem via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). Exemplary Direct Fusions

In some embodiments, a bifunctional protein construct is provided that comprises, from N' to C', a second binding moiety comprising the DLL4 ECD or a variant thereof (e.g., any one of SEQ ID NOs: 126-135 and 260-267); optionally, a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 136 (hereinafter referred to as "DLL4wt-LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 137 (hereinafter referred to as "DLL4v-LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL4 variant ECD, wherein the DLL4 variant ECD comprises the amino acid sequence of SEQ ID NO: 128; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 138 (hereinafter referred to as "DLL4max-LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 139 (hereinafter referred to as "DLL4deimmune (DLL4di)-LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL1 ECD or a variant thereof; optionally, a peptide linker; and a first binding moiety comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 140 (hereinafter referred to as "DLL1wt-LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL3 ECD or a variant thereof; optionally, a peptide linker; and a first binding moiety comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 141 (hereinafter referred to as "DLL3wt-LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag1 ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs: 132-134); optionally, a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5) (e.g., SEQ ID NO: 119).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 142 (hereinafter referred to as "Jag1wt-LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 143 (hereinafter referred to as "Jag1v-LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag2 ECD or a variant thereof; optionally, a peptide linker; and a first binding moiety comprising the LG domain of LAMA2 (e.g., LAMA2 LG4-5). In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising LAMA2 LG4-5, wherein the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 144 (hereinafter referred to as "Jag2wt-LG4-5").

In some embodiments, a bifunctional protein construct is provided that comprises, from N' to C', a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECDs or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267); optionally, a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-LAMA2 scFv (e.g., SEQ ID NOs: 145 or 268).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 146 (hereinafter referred to as "DLL4wt-LG21scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 147 (hereinafter referred to as "DLL4v-LG21 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 148 (hereinafter referred to as "DLL4max-LG21 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 149 (hereinafter referred to as "DLL4di-LG21 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL1 ECD or a variant thereof; optionally, a peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 150 (hereinafter referred to as "DLL1wt-LG21scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL3 ECD or a variant thereof; optionally, a peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 151 (hereinafter referred to as "DLL3wt-LG21scFv").

In some embodiments, a bifunctional protein construct is provided that comprises, from N' to C', a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs: 132-134); optionally, a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-LAMA2 scFv (e.g., SEQ ID NOs: 145 or 268).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 152 (hereinafter referred to as "Jag1wt-LG21scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 153 (hereinafter referred to as "Jag1v-LG21 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag2 ECD or a variant thereof; optionally, a peptide linker; and a first binding moiety comprising an anti-LAMA2 scFv. In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-LAMA2 scFv, wherein the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 154 (hereinafter referred to as "Jag2wt-LG21scFv").

In some embodiments, a bifunctional protein construct is provided that comprises, from N' to C', a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECDs or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267); optionally, a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-matrix glycan scFv (e.g., SEQ ID NO: 155).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix scFv, wherein the anti-matrix scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 156 (hereinafter referred to as "DLL4wt-ADG41 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix scFv, wherein the anti-matrix scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 157 (hereinafter referred to as "DLL4v-ADG41 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix scFv, wherein the anti-matrix scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 158 (hereinafter referred to as "DLL4max-ADG41 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix scFv, wherein the anti-matrix scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 159 (hereinafter referred to as "DLL4di-ADG41 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL1 ECD or a variant thereof; optionally, a peptide linker; and a first binding moiety comprising an anti-matrix scFv. In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix scFv, wherein the anti-matrix scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 160 (hereinafter referred to as "DLL1wt-ADG41scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL3 ECD or a variant thereof; optionally, a peptide linker; and a first binding moiety comprising an anti-matrix scFv. In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix scFv, wherein the anti-matrix scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 161 (hereinafter referred to as "DLL3wt-ADG41scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag1 ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs: 132-134); optionally, a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-matrix scFv (e.g., SEQ ID NO: 155).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix glycan scFv, wherein the anti-matrix glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 162 (hereinafter referred to as "Jag1wt-ADG41 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix scFv, wherein the anti-matrix scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 163 (hereinafter referred to as "Jag1v-ADG41 scFv").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag2 ECD or a variant thereof; optionally, a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338); and a first binding moiety comprising an anti-matrix glycan scFv. In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135; optionally, a peptide linker (e.g., SEQ ID NO: 211); and a first binding moiety comprising an anti-matrix glycan scFv, wherein the anti-matrix glycan scFv comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 164 (hereinafter referred to as "Jag2wt-ADG41scFv").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab (e.g., SEQ ID NO: 165) and a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECDs or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab (e.g., SEQ ID NO: 165) via an optionally present peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding moiety and the anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 167 (hereinafter referred to as "DLL4wt-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding moiety and the anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 168 (hereinafter referred to as "DLL4v-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optionally present peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding moiety and the second polypeptide of the anti-LAMA2 Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 169 (hereinafter referred to as "DLL4max-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optionally present peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding moiety and the anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 170 (hereinafter referred to as "DLL4di-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a DLL1 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a DLL1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optionally present peptide linker (e.g., SEQ ID NO: 211), wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding moiety and the anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 171 (hereinafter referred to as "DLL1wt-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a DLL3 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a DLL3 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optionally present peptide linker (e.g., SEQ ID NO: 211), wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding moiety and the anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 172 (hereinafter referred to as "DLL3wt-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab (e.g., SEQ ID NO: 165) and a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs: 132-134), wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab (e.g., SEQ ID NO: 165) via an optionally present peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optionally present peptide linker (e.g., SEQ ID NO: 211), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding moiety and the anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 173 (hereinafter referred to as "Jag1wt-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding portion and the anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 174 (hereinafter referred to as "Jag1v-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising Jag2 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-LAMA2 Fab and a second binding moiety comprising a Jag2 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-LAMA2 Fab via an optionally present peptide linker (e.g., SEQ ID NO: 211), wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135, and wherein the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165, and a fusion polypeptide comprising a second binding moiety and the anti-LAMA2 Fab second polypeptide, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 175 (hereinafter referred to as "Jag2wt-LG21-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix glycan Fab (e.g., SEQ ID NO: 176) and a second binding moiety comprising a DLL4 ECD or a variant thereof (such as any of the DLL4 ECDs or engineered DLL4 ECDs described herein, e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix glycan Fab (e.g., SEQ ID NO: 176) via an optionally present peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises a first binding moiety comprising an anti-matrix Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 126, and wherein the anti-matrix Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 178 (hereinafter referred to as "DLL4wt-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 127, and wherein the anti-matrix Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 179 (hereinafter referred to as "DLL4v-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 128, and wherein the anti-matrix Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 180 (hereinafter referred to as "DLL4max-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix Fab and a second binding moiety comprising a variant DLL4 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 129, and wherein the anti-matrix Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 181 (hereinafter referred to as "DLL4di-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix Fab and a second binding moiety comprising a DLL1 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix glycan Fab and a second binding moiety comprising a DLL1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix glycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130, and wherein the anti-matrix glycan Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 182 (hereinafter referred to as "DLL1wt-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix Fab and a second binding moiety comprising a DLL3 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix glycan Fab and a second binding moiety comprising a DLL3 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix glycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131, and wherein the anti-matrix glycan Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 183 (hereinafter referred to as "DLL3wt-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix glycan Fab (e.g., SEQ ID NO: 176) and a second binding moiety comprising a Jag1 ECD or a variant thereof (such as any of the Jag1 ECDs described herein, e.g., any of SEQ ID NOs: 132-134), wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix glycan Fab (e.g., SEQ ID NO: 176) via an optionally present peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338).

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix glycan Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix glycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133, and wherein the anti-matrix glycan Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 184 (hereinafter referred to as "Jag1wt-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix glycan Fab and a second binding moiety comprising a variant Jag1 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix glycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 134, and wherein the anti-matrix glycan Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 185 (hereinafter referred to as "Jag1v-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix glycan Fab and a second binding moiety comprising Jag2 ECD or a variant thereof, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix glycan Fab via an optional peptide linker. In some embodiments, a bifunctional protein construct is provided, wherein the bifunctional protein construct comprises: a first binding moiety comprising an anti-matrix glycan Fab and a second binding moiety comprising a Jag2 ECD, wherein the second binding moiety is fused to the N-terminus of the VL of the anti-matrix glycan Fab via an optional peptide linker (e.g., SEQ ID NO: 211), wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135, and wherein the anti-matrix glycan Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the bifunctional protein construct comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176, and a fusion polypeptide comprising a second binding moiety and a second polypeptide of the anti-matrix glycan Fab, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 186 (hereinafter referred to as "Jag2wt-ADG41-Fab").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL4 ECD or a variant thereof (e.g., SEQ ID NO: 339); optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 339; optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising the laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 319 (hereinafter referred to as "DLL4(EGF1-6)-GS linker-laminin-a2 LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C': a second binding moiety comprising a DLL4 ECD or a variant thereof (e.g., SEQ ID NO: 339); optionally a peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); optionally a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 339; optionally a peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); optionally a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 320 (hereinafter referred to as "DLL4(EGF1-6)-HSA-laminin-a2 LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C': a second binding moiety comprising a DLL1 ECD or a variant thereof (e.g., SEQ ID NO: 340); optionally a peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); optionally a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL1 ECD, wherein the variant DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 340; optionally a peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); optionally a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 323 (hereinafter referred to as "DLL1(EGF1-6)-HSA-laminin-a2 LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising Jag1 ECD or a variant thereof (e.g., SEQ ID NO: 341); optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 341; optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising the laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 325 (hereinafter referred to as "Jagged1 (EGF1-6)-laminin-a2 LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C': a second binding moiety comprising Jag1 ECD or a variant thereof (e.g., SEQ ID NO: 341); optionally, a peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 341; optionally a peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); optionally a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising the laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 326 (hereinafter referred to as "Jagged1(EGF1-6)-HSA-laminin-a2 LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising the Jag2 ECD or a variant thereof (e.g., SEQ ID NO: 342); optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising the laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag2 ECD, wherein the variant Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 342; optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising the laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 328 (hereinafter referred to as "Jagged2 (EGF1-6)-laminin-a2 LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C': a second binding moiety comprising Jag2 ECD or a variant thereof (e.g., SEQ ID NO: 342); optionally, a peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant Jag2 ECD, wherein the variant Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 342; optionally a peptide linker (e.g., SEQ ID NO: 337); a carrier protein (e.g., SEQ ID NO: 344); optionally a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain, wherein the laminin-a2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 329 (hereinafter referred to as "Jagged2(EGF1-6)-HSA-laminin-a2 LG4-5").

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a DLL1 ECD or a variant thereof (e.g., SEQ ID NO: 340); optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-a2 LG4-5 domain (e.g., SEQ ID NO: 343).

In some embodiments, a bifunctional protein construct is provided, comprising, from N' to C', a second binding moiety comprising a variant DLL1 ECD, wherein the variant DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 340; optionally, a peptide linker (e.g., SEQ ID NO: 337); and a first binding moiety comprising a laminin-α2 LG4-5 domain, wherein the laminin-α2 LG4-5 domain comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the bifunctional protein construct comprises the amino acid sequence of SEQ ID NO: 322 (hereinafter referred to as "DLL1 (EGF1-6)-laminin-α2 LG4-5"). 2. Fc Domain Fusions

In some embodiments, the bifunctional protein construct comprises an Fc domain, comprising a first unit and a second unit. The Fc domain can be a third moiety connected to the first binding moiety and the second binding moiety. In some embodiments, when the first binding moiety or the second binding moiety is a full-length antibody, the Fc domain can be a portion of the full-length antibody.

In some embodiments, the Fc domain is a variant Fc domain with minimal or no effector function. In some embodiments, the variant Fc domain is derived from IgG1 Fc or IgG4 Fc.

One or more amino acid modifications can be introduced into the Fc domain, thereby generating an Fc domain variant. The Fc domain variant can comprise a human Fc domain sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc domain) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In some embodiments, the Fc domain has some, but not all, effector functions, making it an ideal candidate for applications where the half-life of the antibody portion in vivo is critical and certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In some embodiments, the Fc domain comprises one or more mutations that increase the half-life of the antibody portion in vivo. In some embodiments, the variant Fc domain has increased FcRn binding at pH 6.0. In some embodiments, the variant Fc domain comprises M252Y/S254T/T256E ("YTE") mutations (EU numbering). See, e.g., Dall'Acqua et al., J Immunol ., 169:5171-5180 (2002) and Wang et al., Protein & Cell , 9(1):63-73 (2018). In some embodiments, the variant Fc domain comprises an M428L/N434S mutation. See, for example, Zalevsky et al., Nat Biotechnol ., 28:157-159 (2010) and Wang et al., Protein & Cell , 9(1):63-73 (2018).

In vitro and/or in vivo cytotoxicity assays can be performed to analyze the CDC and/or ADCC activity of the Fc domain. For example, an Fc receptor (FcR) binding assay can be performed to determine whether the antibody has FcγR binding ability (and therefore may have ADCC activity) and/or retains FcRn binding ability. The primary cells that mediate ADCC, namely NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol . 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of molecules of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al. , Proc. Nat'l Acad . Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad . Sci. USA 82:1499-1502 (1985); 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, for example, the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and the CytoTox ^96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model, such as that disclosed in Clynes et al., Proc. Nat'l Acad . Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3c binding ELISAs. FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l . Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those with substitutions at one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include variant Fcs with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine ( U.S. Patent No. 7,332,581 ). In some embodiments, the Fc domain of a bifunctional protein construct comprises one or more of these mutations. In some embodiments, the variant Fc domain comprises an N297A, N297Q, or N297G mutation. See, e.g., Bolt et al., Eur J Immunol ., 23:403-411 (1993); Leabman et al., Mabs , 5:896-903 (2013); Tao and Morrison, J Immunol ., 143:2595-2601 (1989); Walker et al., Biochem J. , 259:347-353 (1989); and Wang et al., Protein & Cell , 9(1):63-73 (2018). In some embodiments, the variant Fc domain comprises an L235E mutation. See, e.g., Alegre et al., J Immunol ., 148:3461-3468 (1992) and Wang et al., Protein & Cell , 9(1):63-73 (2018).

Certain antibody variants with improved or reduced FcR binding have been described. See, for example, U.S. Patent No. 6,737,056; WO 2004/056312; and Shields et al ., J. Biol. Chem. 9(2): 6591-6604 (2001).

In some embodiments, the Fc domain is derived from human IgG1, human IgG2, or human IgG4. In some embodiments, the Fc domain is derived from an IgG1 Fc domain (e.g., a human IgG1 Fc domain). In some embodiments, the human IgG1 Fc comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments, each subunit of the Fc domain comprises a mutation that reduces or disrupts effector function. In some embodiments, effector function is eliminated by a mutation in the constant region that eliminates glycosylation, such as an "effector-less mutation." In one embodiment, the effector-less mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region (EU numbering). See, for example, Shields et al., J. Biol. Chem . 276 (9): 6591-6604 (2001). Alternatively or additionally, effector function can be reduced or eliminated by manufacturing techniques, such as expression in non-glycosylated host cells (e.g., E. coli), or techniques that cause a change in glycosylation pattern that renders it ineffective or less effective in promoting effector function (e.g., Shinkawa et al . , J. Biol . Chem . 278(5): 3466-3473 (2003). In some embodiments, each subunit of the Fc domain comprises L234A/L235A "LALA" mutations (EU numbering). In some embodiments, each subunit of the Fc domain comprises L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises SEQ ID NO:277. In some embodiments, the Fc domain comprises a mutation that shortens the half-life of the bifunctional protein construct in the blood circulation, such as the H435A mutation (EU numbering). In some embodiments, each subunit of the Fc domain (e.g., human IgG1 Fc) comprises L234A/L235A and H435A mutations. In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO:188. In some embodiments, the Fc domain is an IgG4 (e.g., human IgG4) Fc domain. In some embodiments, the Fc domain comprises a mutation that reduces Fab exchange, such as the S228P mutation. In some embodiments, the IgG4 Fc domain comprises the F234A/L235A mutation. See, for example, Xu et al., Cell Immunol ., 200:16-26 (2000) and Wang et al., Protein & Cell , 9(1):63-73 (2018). For other examples of Fc domain variants, see also Duncan and Winter, Nature, 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351.

In some embodiments, when the bifunctional protein construct is a heterodimeric protein comprising two distinct polypeptide chains, the Fc domain may comprise one or more mutations that promote heterodimeric protein assembly. For example, a knob-in-hole is used for heterodimerization of antibody CH3 domains. Knob-in-hole technology has previously been applied to generate full-length human bispecific antibodies with a single common light chain (LC) (Merchant et al., "An efficient route to human bispecific IgG." Nat Biotechnol. 1998;16:677-81; Jackman et al., "Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling." J Biol Chem. 2010;285:20850-9). See also WO1996027011, which is incorporated herein by reference in its entirety for all purposes. All of these mutations can be incorporated into the bifunctional protein constructs described herein.

Thus, for example, in some embodiments, a bifunctional protein construct comprises an Fc domain comprising: a first CH3 domain and a second CH3 domain comprising a button-in-hole (KIH) residue. In some embodiments, the first CH3 domain is altered such that one or more amino acid residues within the CH3/CH3 interface are replaced with one or more amino acid residues having a larger side chain volume, thereby creating a protuberance on the surface of the first CH3 domain that interacts with the second CH3 domain; and the second CH3 domain is altered such that one or more amino acid residues within the CH3/CH3 interface are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity on the surface of the second CH3 domain that interacts with the first CH3 domain. In some embodiments, the protuberance is a knob. In some embodiments, the alteration that creates a knob is T366W. In some embodiments, the cavity is a pore. In some embodiments, the alteration that creates a pore is T366S/L368A/Y407V. Thus, in some embodiments, the Fc domains described herein comprise a Kn2-into-pore mutation, wherein: i) the first subunit of the Fc domain comprises a Kn2 mutation and the second subunit of the Fc domain comprises a pore mutation; or ii) the second subunit of the Fc domain comprises a Kn2 mutation and the first subunit of the Fc domain comprises a pore mutation. In some embodiments, the Kn2 mutation is T366W (EU numbering) and the pore mutation is T366S/L368A/Y407V (EU numbering). Unless otherwise indicated, all amino acid positions within the Fc domain are numbered according to the EU numbering system.

In some embodiments, the bifunctional protein construct comprises an Fc domain comprising mutations that allow purification of an asymmetric Fc domain. In some embodiments, the first Fc domain subunit or the second Fc domain subunit comprises H435R/Y436F mutations (EU numbering). In some embodiments, each Fc domain subunit comprises H435R/Y436F mutations (EU numbering).

Strop et al. (Rinat-Pfizer Inc.) described a method for generating stable bispecific antibodies by separately expressing and purifying two antibodies of interest and then mixing them together under defined redox conditions (J. Mol. Biol. (2012) 420:204-19).

Other heterodimerization domains that have a strong preference for heterodimer formation over homodimer formation can be incorporated into the bifunctional protein constructs of the present invention. Illustrative examples include, but are not limited to, WO2007147901 (Kjærgaard et al., Novo Nordisk, describing ionic interactions); WO2009/089004 (Kannan et al., Amgen, describing electrostatic steering); and WO2010/034605 (Christensen et al., Genentech, describing coiled coils). See also, for example, Pack, P. and Plueckthun, A., Biochemistry 31, 1579-1584 (1992), which describes leucine zippers; or Pack et al., Bio/Technology 11, 1271-1277 (1993), which describes helix-turn-helix motifs. The term "heterodimerization domain" does not exclude additional units besides the two heterodimers in a bifunctional construct. In certain embodiments, a bifunctional protein construct comprises one or more heterodimerization domains.

In some embodiments, when the bifunctional protein construct is a heterodimeric protein comprising two different polypeptide chains, the Fc domain may comprise one or more charge pair mutations that promote heterodimeric protein assembly. In some embodiments, an amino acid residue in a first subunit of the Fc domain is replaced with a positively charged residue (e.g., R, H, or K), and an amino acid residue in a second subunit of the Fc domain is replaced with a negatively charged residue (e.g., D or E). In some embodiments, an amino acid residue in a first subunit of the Fc domain is replaced with a negatively charged residue, and an amino acid residue in a second subunit of the Fc domain is replaced with a positively charged residue. In some embodiments, the amino acid residue at D399 (EU numbering) in the first unimole of the Fc domain is substituted with a positively charged residue, and the amino acid residue at K409 (EU numbering) in the second unimole of the Fc domain is substituted with a negatively charged residue. In some embodiments, the amino acid residue at K409 (EU numbering) in the first unimole of the Fc domain is substituted with a negatively charged residue, and the amino acid residue at D399 (EU numbering) in the second unimole of the Fc domain is substituted with a positively charged residue.

In some embodiments, the Fc domain used in the bifunctional protein constructs described herein comprises the amino acid sequence of any one of SEQ ID NOs: 188, 254, and 277.

In some embodiments, a bifunctional protein construct is provided, comprising: i) one or more units of a first binding moiety that specifically binds to a muscle-specific molecule; ii) a first unit that specifically binds to a first notch receptor and activates a second binding moiety of the first notch receptor; iii) a second unit that specifically binds to a second notch receptor and activates a second binding moiety of the second notch receptor; and iv) an Fc domain comprising the first unit and the second unit; optionally, wherein the first unit of the second binding moiety and the second unit of the second binding moiety each independently comprise an ECD of a notch ligand or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., SEQ ID NO: 1). NOs: 126-129 and 260-267), the nick ligand is selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2. In some embodiments, the first unit of the second binding moiety is the same as the second unit of the second binding moiety. In some embodiments, the first unit of the second binding moiety is different from the second unit of the second binding moiety. One or more units of the first binding moiety can be the same or different; and/or can bind to the same or different muscle-specific molecules. In some embodiments, the bifunctional protein construct further comprises a third unit of the second binding moiety that specifically binds to a third nick receptor and a fourth unit of the second binding moiety that specifically binds to a fourth nick receptor. In some embodiments, the third unit of the second binding moiety and the fourth unit of the second binding moiety each independently comprise an ECD of a nick ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2. The first, second, third, and/or fourth units of the second binding moiety can be the same or different and/or can bind to the same or different muscle-specific molecules. In some embodiments, all four units of the second binding moiety are the same. In some embodiments, at least one of the four units of the second binding moiety is different from the other units. One or more units of the first binding moiety can comprise an antibody portion or a non-antibody portion. Each binding moiety can be independently fused to the N-terminus and/or C-terminus of the Fc domain. The bifunctional protein construct can be a homodimer or a heterodimer.

In some embodiments, a bifunctional protein construct is provided, comprising: i) a first unit that specifically binds to a first binding moiety of a first muscle-specific molecule; ii) a second unit that specifically binds to a first binding moiety of a second muscle-specific molecule; iii) one or more units of the second binding moiety that specifically bind to and activate a notch receptor; and iv) an Fc domain comprising the first and second units. In some embodiments, a bifunctional protein construct is provided, comprising: i) a first unit comprising a first binding moiety of a first antibody portion, the first antibody portion specifically binding to a first muscle-specific molecule; ii) a second unit comprising a first binding moiety of a second antibody portion, the second antibody portion specifically binding to a second muscle-specific molecule; iii) one or more units of a second binding moiety that specifically bind to and activate a notch receptor; and iv) an Fc domain comprising the first unit and the second unit; wherein the first antibody portion and the second antibody portion are each independently selected from the group consisting of: Fab, scFv, and sdAb. In some embodiments, a bifunctional protein construct is provided, comprising: i) a first unit comprising a first binding moiety comprising a first non-antibody portion, wherein the first non-antibody portion specifically binds to a first muscle-specific molecule; ii) a second unit comprising a first binding moiety comprising a second non-antibody portion, wherein the second non-antibody portion specifically binds to a second muscle-specific molecule; iii) one or more units of a second binding moiety that specifically binds to and activates a notch receptor; and iv) an Fc domain comprising the first unit and the second unit; wherein the first non-antibody portion and the second non-antibody portion are each independently selected from the group consisting of the LG domain of laminin, the LG domain of agrin, the LG domain of nidogen, and the LG domain of perlein. In some embodiments, the first non-antibody portion and/or the second non-antibody portion comprises LAMA2 LG4-5, which comprises the amino acid sequence of SEQ ID NO: 118 or 119. In some embodiments, the first unit of the first binding portion comprises two or more first non-antibody portions connected in series, and the second unit of the first binding portion comprises two or more second non-antibody portions connected in series. In some embodiments, the first unit of the first binding portion is the same as the second unit of the first binding portion. In some embodiments, the first unit of the first binding portion is different from the second unit of the first binding portion. One or more units of the second binding portion may be the same or different; and/or may bind to the same or different notched receptors. Each binding portion may be independently fused to the N-terminus and/or C-terminus of the Fc domain. The bifunctional protein construct may be a homodimer or a heterodimer.

In some embodiments, one or more units of the first binding moiety in the bifunctional protein construct comprises (or is) Fab, wherein the Fab is linked to the C-terminus of the subunit of the Fc domain via VH or VL, for example, via an optionally present linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, a bifunctional protein construct is provided, comprising: a first Fab (Fab1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); and a second Fab (Fab2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2), CH1 (H2-CH1), VL (VL2), and CL. (L2-CL); a first unit that specifically binds to a first nick receptor and activates the second binding portion of the first nick receptor; a second unit that specifically binds to a second nick receptor and activates the second binding portion of the second nick receptor; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the second binding portion - optionally a linker - the first unit of the Fc domain - optionally a linker - VH1-(H1-CH1); ii) a second polypeptide , which comprises, from N' to C': the second unit of the second binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - VH2-(H2-CH1); iii) a third polypeptide, which comprises, from N' to C': VL1-(L1-CL); and iv) a fourth polypeptide, which comprises, from N' to C': VL2-(L2-CL); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, a bifunctional protein construct is provided, comprising: a first Fab (Fab1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); and a second Fab (Fab2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2), CH1 (H2-CH1), VL (VL2), and CL. (L2-CL); a first unit that specifically binds to a first nick receptor and activates the second binding portion of the first nick receptor; a second unit that specifically binds to a second nick receptor and activates the second binding portion of the second nick receptor; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the second binding portion - optionally a linker - the first unit of the Fc domain - optionally a linker - VL1-(L1-CL); ii) a second polypeptide, It comprises, from N' to C': the second unit of the second binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - VL2-(L2-CL); iii) a third polypeptide, which comprises, from N' to C': VH1-(H1-CH1); and iv) a fourth polypeptide, which comprises, from N' to C': VH2-(H2-CH1); wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. Fab1 and Fab2 may be the same or different and/or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to the matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, the anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nick ligand or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the nick ligand is selected from the group consisting of: delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any one of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, one or more units of the first binding moiety comprises (or is) an scFv, wherein the scFv is linked to the C-terminus of the subunit of the Fc domain via the VH or VL, such as through an optionally present linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, a bifunctional protein construct is provided, comprising: a first scFv (scFv1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1) and VL (VL1); a second scFv (scFv2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2) and VL (VL2); a first unit that specifically binds to a first notch receptor and activates the second binding portion of the first notch receptor; a second unit that specifically binds to a second notch receptor and activates the second binding portion of the second notch receptor; and a Fv comprising the first unit and the second unit. c domain; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding moiety - optionally a linker - a first unit of the Fc domain - optionally a linker - scFv1 (VH1 - optionally a linker - VL1, or VL1 - optionally a linker - VH1); and ii) a second polypeptide comprising, from N' to C': a second unit of the second binding moiety - optionally a linker - a second unit of the Fc domain - optionally a linker - scFv2 (VH2 - optionally a linker - VL2, or VL2 - optionally a linker - VH2). In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. scFv1 and scFv2 may be the same or different; and/or may bind to the same or different muscle-specific molecules. In some embodiments, both scFv1 and scFv2 specifically bind to matrix glycans. In some embodiments, the anti-matrix glycan scFv1 and/or anti-matrix glycan scFv2 comprise the amino acid sequence of SEQ ID NO: 155. In some embodiments, both scFv1 and scFv2 specifically bind to LAMA2. In some embodiments, the anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nicked receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of: delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any one of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, one or more units of the first binding moiety comprises (or is) an sdAb, wherein the sdAb is linked to the C-terminus of the subunit of the Fc domain, such as through an optionally present linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, a bifunctional protein construct is provided, comprising: a first sdAb that specifically binds to a first muscle-specific molecule; (sdAb1, such as VHH1); a second sdAb (sdAb2, such as VHH2) that specifically binds to a second muscle-specific molecule; a first unit that specifically binds to a first notch receptor and activates a second binding portion of the first notch receptor; a second unit that specifically binds to a second notch receptor and activates a second binding portion of the second notch receptor; and an Fc domain comprising the first unit and the second unit; wherein The bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit of a second binding moiety, optionally a linker, a first unit of an Fc domain, optionally a linker, and sdAb1; and ii) a second polypeptide comprising, from N' to C', a second unit of a second binding moiety, optionally a linker, a second unit of an Fc domain, optionally a linker, and sdAb2. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. sdAb1 and sdAb2 can be the same or different and/or can bind to the same or different muscle-specific molecules. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, one or more units of the first binding moiety comprise (or are) Fab, wherein the Fab is linked to the N-terminus of the subunit of the Fc domain via CH1 or CL, such as through a linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), as appropriate. In some embodiments, a bifunctional protein construct is provided, comprising: a first Fab (Fab1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); and a second Fab (Fab2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2), CH1 (H2-CH1), VL (VL2), and CL. (L2-CL); a first unit that specifically binds to a first nick receptor and activates the second binding portion of the first nick receptor; a second unit that specifically binds to a second nick receptor and activates the second binding portion of the second nick receptor; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', VH1-(H1-CH1)-optionally a linker-the first unit of the Fc domain-optionally a linker-the first unit of the second binding portion; ii) a second polypeptide , which comprises, from N' to C': VH2-(H2-CH1)-optionally a linker-a second unit of the Fc domain-optionally a linker-a second unit of the second binding moiety; iii) a third polypeptide, which comprises, from N' to C': VL1-(L1-CL); and iv) a fourth polypeptide, which comprises, from N' to C': VL2-(L2-CL); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, a bifunctional protein construct is provided, comprising: a first Fab (Fab1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); and a second Fab (Fab2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2), CH1 (H2-CH1), VL (VL2), and CL. (L2-CL); a first unit that specifically binds to a first nick receptor and activates the second binding portion of the first nick receptor; a second unit that specifically binds to a second nick receptor and activates the second binding portion of the second nick receptor; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': VL1-(L1-CL)-optionally a linker-the first unit of the Fc domain-optionally a linker-the first unit of the second binding portion; ii) a second polypeptide comprising, from N' to C' The invention further comprises: a second polypeptide comprising: VL2-(L2-CL)-optionally a linker-an Fc domain; a second polypeptide comprising: a second polypeptide specifically binding to a second binding moiety of a second nicked receptor; iii) a third polypeptide comprising, from N' to C', VH1-(H1-CH1); and iv) a fourth polypeptide comprising, from N' to C', VH2-(H2-CH1); wherein VL1-(L1-CL) forms a Fab1 with VH1-(H1-CH1), and VH2-(H2-CH1) forms a Fab2 with VL2-(L2-CL). In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. Fab1 and Fab2 may be the same or different and/or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to the matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, the anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nick ligand or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the nick ligand is selected from the group consisting of: delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any one of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: i) a first binding moiety comprising a full-length antibody that specifically binds to a muscle-specific molecule, wherein the full-length antibody comprises an Fc domain comprising a first unit and a second unit; ii) a first unit of a second binding moiety fused to the C-terminus of the first unit of the Fc domain (directly or via an optional linker); and iii) a second unit of a second binding moiety fused to the C-terminus of the second unit of the Fc domain (directly or via an optional linker); wherein the first unit of the second binding moiety specifically binds to and activates a first nick receptor, and the second unit of the second binding moiety specifically binds to and activates a second nick receptor. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, one or more units of the first binding moiety comprises (or is) an scFv, wherein the scFv is linked to the N-terminus of the subunit of the Fc domain via the VH or VL, such as through an optionally present linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338). In some embodiments, a bifunctional protein construct is provided, comprising: a first scFv (scFv1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1) and VL (VL1); a second scFv (scFv2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2) and VL (VL2); a first unit that specifically binds to a first notch receptor and activates the second binding portion of the first notch receptor; a second unit that specifically binds to a second notch receptor and activates the second binding portion of the second notch receptor; and an Fc unit comprising the first unit and the second unit. domain; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': scFv1 (VH1-optionally linker-VL1 or VL1-optionally linker-VH1)-optionally linker-first subunit of the Fc domain-optionally linker-first subunit of the second binding moiety; and ii) a second polypeptide comprising, from N' to C': scFv2 (VH2-optionally linker-VL2 or VL2-optionally linker-VH2)-optionally linker-second subunit of the Fc domain-optionally linker-second subunit of the second binding moiety. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. scFv1 and scFv2 may be the same or different; and/or may bind to the same or different muscle-specific molecules. In some embodiments, both scFv1 and scFv2 specifically bind to matrix glycans. In some embodiments, the anti-matrix glycan scFv1 and/or anti-matrix glycan scFv2 comprise the amino acid sequence of SEQ ID NO: 155. In some embodiments, both scFv1 and scFv2 specifically bind to LAMA2. In some embodiments, the anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nicked receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of: delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any one of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, one or more units of the first binding moiety comprise (or are) sdAbs, wherein the sdAbs are linked to the N-terminus of the subunit of the Fc domain, such as through an optionally present linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338). In some embodiments, a bifunctional protein construct is provided, comprising: a first sdAb (sdAb1, e.g., VHH1) that specifically binds to a first muscle-specific molecule; a second sdAb (sdAb2, e.g., VHH2) that specifically binds to a second muscle-specific molecule; a first unit of a second binding moiety that specifically binds to a first nick receptor and activates the first nick receptor; and a second binding moiety that specifically binds to a second nick receptor and activates the second nick receptor. and an Fc domain comprising the first and second units; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', sdAb1 - optionally a linker - the first unit of the Fc domain - optionally a linker - the first unit of the second binding moiety; and ii) a second polypeptide comprising, from N' to C', sdAb2 - optionally a linker - the second unit of the Fc domain - optionally a linker - the second unit of the second binding moiety. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. sdAb1 and sdAb2 can be the same or different and/or can bind to the same or different muscle-specific molecules. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: a first Fab (Fab1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit that specifically binds to a first nick receptor and activates the second binding portion of the first nick receptor; a unit that specifically binds to a second nick receptor and activates the second binding portion of the second nick receptor; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct The antibody comprises: i) a first polypeptide comprising, from N' to C', VH1-(H1-CH1)-optionally a linker-a first subunit of an Fc domain; ii) a second polypeptide comprising, from N' to C', VH2-(H2-CH1)-optionally a linker-a second subunit of an Fc domain; iii) a third polypeptide comprising, from N' to C', VL1-(L1-CL)-optionally a linker-a first subunit of a second binding moiety; and iv) a fourth polypeptide comprising, from N' to C', VL2-(L2-CL)-optionally a linker-a second subunit of a second binding moiety; and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. Fab1 and Fab2 can be the same or different and/or can bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to the matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, the anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding moiety and the second unit of the second binding moiety may be the same or different and/or may bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: i) a first binding moiety comprising a full-length antibody that specifically binds to a muscle-specific molecule; ii) a first unit of a second binding moiety fused to the C-terminus of the first light chain of the full-length antibody (directly or via an optional linker); and iii) a second unit of the second binding moiety fused to the C-terminus of the second light chain of the full-length antibody (directly or via an optional linker); wherein the first unit of the second binding moiety specifically binds to and activates a first nick receptor, and the second unit of the second binding moiety specifically binds to and activates a second nick receptor. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: a first Fab (Fab1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit that specifically binds to a first notch receptor and activates the second binding portion of the first notch receptor; a second unit that specifically binds to a second notch receptor and activates the second binding portion of the second notch receptor; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct The antibody comprises: i) a first polypeptide comprising, from N' to C', VH1-(H1-CH1)-optionally a linker-a first subunit of an Fc domain; ii) a second polypeptide comprising, from N' to C', VH2-(H2-CH1)-optionally a linker-a second subunit of an Fc domain; iii) a third polypeptide comprising, from N' to C', the first subunit of the second binding moiety-optionally a linker-VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C', the second subunit of the second binding moiety-optionally a linker-VL2-(L2-CL); wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. Fab1 and Fab2 can be the same or different and/or can bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to the matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, the anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding moiety and the second unit of the second binding moiety may be the same or different and/or may bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: a first binding moiety comprising a full-length antibody that specifically binds to a muscle-specific molecule; a first unit of a second binding moiety fused to the N-terminus of the first light chain of the full-length antibody (directly or via an optional linker); and a second unit of the second binding moiety fused to the N-terminus of the second light chain of the full-length antibody (directly or via an optional linker); wherein the first unit of the second binding moiety specifically binds to a first nick receptor and activates the first nick receptor, and the second unit of the second binding moiety specifically binds to a second nick receptor and activates the second nick receptor. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: a first Fab (Fab1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); a second Fab (Fab2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2), CH1 (H2-CH1), VL (VL2), and CL (L2-CL); a first unit that specifically binds to a first notch receptor and activates the second binding portion of the first notch receptor; a second unit that specifically binds to a second notch receptor and activates the second binding portion of the second notch receptor; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct The antibody comprises: i) a first polypeptide comprising, from N' to C': the first unit of the second binding moiety - optionally a linker - VH1-(H1-CH1) - optionally a linker - a first subunit of the Fc domain; ii) a second polypeptide comprising, from N' to C': the second unit of the second binding moiety - optionally a linker - VH2-(H2-CH1) - optionally a linker - a second subunit of the Fc domain; iii) a third polypeptide comprising, from N' to C': VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C': VL2-(L2-CL); wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. Fab1 and Fab2 can be the same or different and/or can bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to the matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, the anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The first unit of the second binding moiety and the second unit of the second binding moiety may be the same or different and/or may bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: a first binding moiety comprising a full-length antibody that specifically binds to a muscle-specific molecule; a first unit of a second binding moiety fused to the N-terminus of the first heavy chain of the full-length antibody (directly or via an optional linker); and a second unit of the second binding moiety fused to the N-terminus of the second heavy chain of the full-length antibody (directly or via an optional linker); wherein the first unit of the second binding moiety specifically binds to a first nick receptor and activates the first nick receptor, and the second unit of the second binding moiety specifically binds to a second nick receptor and activates the second nick receptor. In some embodiments, the optional linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or two units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: a first Fab (Fab1) that specifically binds to a first muscle-specific molecule, comprising VH (VH1), CH1 (H1-CH1), VL (VL1), and CL (L1-CL); and a second Fab (Fab2) that specifically binds to a second muscle-specific molecule, comprising VH (VH2), CH1 (H2-CH1), VL (VL2), and CL. (L2-CL); a first unit that specifically binds to a first notch receptor and activates the second binding portion of the first notch receptor; a second unit that specifically binds to a second notch receptor and activates the second binding portion of the second notch receptor; a third unit that specifically binds to a third notch receptor and activates the second binding portion of the third notch receptor; a fourth unit that specifically binds to a fourth notch receptor and activates the second binding portion of the fourth notch receptor; and an Fc domain comprising the first unit and the second unit wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit of a second binding moiety, optionally a linker, VH1-(H1-CH1)-optionally a linker-a first unit of an Fc domain; ii) a second polypeptide comprising, from N' to C', a second unit of a second binding moiety, optionally a linker, VH2-(H2-CH1)-optionally a linker-a second unit of an Fc domain; iii) a second polypeptide comprising, from N' to C', a second unit of a second binding moiety, optionally a linker, VH2-(H2-CH1)-optionally a linker-a second unit of an Fc domain; A tripeptide comprising, from N' to C', the third unit of the second binding moiety, optionally a linker, VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C', the fourth unit of the second binding moiety, optionally a linker, VL2-(L2-CL); wherein VL1-(L1-CL) forms a Fab1 with VH1-(H1-CH1), and VH2-(H2-CH1) forms a Fab2 with VL2-(L2-CL). In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which may be the same or different within the bifunctional protein construct. In some embodiments, the first muscle-specific molecule and the second muscle-specific molecule are independently selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. Fab1 and Fab2 may be the same or different and/or may bind to the same or different muscle-specific molecules. In some embodiments, both Fab1 and Fab2 specifically bind to the matrix glycan Fab. In some embodiments, the anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, both Fab1 and Fab2 specifically bind to LAMA2. In some embodiments, the anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The first unit, second unit, third unit, and/or fourth unit of the second binding moiety may be the same or different; and/or may bind to the same or different nicked receptors. In some embodiments, all four units of the second binding moiety are the same. In some embodiments, at least one of the four units of the second binding moiety is different from the other units. In some embodiments, the second binding moiety (one or more units, or all units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the nicked ligand is selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or more units, or all units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, a bifunctional protein construct is provided, comprising: a first binding moiety comprising a full-length antibody that specifically binds to a muscle-specific molecule; a first unit of a second binding moiety fused to the N-terminus of the first light chain of the full-length antibody (directly or via a linker, if applicable); a second unit of the second binding moiety fused to the N-terminus of the second light chain of the full-length antibody (directly or via a linker, if applicable); a third unit of the second binding moiety fused to the N-terminus of the first heavy chain of the full-length antibody (directly or via a linker, if applicable); and a fourth unit of the second binding moiety, which is fused to the N-terminus of the second heavy chain of the full-length antibody (directly or through a linker, if present); wherein the first unit of the second binding moiety specifically binds to and activates a first nick receptor, the second unit of the second binding moiety specifically binds to and activates a second nick receptor, the third unit of the second binding moiety specifically binds to and activates a third nick receptor, and the fourth unit of the second binding moiety specifically binds to and activates a fourth nick receptor. In some embodiments, the optional linker is a peptide linker (e.g., any one of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and a matrix glycan on α-DG. The first unit, second unit, third unit, and/or fourth unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, all four units of the second binding moiety are the same. In some embodiments, at least one of the four units of the second binding moiety is different from the other units. In some embodiments, the second binding moiety (one or more units, or all units) comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the nicked ligand is selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or more units, or all units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267.

In some embodiments, one or more units of the first binding moiety comprises a non-antibody portion that specifically binds to a muscle-specific molecule, wherein the non-antibody portion is linked to the C-terminus or N-terminus of the Fc domain subunit, for example, via an optionally present linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). For example, in some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion that specifically binds to a first muscle-specific molecule; a second non-antibody portion that specifically binds to a second muscle-specific molecule; a first unit that specifically binds to a first nick receptor and activates the second binding portion of the first nick receptor; a second unit that specifically binds to a second nick receptor and activates the second binding portion of the second nick receptor; and a first The bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - the first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - the second non-antibody moiety. In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion that specifically binds to a first muscle-specific molecule; a second non-antibody portion that specifically binds to a second muscle-specific molecule; a first unit that specifically binds to a first nick receptor and activates the second binding portion of the first nick receptor; a second unit that specifically binds to a second nick receptor and activates the second binding portion of the second nick receptor; and a first unit comprising wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first non-antibody moiety - an optional linker - a first unit of the Fc domain - an optional linker - a first unit of the second binding moiety; and ii) a second polypeptide comprising, from N' to C': a second non-antibody moiety - an optional linker - a second unit of the Fc domain - an optional linker - a second unit of the second binding moiety. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises a protein domain independently selected from the group consisting of a laminin G-like domain (LG domain) of laminin, aggrecan LG domain, nidogen LG domain, perlein LG domain, aggrecan laminin coiled-coil binding domain, and nidogen laminin γ binding domain. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises two or more protein domains (e.g., LG domains) tandemly linked, for example, via an optional linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises LAMA2 LG4-5, which comprises the amino acid sequence of SEQ ID NOs: 118 or 119. In some embodiments, the first non-antibody moiety comprises two LAMA2 LG4-5 linked in tandem, and the second non-antibody moiety comprises two LAMA2 LG4-5 linked in tandem. In some embodiments, the non-antibody moiety comprises a laminin G-like domain (LG domain). In some embodiments, the bifunctional protein construct comprises more than one (e.g., two or three) non-antibody moieties (e.g., LG domains) optionally linked in tandem via a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338). The first non-antibody moiety and the second non-antibody moiety can be the same or different and/or can bind to the same or different muscle-specific molecules. In some embodiments, the optional peptide linker is a peptide linker (e.g., any of SEQ ID NOs: 211, 212, 337, and 338), which can be the same or different within the bifunctional protein construct. The first unit of the second binding moiety and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, the second binding moiety (one or both units) comprises an ECD of a nick ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the nick ligand is selected from the group consisting of: delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any one of SEQ ID NOs: 126-135 and 260-267. Exemplary Fc Domain Fusions

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti-LAMA2 Fab2); a first unit comprising a second binding moiety of DLL4 ECD or a variant thereof; a second unit comprising a second binding moiety of DLL4 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding moiety - an optional linker - a first unit of the Fc domain - an optional linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': a second binding moiety; The invention also provides a method for preparing an anti-LAMA2 antibody comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The anti-LAMA2 antibody comprises ... In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NOs: 188 or 277.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, a bifunctional protein construct is provided comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 189; and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165 (hereinafter referred to as "DLL4wt-Fc-LG21-Fab"). In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4v-Fc-LG21-Fab") is provided, comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 190; and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4max-Fc-LG21-Fab") is provided, comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 191; and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.

In some embodiments, the first and second units of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4di-Fc-LG21-Fab") is provided, comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 192; and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti-LAMA2 Fab2); a first unit comprising a second binding moiety of a DLL1 ECD or a variant thereof; a second unit comprising a second binding moiety of a DLL1 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding moiety - an optional linker - a first unit of the Fc domain - an optional linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': a second binding moiety; The invention also provides a method for preparing an anti-LAMA2 antibody comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The anti-LAMA2 antibody comprises ... In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL1wt-Fc-LG21-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 193; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti-LAMA2 Fab2); a first unit comprising a second binding moiety of a DLL3 ECD or a variant thereof; a second unit comprising a second binding moiety of a DLL3 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding moiety - an optional linker - a first unit of the Fc domain - an optional linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': a second binding moiety; The invention also provides a method for preparing an anti-LAMA2 antibody comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The anti-LAMA2 antibody comprises ... In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL3wt-Fc-LG21-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 194; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti-LAMA2 Fab2); a first unit comprising a second binding moiety of Jag1 ECD or a variant thereof; a second unit comprising a second binding moiety of Jag1 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding moiety - an optional linker - a first unit of the Fc domain - an optional linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': a second binding moiety; The invention also provides a method for preparing an anti-LAMA2 antibody comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The anti-LAMA2 antibody comprises ... In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1wt-Fc-LG21-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 195; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1v-Fc-LG21-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 196; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 Fab (anti-LAMA2 Fab1); a second anti-LAMA2 Fab (anti-LAMA2 Fab2); a first unit comprising a second binding moiety of Jag2 ECD or a variant thereof; a second unit comprising a second binding moiety of Jag2 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding moiety - an optional linker - a first unit of the Fc domain - an optional linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': a second binding moiety; The invention also provides a method for preparing an anti-LAMA2 antibody comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. The anti-LAMA2 antibody comprises ... In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag2wt-Fc-LG21-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 197; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 165.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan Fab (anti-matrix glycan Fab1); a second anti-matrix glycan Fab (anti-matrix glycan Fab2); a first unit comprising a second binding moiety of DLL4 ECD or a variant thereof; a second unit comprising a second binding moiety of DLL4 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': the second unit of the second binding moiety - optionally a linker - Fc The invention also provides a second polypeptide unit comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; a second polypeptide comprising the linker VL2-(L2-CL) and a third polypeptide comprising, from N' to C', VH1-(H1-CH1); and a fourth polypeptide comprising, from N' to C', VH2-(H2-CH1); wherein VL1-(L1-CL) and VH1-(H1-CH1) form anti-matrix glycan Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form anti-matrix glycan Fab2. In some embodiments, anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. Anti-matrix glycan Fab1 and anti-matrix glycan Fab2 may be the same or different. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors.

In some embodiments, the first and second units of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4wt-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 198; and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4v-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 199; and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4max-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 200; and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second units of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4di-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 201; and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan Fab (anti-matrix glycan Fab1); a second anti-matrix glycan Fab (anti-matrix glycan Fab2); a first unit comprising a second binding moiety of DLL1 ECD or a variant thereof; a second unit comprising a second binding moiety of DLL1 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': the second unit of the second binding moiety - optionally a linker - Fc The invention also provides a second polypeptide unit comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; a second polypeptide comprising the linker VL2-(L2-CL) and a third polypeptide comprising, from N' to C', VH1-(H1-CH1); and a fourth polypeptide comprising, from N' to C', VH2-(H2-CH1); wherein VL1-(L1-CL) and VH1-(H1-CH1) form anti-matrix glycan Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form anti-matrix glycan Fab2. In some embodiments, anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. Anti-matrix glycan Fab1 and anti-matrix glycan Fab2 may be the same or different. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL1wt-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 202; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan Fab (anti-matrix glycan Fab1); a second anti-matrix glycan Fab (anti-matrix glycan Fab2); a first unit comprising a second binding moiety of a DLL3 ECD or a variant thereof; a second unit comprising a second binding moiety of a DLL3 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of a second binding moiety - optionally a linker - a first unit of an Fc domain - optionally a linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': a second unit of a second binding moiety - optionally a linker - Fc domain The invention also provides a second polypeptide unit comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; a second polypeptide comprising the linker VL2-(L2-CL) and a third polypeptide comprising, from N' to C', VH1-(H1-CH1); and a fourth polypeptide comprising, from N' to C', VH2-(H2-CH1); wherein VL1-(L1-CL) and VH1-(H1-CH1) form anti-matrix glycan Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form anti-matrix glycan Fab2. In some embodiments, anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. Anti-matrix glycan Fab1 and anti-matrix glycan Fab2 may be the same or different. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL3wt-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 203; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan Fab (anti-matrix glycan Fab1); a second anti-matrix glycan Fab (anti-matrix glycan Fab2); a first unit comprising a second binding moiety of Jag1 ECD or a variant thereof; a second unit comprising a second binding moiety of Jag1 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding moiety - optionally a linker - a first unit of the Fc domain - optionally a linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': a second unit of the second binding moiety - optionally a linker - Fc domain The invention also provides a second polypeptide unit comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; a second polypeptide comprising the linker VL2-(L2-CL) and a third polypeptide comprising, from N' to C', VH1-(H1-CH1); and a fourth polypeptide comprising, from N' to C', VH2-(H2-CH1); wherein VL1-(L1-CL) and VH1-(H1-CH1) form anti-matrix glycan Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form anti-matrix glycan Fab2. In some embodiments, anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. Anti-matrix glycan Fab1 and anti-matrix glycan Fab2 may be the same or different. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1wt-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 204; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1v-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 205; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan Fab (anti-matrix glycan Fab1); a second anti-matrix glycan Fab (anti-matrix glycan Fab2); a first unit comprising a second binding moiety of Jag2 ECD or a variant thereof; a second unit comprising a second binding moiety of Jag2 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C': the second unit of the second binding moiety - optionally a linker - Fc domain The invention also provides a second polypeptide unit comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; a second polypeptide comprising the linker VL2-(L2-CL) and a third polypeptide comprising, from N' to C', VH1-(H1-CH1); and a fourth polypeptide comprising, from N' to C', VH2-(H2-CH1); wherein VL1-(L1-CL) and VH1-(H1-CH1) form anti-matrix glycan Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form anti-matrix glycan Fab2. In some embodiments, anti-matrix glycan Fab1 and/or anti-matrix glycan Fab2 comprise: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. Anti-matrix glycan Fab1 and anti-matrix glycan Fab2 may be the same or different. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag2wt-Fc-ADG41-Fab") is provided, comprising: a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 206; and a third polypeptide and a fourth polypeptide, each comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit comprising a second binding moiety of DLL4 ECD or a variant thereof; a second unit comprising a second binding moiety of DLL4 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - anti-LAMA2 scFv1; and ii) a second polypeptide comprising, from N' to C', a second unit of a second binding moiety, optionally a linker, a second unit of an Fc domain, optionally a linker, and an anti-LAMA2 scFv2. The anti-LAMA2 scFv1 and anti-LAMA2 scFv2 may be the same or different. In some embodiments, the anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprise a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. The first and second units of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188 or 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NO: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4wt-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 223. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4wt-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 224. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4max-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 225. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second units of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4di-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 226. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 260. In some embodiments, a bifunctional protein construct (hereinafter designated "huD4v_G2S_F81L_H168Y-Fc(AAG)-LG21scFvDS") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 269. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 261. In some embodiments, a bifunctional protein construct (hereinafter designated "huD4v11_G2S_T52N_F81L_T135N_H168Y-Fc(AAG)-LG21scFvDS") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 270. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 262. In some embodiments, a bifunctional protein construct (hereinafter designated "huD4v12_G2S_R66S_F81L_H168Y-Fc(AAG)-LG21scFvDS") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 271. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 263. In some embodiments, a bifunctional protein construct (hereinafter designated "huD4v13_G2S_E14H_T52N_R66T_P76L_F81L_T135N_H168Y_N231D-Fc(AAG)-LG21scFvDS") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 272. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 264. In some embodiments, a bifunctional protein construct (hereinafter designated "huD4v14_G2S_T52N_R66T_P76L_F81L_T135N_H168Y_Q220H_N260D-Fc(AAG)-LG21scFvDS") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 273. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 265. In some embodiments, a bifunctional protein construct (hereinafter designated "huD4v22_G2S_R66S_F81L_H168Y-Fc(AAG)-LG21scFvDS") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 274. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 266. In some embodiments, a bifunctional protein construct (hereinafter designated "huD4v23_G2S_E14H_R66T_P76L_F81L_H168Y_N231D-FcAAG-LG21DS") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 275. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 267. In some embodiments, a bifunctional protein construct (hereinafter designated "huD4v24_G2S_R66T_P76L_F81L_H168Y_Q220H_N260D-FcAAG-LG21DS") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 276. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit comprising a second binding moiety of DLL1 ECD or a variant thereof; a second unit comprising a second binding moiety of DLL1 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and anti-LAMA2; scFv1; and ii) a second polypeptide comprising, from N' to C', a second unit of a second binding moiety, optionally a linker, a second unit of an Fc domain, optionally a linker, and an anti-LAMA2 scFv2. The anti-LAMA2 scFv1 and the anti-LAMA2 scFv2 may be the same or different. In some embodiments, the anti-LAMA2 scFv1 and/or the anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprise a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. The first unit and the second unit of the second binding moiety may be the same or different; and/or may bind to the same or different nicked receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL1wt-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 227. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit comprising a second binding moiety of a DLL3 ECD or a variant thereof; a second unit comprising a second binding moiety of a DLL3 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and anti-LAMA2; scFv1; and ii) a second polypeptide comprising, from N' to C', a second unit of a second binding moiety, optionally a linker, a second unit of an Fc domain, optionally a linker, and an anti-LAMA2 scFv2. The anti-LAMA2 scFv1 and the anti-LAMA2 scFv2 may be the same or different. In some embodiments, the anti-LAMA2 scFv1 and/or the anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprise a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. The first unit and the second unit of the second binding moiety may be the same or different; and/or may bind to the same or different nicked receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL3wt-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit comprising a second binding moiety of Jag1 ECD or a variant thereof; a second unit comprising a second binding moiety of Jag1 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': a first unit of the second binding moiety, optionally a linker, a first unit of the Fc domain, optionally a linker, and anti-LAMA2; scFv1; and ii) a second polypeptide comprising, from N' to C', a second unit of a second binding moiety, optionally a linker, a second unit of an Fc domain, optionally a linker, and an anti-LAMA2 scFv2. The anti-LAMA2 scFv1 and the anti-LAMA2 scFv2 may be the same or different. In some embodiments, the anti-LAMA2 scFv1 and/or the anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprise a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. The first unit and the second unit of the second binding moiety may be the same or different; and/or may bind to the same or different nicked receptors. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1wt-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 229. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1v-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 230. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-LAMA2 scFv (anti-LAMA2 scFv1); a second anti-LAMA2 scFv (anti-LAMA2 scFv2); a first unit comprising a second binding moiety of Jag2 ECD or a variant thereof; a second unit comprising a second binding moiety of Jag2 ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C': the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - anti-LAMA2 scFv1; and ii) a second polypeptide comprising, from N' to C', a second unit of a second binding moiety, optionally a linker, a second unit of an Fc domain, optionally a linker, and an anti-LAMA2 scFv2. The anti-LAMA2 scFv1 and the anti-LAMA2 scFv2 may be the same or different. In some embodiments, the anti-LAMA2 scFv1 and/or the anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprise a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. The first unit and the second unit of the second binding moiety may be the same or different; and/or may bind to the same or different nicked receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag2wt-Fc-LG21scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 231. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan scFv (anti-matrix glycan scFv1); a second anti-matrix glycan scFv (anti-matrix glycan scFv2); a first unit comprising a second binding moiety of a DLL4 ECD or a variant thereof; and a second unit comprising a DLL4 ECD or a variant thereof. The bifunctional protein construct comprises: a second unit of the second binding moiety of an ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv1; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv2. The anti-matrix glycan scFv1 and the anti-matrix glycan scFv2 may be the same or different. In some embodiments, the anti-matrix glycan scFv1 and/or anti-matrix glycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188 or 277. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4wt-Fc-ADG41scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 232. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4v-Fc-ADG41scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 233. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4max-Fc-ADG41scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 234. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4di-Fc-ADG41scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 235. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan scFv (anti-matrix glycan scFv1); a second anti-matrix glycan scFv (anti-matrix glycan scFv2); a first unit comprising a second binding moiety of a DLL1 ECD or a variant thereof; and a second unit comprising a DLL1 ECD or a variant thereof. The bifunctional protein construct comprises: a second unit of the second binding moiety of an ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv1; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv2. The anti-matrix glycan scFv1 and the anti-matrix glycan scFv2 may be the same or different. In some embodiments, the anti-matrix glycan scFv1 and/or anti-matrix glycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL1wt-Fc-ADG41 scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 236. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan scFv (anti-matrix glycan scFv1); a second anti-matrix glycan scFv (anti-matrix glycan scFv2); a first unit comprising a second binding moiety of a DLL3 ECD or a variant thereof; and a second unit comprising a DLL3 The bifunctional protein construct comprises: a second unit of the second binding moiety of an ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv1; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv2. The anti-matrix glycan scFv1 and the anti-matrix glycan scFv2 may be the same or different. In some embodiments, the anti-matrix glycan scFv1 and/or anti-matrix glycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL3wt-Fc-ADG41 scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 237. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan scFv (anti-matrix glycan scFv1); a second anti-matrix glycan scFv (anti-matrix glycan scFv2); a first unit comprising a second binding moiety of Jag1 ECD or a variant thereof; and a second unit comprising Jag1 ECD or a variant thereof. The bifunctional protein construct comprises: a second unit of the second binding moiety of an ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv1; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv2. The anti-matrix glycan scFv1 and the anti-matrix glycan scFv2 may be the same or different. In some embodiments, the anti-matrix glycan scFv1 and/or anti-matrix glycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1wt-Fc-ADG41scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1v-Fc-ADG41scFv") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first anti-matrix glycan scFv (anti-matrix glycan scFv1); a second anti-matrix glycan scFv (anti-matrix glycan scFv2); a first unit comprising a second binding moiety of Jag2 ECD or a variant thereof; and a second unit comprising Jag2 The bifunctional protein construct comprises: a second unit of the second binding moiety of an ECD or a variant thereof; and an Fc domain comprising the first unit and the second unit; wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the second binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv1; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - an anti-matrix glycan scFv2. The anti-matrix glycan scFv1 and the anti-matrix glycan scFv2 may be the same or different. In some embodiments, the anti-matrix glycan scFv1 and/or anti-matrix glycan scFv2 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag2wt-Fc-ADG41scFv") is provided, wherein the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each of which comprises the amino acid sequence of SEQ ID NO: 240. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising LAMA2 LG4-5; a second non-antibody portion comprising LAMA2 LG4-5; a first unit comprising a second binding portion comprising DLL4 ECD or a variant thereof; and a second unit comprising DLL4 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NOs: 188 or 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4wt-Fc-LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4v-Fc-LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4max-Fc-LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 243. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the first and second subunits of the second binding moiety each comprise a variant DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 129. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4di-Fc-LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 244. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising LAMA2 LG4-5; a second non-antibody portion comprising LAMA2 LG4-5; a first unit comprising a second binding portion comprising DLL1 ECD or a variant thereof; and a second unit comprising DLL1. The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL1wt-Fc-LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each of which comprises the amino acid sequence of SEQ ID NO: 245. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising LAMA2 LG4-5; a second non-antibody portion comprising LAMA2 LG4-5; a first unit comprising a second binding portion comprising DLL3 ECD or a variant thereof; and a second unit comprising DLL3 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL3wt-Fc-LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each of which comprises the amino acid sequence of SEQ ID NO: 246. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising LAMA2 LG4-5; a second non-antibody portion comprising LAMA2 LG4-5; a first unit comprising a second binding portion comprising Jag1 ECD or a variant thereof; and a second unit comprising Jag1 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1wt-Fc-LG4-5") is provided, wherein the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 247. In some embodiments, the first unit and the second unit of the second binding moiety each comprise a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jag1v-Fc-LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 248. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising LAMA2 LG4-5; a second non-antibody portion comprising LAMA2 LG4-5; a first unit comprising a second binding portion comprising Jag2 ECD or a variant thereof; and a second unit comprising Jag2 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct is provided (hereinafter referred to as "Jag2wt-Fc-LG4-5"), which comprises a first polypeptide and a second polypeptide, each of which comprises the amino acid sequence of SEQ ID NO: 249. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising laminin-a2 LG4-5; a second non-antibody portion comprising laminin-a2 LG4-5; a first unit comprising a second binding portion of DLL4 ECD or a variant thereof; and a second unit comprising DLL4. The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises DLL4 ECD or a variant thereof, wherein the DLL4 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 339. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4(EGF1-6)-hIgG1-Fc-laminin-a2 LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 321. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 345. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising laminin-a2 LG4-5; a second non-antibody portion comprising laminin-a2 LG4-5; a first unit comprising a second binding portion comprising DLL1 ECD or a variant thereof; and a second unit comprising DLL1 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises DLL1 ECD or a variant thereof, wherein the DLL1 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 340. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL1(EGF1-6)-hIgG1-Fc-laminin-a2 LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 324. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 345. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising laminin-a2 LG4-5; a second non-antibody portion comprising laminin-a2 LG4-5; a first unit comprising a second binding portion comprising Jag1 ECD or a variant thereof; and a second unit comprising Jag1 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag1 ECD or a variant thereof, wherein the Jag1 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 341. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jagged1 (EGF1-6)-hIgG1-Fc-laminin-a2 LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 327. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 345. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising laminin-a2 LG4-5; a second non-antibody portion comprising laminin-a2 LG4-5; a first unit comprising a second binding portion comprising Jag2 ECD or a variant thereof; and a second unit comprising Jag2 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD or a variant thereof, wherein the Jag2 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 342. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jagged-2 (EGF1-6)-hIgG1-Fc-laminin-a2 LG4-5") is provided, wherein the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each of which comprises the amino acid sequence of SEQ ID NO: 330. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 345. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising laminin-a2 LG4-5; a second non-antibody portion comprising laminin-a2 LG4-5; a first unit comprising a second binding portion of DLL4 ECD or a variant thereof; and a second unit comprising DLL4. The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises DLL4 ECD or a variant thereof, wherein the DLL4 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 339. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL4(EGF1-6)-hIgG4-Fc-laminin-a2 LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 331. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 346. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising laminin-a2 LG4-5; a second non-antibody portion comprising laminin-a2 LG4-5; a first unit comprising a second binding portion comprising DLL1 ECD or a variant thereof; and a second unit comprising DLL1 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises DLL1 ECD or a variant thereof, wherein the DLL1 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 340. The first unit and the second unit of the second binding moiety can be the same or different and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "DLL1(EGF1-6)-hIgG4-Fc-laminin-a2 LG4-5") is provided, comprising a first polypeptide and a second polypeptide, each comprising the amino acid sequence of SEQ ID NO: 332. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 346. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising laminin-a2 LG4-5; a second non-antibody portion comprising laminin-a2 LG4-5; a first unit comprising a second binding portion comprising Jag1 ECD or a variant thereof; and a second unit comprising Jag1 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag1 ECD or a variant thereof, wherein the Jag1 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 341. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jagged1 (EGF1-6)-hIgG4-Fc-laminin-a2 LG4-5") is provided, wherein the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each of which comprises the amino acid sequence of SEQ ID NO: 333. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 346. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: a first non-antibody portion comprising laminin-a2 LG4-5; a second non-antibody portion comprising laminin-a2 LG4-5; a first unit comprising a second binding portion comprising Jag2 ECD or a variant thereof; and a second unit comprising Jag2 The bifunctional protein construct comprises: a first polypeptide comprising, from N' to C', the first unit of the second binding moiety, optionally a linker, the first unit of the Fc domain, optionally a linker, and a first non-antibody moiety; and ii) a second polypeptide comprising, from N' to C', the second unit of the second binding moiety, optionally a linker, the second unit of the Fc domain, optionally a linker, and a second non-antibody moiety. The first non-antibody moiety and the second non-antibody moiety may be the same or different. In some embodiments, the first non-antibody moiety and/or the second non-antibody moiety comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD or a variant thereof, wherein the Jag2 ECD or a variant thereof comprises the amino acid sequence of SEQ ID NO: 342. The first unit and the second unit of the second binding moiety can be the same or different; and/or can bind to the same or different nick receptors. In some embodiments, a bifunctional protein construct (hereinafter referred to as "Jagged2 (EGF1-6)-hIgG4-Fc-laminin-a2 LG4-5") is provided, wherein the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each of which comprises the amino acid sequence of SEQ ID NO: 334. In some embodiments, each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 346. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the bifunctional construct comprises the amino acid sequence of any one of SEQ ID NOs: 250-252.

In some embodiments, a bifunctional construct provided herein may comprise: a first unit comprising a first binding moiety of two or more LAMA2 LG4-5 linked in tandem, and a second unit comprising a first binding moiety of two or more LAMA2 LG4-5 linked in tandem. The first unit and the second unit of the first binding moiety may be the same or different. Heterodimer Fusions

The bifunctional protein constructs described herein (e.g., comprising an Fc domain, the Fc domain comprising a first unit and a second unit) may comprise two or more different polypeptide chains. In some embodiments, the bifunctional protein construct is a heterodimer. In some embodiments, a bifunctional protein construct is provided, comprising: a first binding portion (e.g., scFv, Fab, sdAb, or a non-antibody portion) that specifically binds to a muscle-specific molecule; a second binding portion (e.g., scFv, Fab, sdAb, or a non-antibody portion) that specifically binds to a notch receptor and activates the notch receptor; and an Fc domain comprising a first unit and a second unit; wherein: i) the first binding portion In some embodiments, the Fc domain comprises a K-in hole mutation and/or a charge pair mutation. In some embodiments, the Fc domain comprises a K-in hole mutation, wherein: i) the first Fc domain comprises a K-in hole mutation, and the second Fc domain comprises a hole mutation; or ii) the second Fc domain comprises a K-in hole mutation, and the first Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering), and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, each subunit of the Fc domain further comprises L234A/L235A/H435A mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the bifunctional protein construct comprises an Fc domain comprising a first subunit and a second subunit, wherein one subunit of the Fc domain is fused (at the N-terminus, C-terminus, or both) to a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to a muscle-specific molecule, and the other subunit of the Fc domain is fused (at the N-terminus, C-terminus, or both) to a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to and activates the notch receptor. The bifunctional protein construct may comprise two or more subunits of the first binding moiety and/or two or more subunits of the second binding moiety. The two or more subunits of the first binding moiety may be the same or different. The two or more subunits of the second binding moiety may be the same or different. In some embodiments, a bifunctional protein construct is provided, comprising: a first binding moiety comprising a Fab that specifically binds to a muscle-specific molecule; a second binding moiety (e.g., comprising a Notch ligand ECD or variant) that specifically binds to and activates a Notch receptor; and an Fc domain comprising a first subunit and a second subunit; wherein the Fab comprises: a first polypeptide comprising VH-CH1 and a second polypeptide comprising VL-CL, wherein the C-terminus of the CH1 or CL of the Fab is fused to the N-terminus of the first subunit of the Fc domain via a first linker, and wherein the second binding moiety is fused to the N-terminus of the second subunit of the Fc domain via a second linker, if appropriate. In some embodiments, the muscle-specific molecule is selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. In some embodiments, the first binding moiety comprises an anti-matrix glycan Fab, such as an anti-matrix glycan Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the first binding moiety comprises an anti-LAMA2 Fab, such as an anti-LAMA2 Fab comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the second binding moiety comprises an ECD of a nick ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the Fc domain comprises a knob-into-hole mutation, wherein: i) the first subunit of the Fc domain comprises a knob mutation and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, each subunit of the Fc domain further comprises L234A/L235A/H435A mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises the L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: i) a first unit that specifically binds to a first nick receptor and activates a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) of the first nick receptor; ii) a second unit that specifically binds to a second nick receptor and activates a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) of the second nick receptor; iii) a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to a muscle-specific molecule. v, Fab, sdAb or non-antibody portion); and iv) an Fc domain comprising a first unit and a second unit; wherein the first unit of the second binding moiety is fused to the N-terminus of the first unit of the Fc domain via a first linker, which is optionally present, and the second unit of the second binding moiety is fused to the N-terminus of the second unit of the Fc domain via a second linker, which is optionally present; and wherein the first binding moiety is fused to the C-terminus of the first unit of the Fc domain or the second unit of the Fc domain via a third linker, which is optionally present. In some embodiments, the bifunctional protein construct comprises: i) a first unit that specifically binds to a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) of a first muscle-specific molecule; and ii) a second unit that specifically binds to a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) of a second muscle-specific molecule; wherein the first unit of the first binding moiety is fused to the C-terminus of the first unit of the Fc domain via an optional third linker, and the second unit of the first binding moiety is fused to the C-terminus of the second unit of the Fc domain via an optional fourth linker. In some embodiments, the optional linker is a peptide linker, which can be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is independently selected from the group consisting of LAMA2, CDH15, α-DG, and basal glycans on α-DG. In some embodiments, the second binding moiety (one or two units) comprises the ECD of a nick ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the Fc domain comprises a knob-into-hole mutation, wherein: i) the first subunit of the Fc domain comprises a knob mutation and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, each subunit of the Fc domain further comprises L234A/L235A/H435A mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: one or more units of a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to one or more muscle-specific molecules; a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to a notch receptor and activates the notch receptor; and an Fc domain comprising a first unit and a second unit; wherein the second binding moiety is fused to the N-terminus of the first unit of the Fc domain via a linker, if applicable; and wherein each of the one or more units of the first binding moiety is independently fused via a linker, if applicable, to one of: i) the C-terminus of the first unit of the Fc domain; ii) the N-terminus of the second unit of the Fc domain; and iii) the C-terminus of the second unit of the Fc domain. In some embodiments, the optional linker is a peptide linker that can be the same or different within the bifunctional protein construct. In some embodiments, the one or more muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. In some embodiments, the second binding moiety comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the Fc domain comprises a knob-into-hole mutation, wherein: i) the first subunit of the Fc domain comprises a knob mutation and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, each subunit of the Fc domain further comprises L234A/L235A/H435A mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises the L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: one or more units of a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to one or more muscle-specific molecules; a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to a notch receptor and activates the notch receptor; and an Fc domain comprising a first unit and a second unit; wherein the second binding moiety is fused to the N-terminus of the second unit of the Fc domain via a linker, if applicable; and wherein each of the one or more units of the first binding moiety is independently fused via a linker, if applicable, to one of: i) the N-terminus of the first unit of the Fc domain; ii) the C-terminus of the first unit of the Fc domain; and iii) the C-terminus of the second unit of the Fc domain. In some embodiments, the optional linker is a peptide linker that can be the same or different within the bifunctional protein construct. In some embodiments, the one or more muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and matrix glycans on α-DG. In some embodiments, the second binding moiety comprises an ECD of a nicked ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety comprises the amino acid sequence of any one of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the Fc domain comprises a knob-into-hole mutation, wherein: i) the first subunit of the Fc domain comprises a knob mutation and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, each subunit of the Fc domain further comprises L234A/L235A/H435A mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises the L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the linker, if present, comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: i) a first unit that specifically binds to a first nick receptor and activates a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) of the first nick receptor; ii) a second unit that specifically binds to a second nick receptor and activates a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) of the second nick receptor; iii) a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to a muscle-specific molecule. v, Fab, sdAb or non-antibody portion); and iv) an Fc domain comprising a first unit and a second unit; wherein the first unit of the second binding moiety is fused to the C-terminus of the first unit of the Fc domain via a first linker, which is optionally present, and the second unit of the second binding moiety is fused to the C-terminus of the second unit of the Fc domain via a second linker, which is optionally present; and wherein the first binding moiety is fused to the N-terminus of the first unit of the Fc domain or the second unit of the Fc domain via a third linker, which is optionally present. In some embodiments, the bifunctional protein construct comprises: i) a first unit that specifically binds to a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) of a first muscle-specific molecule; and ii) a second unit that specifically binds to a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) of a second muscle-specific molecule; wherein the first unit of the first binding moiety is fused to the N-terminus of the first unit of the Fc domain via an optional third linker, and the second unit of the first binding moiety is fused to the N-terminus of the second unit of the Fc domain via an optional fourth linker. In some embodiments, the optional linker is a peptide linker, which can be the same or different within the bifunctional protein construct. In some embodiments, the muscle-specific molecule is independently selected from the group consisting of LAMA2, CDH15, α-DG, and basal glycans on α-DG. In some embodiments, the second binding moiety (one or two units) comprises the ECD of a nick ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety (one or two units) comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the Fc domain comprises a knob-into-hole mutation, wherein: i) the first subunit of the Fc domain comprises a knob mutation and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, each subunit of the Fc domain further comprises L234A/L235A/H435A mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: one or more units of a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to one or more muscle-specific molecules; a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to a notch receptor and activates the notch receptor; and an Fc domain comprising a first unit and a second unit; wherein the second binding moiety is fused to the C-terminus of the first unit of the Fc domain via an optional linker; and wherein each of the one or more units of the first binding moiety is independently fused via an optional linker to one of the following: i) the N-terminus of the first unit of the Fc domain; ii) the N-terminus of the second unit of the Fc domain; and iii) the C-terminus of the second unit of the Fc domain. In some embodiments, the one or more muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and basal glycans on α-DG. In some embodiments, the second binding moiety comprises the ECD of a nick ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the Fc domain comprises a knob-into-hole mutation, wherein: i) the first subunit of the Fc domain comprises a knob mutation and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, each subunit of the Fc domain further comprises L234A/L235A/H435A mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, a bifunctional protein construct is provided, comprising: one or more units of a first binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to one or more muscle-specific molecules; a second binding moiety (e.g., scFv, Fab, sdAb, or non-antibody moiety) that specifically binds to a notch receptor and activates the notch receptor; and an Fc domain comprising a first unit and a second unit; wherein the second binding moiety is fused to the C-terminus of the second unit of the Fc domain via a linker, if applicable; and wherein each of the one or more units of the first binding moiety is independently fused via a linker, if applicable, to one of: i) the N-terminus of the first unit of the Fc domain; ii) the C-terminus of the first unit of the Fc domain; and iii) the N-terminus of the second unit of the Fc domain. In some embodiments, the one or more muscle-specific molecules are independently selected from the group consisting of LAMA2, CDH15, α-DG, and basal glycans on α-DG. In some embodiments, the second binding moiety comprises the ECD of a nick ligand, or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), selected from the group consisting of delta-like 1 (DLL1), DLL3, DLL4, Jag1, and Jag2. In some embodiments, the second binding moiety comprises the amino acid sequence of any of SEQ ID NOs: 126-135 and 260-267. In some embodiments, the Fc domain comprises a knob-into-hole mutation, wherein: i) the first subunit of the Fc domain comprises a knob mutation and the second subunit of the Fc domain comprises a hole mutation; or ii) the second subunit of the Fc domain comprises a knob mutation and the first subunit of the Fc domain comprises a hole mutation. In some embodiments, the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). In some embodiments, each subunit of the Fc domain further comprises L234A/L235A/H435A mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and wherein each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, each subunit of the Fc domain comprises L234A/L235A/P329G "LALALPG" mutations (EU numbering). In some embodiments, the Fc domain is derived from human IgG1, and each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the optional linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338.

In some embodiments, the bifunctional protein constructs provided herein comprise i) one or more units of a second binding moiety, each independently comprising an ECD of a Notch ligand or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the Notch ligand is selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and ii) one or more units of a first binding moiety, each independently comprising an antibody portion that specifically binds to a muscle-specific molecule, optionally wherein the antibody portion is independently selected from the group consisting of an scFv, a Fab, and an sdAb. In some embodiments, i) two or more units of the second binding moiety are identical; and/or ii) two or more units of the first binding moiety are identical. In some embodiments, i) two or more units of the second binding moiety are different; and/or ii) at least one of the two or more units of the first binding moiety is different from the other units.

For Fc-containing bifunctional protein constructs comprising two or more different polypeptide chains (e.g., heterodimeric bifunctional protein constructs), Fc domains comprising knob-in-hole mutations and/or charge pair mutations can be used. In some embodiments, the Fc domain comprises charge pair mutations, wherein an amino acid residue in a first subunit of the Fc domain is substituted with a positively charged residue, and an amino acid residue in a second subunit of the Fc domain is substituted with a negatively charged residue. In some embodiments, the Fc domain comprises charge pair mutations, wherein an amino acid residue in a first subunit of the Fc domain is substituted with a negatively charged residue, and an amino acid residue in a second subunit of the Fc domain is substituted with a positively charged residue. In some embodiments, the Fc domain comprises a charged pair mutation, wherein the amino acid residue at D399 (EU numbering) in the first unimole of the Fc domain is substituted with a positively charged residue, and the amino acid residue at K409 (EU numbering) in the second unimole of the Fc domain is substituted with a negatively charged residue. In some embodiments, the Fc domain comprises a charged pair mutation, wherein the amino acid residue at K409 (EU numbering) in the first unimole of the Fc domain is substituted with a negatively charged residue, and the amino acid residue at D399 (EU numbering) in the second unimole of the Fc domain is substituted with a positively charged residue. In some embodiments, the bifunctional protein construct comprises a first subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 348 and a second subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 255. In some embodiments, the bifunctional protein construct comprises a first subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 255 and a second subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 348. In some embodiments, the bifunctional protein construct comprises a first subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 256 and a second subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 257. In some embodiments, the bifunctional protein construct comprises: a first subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 257 and a second subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 256. In some embodiments, the bifunctional protein construct comprises: a first subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 258 and a second subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 259. In some embodiments, the bifunctional protein construct comprises: a first subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 259 and a second subunit comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 258. A. First Binding Moiety Muscle-Specific Molecules That Specifically Bind to Muscle-Specific Molecules

The muscle-specific molecule enables preferential delivery and anchoring of the bifunctional protein construct to muscle tissue. The first binding moiety of the bifunctional protein construct described herein specifically binds to the muscle-specific molecule. In some embodiments, the muscle-specific molecule is expressed only in muscle cells or tissue. In some embodiments, the muscle-specific molecule is a molecule that is primarily expressed in muscle cells or tissue compared to cells or tissue of non-muscle organs. In some embodiments, the muscle-specific molecule is also expressed in cells or tissue of non-muscle organs. In some embodiments, the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or in the basal lamina. In some embodiments, the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlein, and M-calcium adhesion protein (CDH15).

In some embodiments, the target antigen is a component of the dystrophin-related glycoprotein complex (DGC). In some embodiments, the target antigen is selected from the group consisting of α-dystrophin (α-DG), β-DG, laminin, perlein, collagen (e.g., type IV collagen), α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, ε-sarcoglycan, ζ-sarcoglycan, disycan, myosin, and a matrix glycan on α-DG. In some embodiments, the target antigen is laminin subunit α-2 (LAMA2), ADG41, CDH15, α-DG, or a matrix glycan on α-DG.

The first binding moiety can comprise an antibody portion or a non-antibody portion that specifically binds to a muscle-specific molecule. In some embodiments, the antibody portion is selected from the group consisting of a full-length antibody, a Fab, a Fab', a F(ab') ₂ , a scFv, and an sdAb. A list of antibody portions that specifically bind to muscle-specific molecules is presented in Table 8. Table 8 : Antibody or non-antibody portions that bind to muscle-specific molecules Muscle-targeted antibodies muscle-specific protein Epitope or epitope region Laminin-211 antibody Laminin-211 α-2 chain α-2 chain Anti-laminin a-2 from Abnova, pure 2D4 Laminin-211 α-2 chain Amino acid sequence of human laminin-α-2 3013~3122 LAMA2 monoclonal antibody (M02), pure 1A7 Laminin-211 α-2 chain Amino acid sequence of human laminin-α-2 3013~3122 LAMA2 mAb, pure CL3450 Laminin-211 α-2 chain In the LG1 domain Laminin-α-2 mAb from U.S. Patent Publication No. US2018/0237511 Laminin-211 α-2 chain LG-4-5 domain α-Myocardial ischemia antibody α-Myocardial ischemia α-Myocardial ischemia Matrix Glycan Antibody IIH6 O-glycans on α-myosin O-glycans on α-myosin Matrix Glycan Antibody VIA-4 O-glycans on α-myosin O-glycans on α-myosin beta-Myocardial ischemia antibody beta-Myocardial ischemia extracellular domain β-Myocardial ischemia mAb in U.S. Patent Publication No. US2018/0237511 beta-Myocardial ischemia Extracellular domain of β-myotrophin α-sarcoglycan antibody α-sarcoglycan extracellular domain β-sarcoglycan antibodies β-sarcoglycan extracellular domain γ-sarcoglycan antibody γ-sarcoglycan extracellular domain δ-sarcoglycan antibody δ-sarcoglycan extracellular domain ε-sarcoglycan antibody ε-sarcoglycan extracellular domain ζ-sarcoglycan antibody ζ-sarcoglycan extracellular domain Myosin antibody Myosin extracellular domain Disaccharide chain proteoglycan antibody Disaccharide chain proteoglycan Disaccharide chain proteoglycan Targeted muscle replacement Functional domain Combined with matching items Laminin-111 α-1 chain LG domain 1-5 O-glycans on α-myosin Laminin-211 α-2 chain LG domain 1-5 O-glycans on α-myosin Laminin-411 α-4 chain LG domain 1-5 O-glycans on α-myosin Laminin-511 α-5 chain LG domain 1-5 O-glycans on α-myosin Pearlin LG domain O-glycans on α-myosin Aggregated proteins LG domain O-glycans on α-myosin Aggregated proteins N-terminal domain Laminin coiled-coil domain Nestin Laminin γ-ear binding domain Laminin γ Anti- LAMA2 antibody part

In some embodiments, the first binding moiety comprises an anti-LAMA2 antibody portion that specifically binds to LAMA2. Any of the anti-LAMA2 antibody portions described herein can be used in the bifunctional protein constructs described herein. The components described herein for binding to LAMA2 can be any of the anti-LAMA2 antibody portions described herein and their functional equivalents. This application also provides novel anti-LAMA2 antibody portions that can be used for purposes other than as components of the bifunctional protein constructs described herein.

In some embodiments, an anti-LAMA2 antibody construct is provided, which comprises any one of the anti-LAMA2 antibody portions described herein (or consists essentially of or consists of). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (such as bispecific). Multispecific antibodies have binding specificity for at least two different antigens or antigenic determinants. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is ChIP-grade, or at least IP-grade or Western blot-grade. The antibody can react with a protein of interest from any organism, such as humans, mice, rabbits, rats, monkeys, flies, zebrafish, chickens, worms, bacteria, etc. In some embodiments, the antibody is a chimeric antibody, a mouse antibody, a rabbit antibody, a rat antibody, a sheep antibody, a human antibody, a partially humanized antibody, a fully humanized antibody, a semisynthetic antibody, or a fully synthetic antibody.

Laminin is a glycoprotein found in the extracellular matrix of mammals. It is a heterotrimeric protein composed of α, β, and γ chains. It is a major component of the basal lamina and plays a role in cell differentiation, migration, and adhesion. LAMA2 is the α2 chain of laminin-2.

The anti-LAMA2 antibody moieties described herein can specifically bind to LAMA2 from any source, including but not limited to dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, LAMA2 is human LAMA2.

In some embodiments, the anti-LAMA2 antibody portion binds only to human LAMA2 and does not bind to LAMA2 from another species. In some embodiments, the anti-LAMA2 antibody portion has cross-species reactivity with LAMA2 other than human LAMA2, such as mouse or cynomolgus macaque LAMA2. Antibody portions with cross-species reactivity can facilitate the extrapolation of animal data to human clinical trials.

The anti-LAMA2 antibody portion can have any antibody format. In some embodiments, the anti-LAMA2 antibody portion comprises a VH and a VL.

In some embodiments, an anti-LAMA2 antibody portion is provided, comprising: i) a VH comprising H-CDR1, H-CDR2, and H-CDR3, each comprising a sequence having SEQ ID NO: 49, wherein the amino acid sequences of H-CDR1 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), H-CDR2 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), and H-CDR3 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)) within a reference VH having the amino acid sequence shown in 49; and ii) a VL comprising L-CDR1, L-CDR2, and L-CDR3, each comprising a sequence having SEQ ID NO: The amino acid sequences of L-CDR1 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), L-CDR2 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), and L-CDR3 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)) within the reference VL of the amino acid sequence shown in 50.

In some embodiments, an anti-LAMA2 antibody portion is provided, comprising a VH and a VL, wherein: (i) the VH comprises: an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (ii) the VL comprises: an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions). NO: 4, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) L-CDR1; an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions).

In some embodiments (independent of or in addition to the CDR sequences described above), an anti-LAMA2 antibody portion is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to SEQ ID NO: 49; and a VL comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to SEQ ID NO: 50. In some embodiments, the one or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in one or more CDRs. In some embodiments, the one or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in one or more framework regions (FRs). In some embodiments, the two or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in both CDRs and FRs.

In some embodiments, an anti-LAMA2 antibody portion is provided, comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 49, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the anti-LAMA2 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments, an anti-LAMA2 antibody portion is provided, comprising: i) a VH comprising H-CDR1, H-CDR2, and H-CDR3, each comprising a sequence having SEQ ID NOs: 54-64, wherein the amino acid sequences of H-CDR1 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), H-CDR2 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), and H-CDR3 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)) within a reference VH of the amino acid sequence shown in any one of NOs: 54-64; and ii) a VL comprising L-CDR1, L-CDR2, and L-CDR3, each comprising a sequence having SEQ ID NO: 54-64. The amino acid sequences of L-CDR1 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), L-CDR2 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), and L-CDR3 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)) within the reference VL of the amino acid sequence shown in any one of NOs: 65-73.

In some embodiments, an anti-LAMA2 antibody portion is provided, comprising a VH and a VL, wherein: (i) the VH comprises: an H-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 7, 13, 16, 19, 22, 25, 28, 31, and 34, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); an H-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 14, 17, 20, 23, 26, 29, 32, and 35, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and a VL comprising SEQ ID NOs: 9, 15, 18, 21, 24, 27, 30, 33 and 36, or a variant thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); and (ii) the VL comprises: an L-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 10, 37, 40, 43 and 46, or a variant thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); a CDR2 comprising SEQ ID NOs: 10, 37, 40, 43 and 46, or a variant thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); a CDR3 comprising SEQ ID NOs: 10, 37, 40, 43 and 46, or a variant thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); 11, 38, 41, 44 and 47, or a variant thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions) L-CDR2; and an L-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 12, 39, 42, 45 and 48, or a variant thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions).

In some embodiments (independent of or in addition to the CDR sequences described above), an anti-LAMA2 antibody portion is provided, comprising: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 54-64, or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to any one of SEQ ID NOs: 54-64; and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 65-73, or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to any one of SEQ ID NOs: 65-73. In some embodiments, the one or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in one or more CDRs. In some embodiments, the one or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in one or more framework regions (FRs). In some embodiments, the two or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in both CDRs and FRs.

In some embodiments, an anti-LAMA2 antibody portion is provided that comprises: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 7, 13, 16, 19, 22, 25, 28, 31, and 34; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 14, 17, 20, 23, 26, 29, 32, and 35; an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 9, 15, 18, 21, 24, 27, 30, 33, and 36; an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 10, 37, 40, 43, and 46; an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11, 38, 41, 44, and 47; and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 11, 38, 41, 44, and 47. 12, 39, 42, 45, and 48. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of any one of SEQ ID NOs: 54-64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of any one of SEQ ID NOs: 65-73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 54, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 55, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 56, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 57, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1 (SEQ ID NO: 7), HC-CDR2 (SEQ ID NO: 26), and HC-CDR3 (SEQ ID NO: 9) of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1 (SEQ ID NO: 10), LC-CDR2 (SEQ ID NO: 11), and LC-CDR3 (SEQ ID NO: 12) of a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 59, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 60, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 61, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 62, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 63, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 64, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-LAMA2 antibody portion comprises: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 54-64, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 65-73. In some embodiments, the anti-LAMA2 antibody portion comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the anti-LAMA2 antibody portion comprises a VH selected from any one of the exemplary VHs in Table 2 and a VL selected from any one of the exemplary VLs in Table 3. Table 2: Exemplary anti-LAMA2 VHs Notes SEQ ID NO anti-lama2 LG21 huVHpar 54 anti-lama2 LG21 huVH1 55 anti-lama2 LG21 huVH2 56 anti-lama2 LG21 huVH3 57 anti-lama2 LG21 huVH4 58 anti-lama2 LG21 huVH5 59 anti-lama2 LG21 huVH6 60 anti-lama2 LG21 huVH7 61 anti-lama2 LG21 huVH8 62 anti-lama2 LG21 huVH9 63 anti-lama2 LG21 huVH10 64 Table 3: Exemplary anti-LAMA2 VLs Notes SEQ ID NO anti-lama2 LG21 huVLpar 65 anti-lama2 LG21 huVL1 66 anti-lama2 LG21 huVL2 67 anti-lama2 LG21 huVL3 68 anti-lama2 LG21 huVL4 69 anti-lama2 LG21 huVL5 70 anti-lama2 LG21 huVL6 71 anti-lama2 LG21 huVL7 72 anti-lama2 LG21 huVL8 73

In some embodiments, the anti-LAMA2 antibody portion comprises a VH paired with a VL as shown in Table 4. In some embodiments, the anti-LAMA2 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 65. Table 4: Exemplary VH/VL combinations for anti-LAMA2 antibody portions Pairing VH VL 1 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 2 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 3 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 4 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 5 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 6 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 7 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 8 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 9 Anti-lama2 LG21 huVHpar (SEQ ID NO: 54) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 10 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 11 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 12 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 13 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 14 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 15 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 16 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 17 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 18 Anti-lama2 LG21 huVH1 (SEQ ID NO: 55) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 19 Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 20 Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) twenty one Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) twenty two Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) twenty three Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) twenty four Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 25 Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 26 Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 27 Anti-lama2 LG21 huVH2 (SEQ ID NO: 56) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 28 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 29 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 30 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 31 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 32 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 33 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 34 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 35 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 36 Anti-lama2 LG21 huVH3 (SEQ ID NO: 57) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 37 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 38 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 39 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 40 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 41 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 42 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 43 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 44 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 45 Anti-lama2 LG21 huVH4 (SEQ ID NO: 58) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 46 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 47 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 48 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 49 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 50 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 51 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 52 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 53 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 54 Anti-lama2 LG21 huVH5 (SEQ ID NO: 59) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 55 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 56 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 57 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 58 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 59 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 60 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 61 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 62 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 63 Anti-lama2 LG21 huVH6 (SEQ ID NO: 60) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 64 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 65 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 66 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 67 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 68 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 69 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 70 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 71 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 72 Anti-lama2 LG21 huVH7 (SEQ ID NO: 61) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 73 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 74 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 75 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 76 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 77 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 78 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 79 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 80 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 81 Anti-lama2 LG21 huVH8 (SEQ ID NO: 62) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 82 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 83 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 84 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 85 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 86 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 87 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 88 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 89 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 90 Anti-lama2 LG21 huVH9 (SEQ ID NO: 63) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73) 91 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVLpar (SEQ ID NO: 65) 92 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVL1 (SEQ ID NO: 66) 93 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVL2 (SEQ ID NO: 67) 94 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVL3 (SEQ ID NO: 68) 95 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVL4 (SEQ ID NO: 69) 96 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVL5 (SEQ ID NO: 70) 97 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVL6 (SEQ ID NO: 71) 98 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVL7 (SEQ ID NO: 72) 99 Anti-lama2 LG21 huVH10 (SEQ ID NO: 64) Anti-lama2 LG21 huVL8 (SEQ ID NO: 73)

In some embodiments, the anti-LAMA2 antibody portion is an anti-LAMA2 scFv. In some embodiments, the anti-LAMA2 scFv comprises, from N' to C', VH-, optionally, a linker-VL. In some embodiments, the anti-LAMA2 scFv comprises, from N' to C', VL-, optionally, a linker-VH. Any suitable linker (e.g., see "Linker" subsection below) can be used herein, including but not limited to SEQ ID NO: 212. In some embodiments, the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-LAMA2 scFv comprises the amino acid sequence of SEQ ID NO: 145 or 268.

In some embodiments, the anti-LAMA2 antibody portion is an anti-LAMA2 Fab. In some embodiments, the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 52 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 53. In some embodiments, the anti-LAMA2 Fab comprises: a first polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 55-64 and a second polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 65-73. Anti-matrix polysaccharide antibody portion

In some embodiments, the first binding moiety comprises an anti-matrix glycan antibody portion that specifically binds to a matrix glycan on α-dystroglycan (α-DG). Any of the anti-α-DG antibody portions described herein can be used in the bifunctional protein constructs described herein. The components described herein for binding to matrix glycans can be any of the anti-matrix glycan antibody portions described herein and their functional equivalents. This application also provides novel anti-matrix glycan antibody portions that can be used for purposes other than as components of the bifunctional protein constructs described herein.

In some embodiments, an anti-matrix glycan antibody construct is provided that comprises (or consists essentially of, or consists of) any of the anti-matrix glycan antibody moieties described herein. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). Multispecific antibodies have binding specificity for at least two different antigens or antigenic determinants. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is ChIP-grade, or at least IP-grade or Western blot-grade. The antibody can react with a protein of interest from any organism, such as human, mouse, rabbit, rat, monkey, fruit fly, zebrafish, chicken, worm, bacteria, etc. In some embodiments, the antibody is a chimeric antibody, a mouse antibody, a rabbit antibody, a rat antibody, a goat antibody, a human antibody, a partially humanized antibody, a fully humanized antibody, a semisynthetic antibody, or a fully synthetic antibody.

α-Dystrophin (α-DG) is a laminin-binding protein and a component of the dystrophin-related glycoprotein complex (DGC). It is uniquely modified with alternating xylose and glucuronic acid subunits at the O-mannose site, termed stroma. Strooma is a receptor for proteins containing the laminin-G domain. Strooma connects α-DG to the basement membrane.

The anti-matrix glycan antibody moieties described herein can specifically bind to matrix glycans derived from any source, including but not limited to canine, feline, porcine, bovine, ovine, goat, horse, rat, rabbit, hamster, guinea pig, monkey, mouse, and human. In some embodiments, the matrix glycans are human matrix glycans.

In some embodiments, the anti-matrix glycan antibody portion binds only to human matrix glycans and does not bind to matrix glycans from another species. In some embodiments, the anti-matrix glycan antibody portion has cross-species reactivity with matrix glycans other than human matrix glycans, such as mouse or cynomolgus macaque macaque glycans. Antibody portions with cross-species reactivity can facilitate the extrapolation of animal data to human clinical trials.

The anti-matrix glycan antibody portion can have any antibody format. In some embodiments, the anti-matrix glycan antibody portion comprises VH and VL.

In some embodiments, the anti-matrix antibody portion comprises: i) a VH comprising H-CDR1, H-CDR2, and H-CDR3, each comprising a sequence having SEQ ID NOs: 89-93; and ii) an amino acid sequence of H-CDR1 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), H-CDR2 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), and H-CDR3 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)) within a reference VH having an amino acid sequence as shown in any one of NOs: 89-93; and ii) a VL comprising L-CDR1, L-CDR2, and L-CDR3, each comprising a sequence having SEQ ID NO: The amino acid sequences of L-CDR1 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), L-CDR2 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), and L-CDR3 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)) within the reference VL of the amino acid sequence shown in any one of NOs: 94-98.

In some embodiments, the anti-matrix antibody portion comprises a VH and a VL, wherein: (i) the VH comprises: an H-CDR1 comprising an amino acid sequence of SEQ ID NO: 74 or 80, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); an H-CDR2 comprising an amino acid sequence of SEQ ID NO: 75 or 81, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and an H-CDR3 comprising an amino acid sequence of SEQ ID NO: 76 or 82, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (ii) the VL comprises: an H-CDR1 comprising an amino acid sequence of SEQ ID NO: 76 or 82, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); NOs: 79, 83 and 86, or variants thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions) L-CDR1; L-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 78, 84 and 87, or variants thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions); and L-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 79, 85 and 88, or variants thereof comprising up to 3 (e.g., 3, 2 or 1) amino acid changes (e.g., insertions, deletions and/or substitutions, such as conservative substitutions).

In some embodiments (independent of or in addition to the CDR sequences described above), the antimatriglycoside portion comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93, or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence identity to any one of SEQ ID NOs: 89-93; and a VL comprising the amino acid sequence of SEQ ID NOs: 94-98, or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence identity to any one of SEQ ID NOs: 94-98. In some embodiments, the one or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in one or more CDRs. In some embodiments, the one or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in one or more framework regions (FRs). In some embodiments, the two or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in both CDRs and FRs.

In some embodiments, the anti-matrix glycan antibody portion comprises: a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74 or 80; a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75 or 81; a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76 or 82; a LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 79, 83, and 86; a LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 78, 84, and 87; and a LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 79, 85, and 88. In some embodiments, the anti-matrix antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94-98. In some embodiments, the anti-matrix antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 89, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 91, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 92, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, an anti-matrix antibody portion is provided, comprising: HC-CDR1, HC-CDR2, and HC-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 93, and LC-CDR1, LC-CDR2, and LC-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, an anti-matrix antibody portion is provided, comprising: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 89-93 and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 94-98. In some embodiments, the anti-matrix antibody portion comprises: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, an anti-matrix glycan antibody portion is provided, comprising: a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 83, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the anti-matrix antibody portion comprises a VH selected from any one of the exemplary VHs in Table 5 and a VL selected from any one of the exemplary VLs in Table 6. Table 5: Exemplary anti-ADG41 VHs Notes SEQ ID NO anti-ADG41 huVH1 90 anti-ADG41 huVH2 91 anti-ADG41 huVH3 92 anti-ADG41 huVH4 93 Table 6: Exemplary anti-ADG4 VL Notes SEQ ID NO anti-ADG41 huVL1 95 anti-ADG41 huVL2 96 anti-ADG41 huVL3 97 anti-ADG41 huVL4 98

In some embodiments, an anti-matrix antibody portion is provided that comprises a VH paired with a VL as shown in Table 7. In some embodiments, an anti-matrix antibody portion is provided that comprises: a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 95. Table 7: Exemplary VH/VL combinations for anti-ADG41 antibody portions Pairing VH VL 1 Anti-ADG41 huVH1 (SEQ ID NO: 90) Anti-ADG41 huVL1 (SEQ ID NO: 95) 2 Anti-ADG41 huVH1 (SEQ ID NO: 90) Anti-ADG41 huVL2 (SEQ ID NO: 96) 3 Anti-ADG41 huVH1 (SEQ ID NO: 90) Anti-ADG41 huVL3 (SEQ ID NO: 97) 4 Anti-ADG41 huVH1 (SEQ ID NO: 90) Anti-ADG41 huVL4 (SEQ ID NO: 98) 5 Anti-ADG41 huVH2 (SEQ ID NO: 91) Anti-ADG41 huVL1 (SEQ ID NO: 95) 6 Anti-ADG41 huVH2 (SEQ ID NO: 91) Anti-ADG41 huVL2 (SEQ ID NO: 96) 7 Anti-ADG41 huVH2 (SEQ ID NO: 91) Anti-ADG41 huVL3 (SEQ ID NO: 97) 8 Anti-ADG41 huVH2 (SEQ ID NO: 91) Anti-ADG41 huVL4 (SEQ ID NO: 98) 9 Anti-ADG41 huVH3 (SEQ ID NO: 92) Anti-ADG41 huVL1 (SEQ ID NO: 95) 10 Anti-ADG41 huVH3 (SEQ ID NO: 92) Anti-ADG41 huVL2 (SEQ ID NO: 96) 11 Anti-ADG41 huVH3 (SEQ ID NO: 92) Anti-ADG41 huVL3 (SEQ ID NO: 97) 12 Anti-ADG41 huVH3 (SEQ ID NO: 92) Anti-ADG41 huVL4 (SEQ ID NO: 98) 13 Anti-ADG41 huVH4 (SEQ ID NO: 93) Anti-ADG41 huVL1 (SEQ ID NO: 95) 14 Anti-ADG41 huVH4 (SEQ ID NO: 93) Anti-ADG41 huVL2 (SEQ ID NO: 96) 15 Anti-ADG41 huVH4 (SEQ ID NO: 93) Anti-ADG41 huVL3 (SEQ ID NO: 97) 16 Anti-ADG41 huVH4 (SEQ ID NO: 93) Anti-ADG41 huVL4 (SEQ ID NO: 98)

In some embodiments, the anti-matrix glycan antibody portion is an anti-matrix glycan scFv. In some embodiments, the anti-matrix glycan scFv comprises, from N' to C', VH - optionally a linker - VL. In some embodiments, the anti-matrix glycan scFv comprises, from N' to C', VL - optionally a linker - VH. Any suitable linker (e.g., see the "Linker" section below) can be used herein, including but not limited to SEQ ID NO: 212. In some embodiments, the anti-matrix glycan antibody portion comprises the amino acid sequence of SEQ ID NO: 99.

In some embodiments, the anti-matrix glycosylated antibody portion is an anti-matrix glycosylated Fab. In some embodiments, the anti-matrix glycosylated Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 100 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 101. Anti- CDH15 Antibody Portion

In some embodiments, the first binding moiety comprises an anti-CDH15 antibody portion that specifically binds to CDH15. Any of the anti-CDH15 antibody portions described herein can be used in the bifunctional protein constructs described herein. The components described herein for binding to CDH15 can be any of the anti-CDH15 antibody portions described herein and their functional equivalents. This application also provides novel anti-CDH15 antibody portions that can be used for purposes other than as components of the bifunctional protein constructs described herein.

Calcadhesin-15 (CDH15) is a member of the calpain superfamily. Calcadhesins consist of an extracellular domain containing five calpain domains, a transmembrane region, and a conserved cytoplasmic domain. CDH15 is essential for myogenesis and may serve as a trigger for terminal muscle cell differentiation.

The anti-CDH15 antibody portions described herein can specifically bind to CDH15 from any source, including but not limited to canine, feline, porcine, bovine, ovine, goat, horse, rat, rabbit, hamster, guinea pig, monkey, mouse, and human. In some embodiments, CDH15 is human CDH15.

In some embodiments, the anti-CDH15 antibody portion binds only to human CDH15 and does not bind to CDH15 from another species. In some embodiments, the anti-CDH15 antibody portion has cross-species reactivity with CDH15 other than human CDH15, such as mouse or cynomolgus macaque CDH15. Antibody portions with cross-species reactivity can facilitate the extrapolation of animal data to human clinical trials.

The anti-CDH15 antibody portion can have any antibody format. In some embodiments, the anti-CDH15 antibody portion comprises a VH and a VL.

In some embodiments, the anti-CDH15 antibody portion comprises: i) a VH comprising H-CDR1, H-CDR2, and H-CDR3, each comprising a sequence having SEQ ID NO: 108; and ii) the amino acid sequences of H-CDR1 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), H-CDR2 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), and H-CDR3 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)) within a reference VH of the amino acid sequence shown in SEQ ID NO: 108; and ii) a VL comprising L-CDR1, L-CDR2, and L-CDR3, each comprising a sequence having SEQ ID NO: The amino acid sequences of L-CDR1 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), L-CDR2 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)), and L-CDR3 (or variants thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions)) within the reference VL of the amino acid sequence shown in 109.

In some embodiments, the anti-CDH15 antibody portion comprises a VH and a VL, wherein: (i) the VH comprises: an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 102, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 103, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and (ii) the VL comprises: an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions). An L-CDR1 comprising the amino acid sequence of SEQ ID NO: 105, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 106, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions); and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 107, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions).

In some embodiments (independent of or in addition to the CDR sequences described above), the anti-CDH15 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 108, or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence identity to SEQ ID NO: 108; and a VL comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence identity to SEQ ID NO: 109. In some embodiments, the one or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in one or more CDRs. In some embodiments, the one or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in one or more framework regions (FRs). In some embodiments, the two or more amino acid changes (e.g., insertions, deletions, and/or substitutions, such as conservative substitutions) are in CDRs and FRs.

In some embodiments, the anti-CDH15 antibody portion comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 102, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 103, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 105, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 107. In some embodiments, the anti-CDH15 antibody portion comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of VH comprising the amino acid sequence of SEQ ID NO: 108, and LC-CDR1, LC-CDR2, and LC-CDR3 of VL comprising the amino acid sequence of SEQ ID NO: 109. In some embodiments, the anti-CDH15 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 108 and a VL comprising the amino acid sequence of SEQ ID NO: 109.

In some embodiments, the anti-CDH15 antibody portion is an anti-CDH15 scFv. In some embodiments, the anti-CDH15 scFv comprises, from N' to C', VH - optionally a linker - VL. In some embodiments, the anti-CDH15 scFv comprises, from N' to C', VL - optionally a linker - VH. Any suitable linker (e.g., see the "Linker" section below) can be used herein, including but not limited to SEQ ID NO: 212. In some embodiments, the anti-CDH15 scFv comprises the amino acid sequence of SEQ ID NO: 110.

In some embodiments, the anti-CDH15 antibody portion is an anti-CDH15 Fab. In some embodiments, the anti-CDH15 Fab comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 111 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 112. Non-antibody portion

In some embodiments, the first binding moiety is a non-antibody binding moiety that specifically binds to a muscle-specific molecule, such as a protein domain of an extracellular matrix (ECM) protein that can bind to matrix glycans (e.g., a laminin G-like domain (LG domain)). The components described herein for binding to muscle tissue-specific molecules can be any of the non-antibody binding moieties described herein and their functional equivalents. A list of non-antibody moieties that specifically bind to muscle-specific molecules is presented in Table 8 .

In some embodiments, the non-antibody portion comprises a protein domain selected from the group consisting of the LG domain of laminin, the LG domain of agrin, the LG domain of nidogen, the LG domain of perlein, the laminin coiled-coil binding domain of agrin, and the laminin gamma binding domain of nidogen.

In some embodiments, the non-antibody portion provided herein comprises the LG domain of laminin. In some embodiments, the non-antibody portion comprises the LG domain of any of laminin subunit alpha-1 (LAMA1), LAMA2, laminin subunit alpha-3 (LAMA3), laminin subunit alpha-4 (LAMA4), or laminin subunit alpha-5 (LAMA5). In some embodiments, the non-antibody portion comprises the amino acid sequence of any of SEQ ID NOs: 118-124. In some embodiments, the non-antibody portion is a variant comprising a sequence having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to any of SEQ ID NOs: 118-124. In some embodiments, the non-antibody portion comprises the LG domain of LAMA2. In some embodiments, the non-antibody portion comprises the LG4-5 domain of LAMA2 (LAMA2 LG4-5). In some embodiments, the LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 118 or 119.

In some embodiments, the non-antibody portion comprises the LG domain of perlein. In some embodiments, the non-antibody portion comprises the amino acid sequence of SEQ ID NO: 120.

In some embodiments, the non-antibody portion comprises the LG domain of aggregin. In some embodiments, the non-antibody portion comprises the amino acid sequence of SEQ ID NO: 121.

In some embodiments, the non-antibody portion comprises the laminin coiled-coil binding domain of agrin. In some embodiments, the laminin coiled-coil binding domain of agrin comprises the amino acid sequence of SEQ ID NO: 122. In some embodiments, the non-antibody portion comprises the laminin α, β, and/or γ binding domains of agrin. In some embodiments, the non-antibody portion comprises amino acids 1-271 of human agrin.

In some embodiments, the non-antibody portion comprises the laminin gamma binding domain of nidogen. In some embodiments, the laminin gamma binding domain of nidogen comprises the amino acid sequence of SEQ ID NO: 123 or 124. In some embodiments, the non-antibody portion comprises the C-terminal domain of human nidogen-1. In some embodiments, the C-terminal domain of human nidogen-1 comprises amino acids 390-1247 of human nidogen-1. In some embodiments, the non-antibody portion comprises the C-terminal domain of human nidogen-2. In some embodiments, the C-terminal domain of human nidogen-2 comprises amino acids 515-1372 of human nidogen-2 . B. A second binding portion that specifically binds to and activates the nick receptor

The Notch signaling pathway plays a critical role in cell fate determination and differentiation in many tissues during embryonic and postnatal development. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscle. A pathological hallmark of Duchenne muscular dystrophy (DMD) is the progressive depletion of satellite cells, leading to a failure of muscle repair. Satellite cells from mdx mice, a DMD mouse model, have been found to have reduced Notch signaling activation, which has been shown to be required for maintaining satellite cell quiescence and self-renewal (see, e.g., Bjornson et al., Stem Cells . 2012, 30(2):232-42; Mourikis et al., Stem Cells . 2012, 30(2):243-52). More importantly and therapeutically relevant, they showed in mouse models that constitutive activation of Notch is sufficient to rescue the self-renewal deficiency of mdx satellite cells, suggesting that enhanced Notch signaling in muscle could be a therapeutic strategy for treating muscular dystrophy (see, e.g., Bjornson et al., Stem Cells . 2012, 30(2):232-42; Mourikis et al., Stem Cells . 2012, 30(2):243-52). Nevertheless, constitutive activation of Notch signaling in transgenic mice harboring the Notch intracellular C-terminal domain (NICD) inhibits myoblast differentiation and muscle repair (see, e.g., Wen et al., Mol Cell Biol . 2012, 32(12):2300-11). Therefore, in order to manipulate notch signaling to enhance satellite cell renewal while maintaining myoblast differentiation for muscle regeneration and repair as a therapeutic approach for muscular dystrophy, spatiotemporal control is required at the right place at the right time.

Four mammalian Notch receptors have been identified (Notch 1, Notch 2, Notch 3, and Notch 4). Notch receptors are activated by single-pass transmembrane ligands of the Delta, Serrated, Lag-2 (DSL) family, which includes Delta-like 1 (DLL1; e.g., GenBank Accession No. NP005609), Delta-like 3 (DLL3, e.g., GenBank Accession No. NP_982353.1 or NP_058637.1), Delta-like 4 (DLL4; e.g., GenBank Accession No. NP_061947.1), Jagged-1 (Jag1; e.g., GenBank Accession No. AAC51731), and Jagged-2 (Jag2; e.g., GenBank Accession No. AAD15562). These ligands are characterized by an N-terminal Notch ligand (MNNL) region, a Delta/Serrate/LAG-2 (DSL) domain, and tandem epidermal growth factor (EGF)-like repeats within the extracellular domain. Interaction between the Notch receptor and the ligand's extracellular domain triggers cleavage of the Notch by furin-like convertases, ADAM (a disintegrin and metalloprotease) metalloproteinases, and/or gamma-secretase. γ-Secretase-mediated cleavage generates and releases the Notch intracellular domain (NICD), which translocates to the nucleus, where it binds to the transcription factor CSL (CBF1, Suppressor of Hairless, Lag-1) to induce expression of downstream target genes. See, for example, Chillakuri et al., Semin Cell Dev Biol ., 2012, 23(4):421-8; Sekine et al., Arthritis Res Ther ., 2012, 14(2):R45.

DLL1, DLL3, and DLL4 are members of the delta family of Notch ligands. DLL1 is a type I cell surface protein composed of an MNNL domain, a DSL domain, and eight EGF-like repeats within the extracellular domain. The first two EGF-like domains of DLL1 are unusual in that they contain very short loop sequences that resemble motifs found in the OSM-11 protein of the nematode C. elegans . In addition, DLL1 contains a PDZ-ligand domain on the cytoplasmic side of the ligand. In human DLL1, the MNNL domain is amino acids 18-176, the DSL domain is amino acids 177-221, EGF-like repeat 1 is amino acids 226-254, EGF-like repeat 2 is amino acids 257-285, EGF-like repeat 3 is amino acids 292-325, EGF-like repeat 4 is amino acids 332-363, EGF-like repeat 5 is amino acids 370-402, EGF-like repeat 6 is amino acids 409-440, EGF-like repeat 7 is amino acids 447-478, and EGF-like repeat 8 is amino acids 485-516. See, e.g., Hirano et al., eLife , 2020, 9:e50979; and Chillakuri et al., Semin Cell Dev Biol ., 2012, 23(4):421-8.

DLL3 is the shortest of the mammalian delta-like ligands and contains an MNNL domain, a DSL domain, and six EGF-like repeats within the extracellular domain. In human DLL3, the MNNL domain is amino acids 27-175, the DSL domain is amino acids 176-215, EGF-like repeat 1 is amino acids 216-249, EGF-like repeat 2 is amino acids 274-310, EGF-like repeat 3 is amino acids 312-351, EGF-like repeat 4 is amino acids 353-389, EGF-like repeat 5 is amino acids 391-427, and EGF-like repeat 6 is amino acids 429-465. See, e.g., Ladi et al., J Cell Biol ., 2005, 170(6): 983-992.

DLL4 is a type I cell surface protein composed of an MNNL domain, a DSL domain, and eight EGF-like repeats within the extracellular domain. DLL4 contains a PDZ-ligand domain on the cytoplasmic side of the ligand. In human DLL4, the MNNL domain is amino acids 27-172, the DSL domain is amino acids 173-217, EGF-like repeat 1 is amino acids 218-251, EGF-like repeat 2 is amino acids 252-282, EGF-like repeat 3 is amino acids 284-322, EGF-like repeat 4 is amino acids 324-360, EGF-like repeat 5 is amino acids 362-400, EGF-like repeat 6 is amino acids 402-438, EGF-like repeat 7 is amino acids 440-476, and EGF-like repeat 8 is amino acids 480-518. See, for example, Hirano et al., eLife , 2020, 9:e50979 and Chillakuri et al., Semin Cell Dev Biol ., 2012, 23(4):421-8.

Jag1 and Jag2 are members of the Serrate family of Notch ligands. The Serrate family differs from the Delta family in that they contain a cysteine-rich domain (CRD) following the EGF-like repeats. Jag1 and Jag2 are composed of an MNNL domain, a DSL domain, 16 EGF-like repeats, and a CRD within the extracellular domain. The first two EGF-like domains in Jag1 and Jag2 are unusual, as they contain very short loop sequences that resemble motifs found in the OSM-11 protein from the nematode C. elegans. Additionally, Jag1 contains a PDZ-ligand domain on the cytoplasmic side of the ligand. See, for example, Chillakuri et al., Semin Cell Dev Biol ., 2012, 23(4):421-8 and Kume, J Angiogenes Res. , 2009, 1:8.

In human Jag1, the MNNL domain is amino acids 34-184, the DSL domain is amino acids 185-229, the EGF-like repeat 1 is amino acids 230-263, the EGF-like repeat 2 is amino acids 264-294, the EGF-like repeat 3 is amino acids 296-334, the EGF-like repeat 4 is amino acids 336-372, the EGF-like repeat 5 is amino acids 374-410, the EGF-like repeat 6 is amino acids 412-448, and the EGF-like repeat 7 is amino acids 4 The EGF-like repeat sequence is composed of amino acids 50-485, EGF-like repeat sequence 8 is composed of amino acids 487-523, EGF-like repeat sequence 9 is composed of amino acids 525-561, EGF-like repeat sequence 10 is composed of amino acids 586-627, EGF-like repeat sequence 11 is composed of amino acids 629-665, EGF-like repeat sequence 12 is composed of amino acids 667-703, EGF-like repeat sequence 13 is composed of amino acids 705-741, EGF-like repeat sequence 14 is composed of amino acids 744-780, and EGF-like repeat sequence 15 is composed of amino acids 782-818. EGF-like repeat sequence 16 is composed of amino acids 820-856, and the CRD domain is composed of amino acids 857-1067.

For human Jag2, the MNNL domain is amino acids 27-195, the DSL domain is amino acids 196-240, the EGF-like repeat 1 is amino acids 241-274, the EGF-like repeat 2 is amino acids 275-305, the EGF-like repeat 3 is amino acids 307-345, the EGF-like repeat 4 is amino acids 347-383, the EGF-like repeat 5 is amino acids 385-421, the EGF-like repeat 6 is amino acids 423-459, and the EGF-like repeat 7 is amino acids 461- 496, EGF-like repeat sequence 8 is amino acids 498-534, EGF-like repeat sequence 9 is amino acids 536-572, EGF-like repeat sequence 10 is amino acids 574-634, EGF-like repeat sequence 11 is amino acids 636-672, EGF-like repeat sequence 12 is amino acids 674-710, EGF-like repeat sequence 13 is amino acids 712-748, EGF-like repeat sequence 14 is amino acids 751-787, and EGF-like repeat sequence 15 is amino acids 789-825. EGF-like repeat sequence 16 is amino acids 827-863, and the CRD domain is amino acids 864-1080. Non-antibody portion

In some embodiments, the second binding moiety of any of the bifunctional protein constructs provided herein comprises an ECD of a nick ligand or a variant thereof, such as any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267), wherein the nick ligand is selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2. The means described herein for binding to a nick receptor can be any of the nick ligand ECDs or variants described herein, and functional equivalents thereof. In some embodiments, the nick ligand ECD or variant thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 EGF-like domains. In some embodiments, the DLL1 ECD comprises an EGF1-8 domain. In some embodiments, the DLL3 ECD comprises an EGF1-6 domain. In some embodiments, the DLL4 ECD comprises an EGF1-8 domain. In some embodiments, the Jag1 ECD comprises an EGF1-16 domain. In some embodiments, the Jag2 ECD comprises an EGF1-16 domain. In some embodiments, the ECD of a nicked ligand, or a variant thereof, comprises a C2 (MNNL) domain. In some embodiments, the ECD of a nicked ligand, or a variant thereof, comprises a DSL domain. In some embodiments, the ECD of a nicked ligand, or a variant thereof, comprises an EGF1-3 domain, an EGF1-4 domain, an EGF1-4 domain, an EGF1-5 domain, or an EGF-1-6 domain. In some embodiments, the ECD of a nicked ligand, or a variant thereof, comprises an EGF1-5 domain. In some embodiments, the ECD of the Notch ligand or a variant thereof comprises a C2(MNNL)-DSL-EGF1-3, C2(MNNL)-DSL-EGF1-4, C2(MNNL)-DSL-EGF1-5, or C2(MNNL)-DSL-EGF1-6 domain.

In some embodiments, the non-antibody moiety binds to a human notch receptor described herein and can be any of the notch ligand ECDs or variants described herein, and functional equivalents thereof. In some embodiments, the non-antibody moiety has cross-species reactivity with a notch receptor described herein and can be any of the notch ligand ECDs or variants described herein, and functional equivalents thereof, such as mouse, rat, or cynomolgus macaque non-antibody moieties. Antibody moieties with cross-species reactivity can facilitate the extrapolation of animal data to human clinical trials.

This application provides engineered DLL4 ECDs that can be incorporated into the bifunctional proteins described herein. In some embodiments, an engineered DLL4 ECD is provided, wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of T52N and T135N, and wherein the amino acid positions are relative to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, the engineered DLL4 ECD further comprises a mutation at one or more amino acid positions selected from the group consisting of G2, E14, R66, P80, F81, H168, Q220, N231, and N260. In some embodiments, the additional mutations are selected from the group consisting of: G2S, E14H, R66S, R66T, P80L, F81L, H168Y, Q220H, N231D, and N260D. In some embodiments, the engineered DLL4 ECD comprises mutations selected from the group consisting of: (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (v) G2S, T52N, F81L, T135N, and H168Y; In some embodiments, the engineered DLL4 ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 261-264.

In some embodiments, an engineered DLL4 ECD is provided, wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of: G2, E14, T52, R66, P80, F81, T135, H168, Q220, N231, and N260, and wherein the amino acid position is relative to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, the mutation is selected from the group consisting of: G2S, E14H, T52N, R66S, R66T, P80L, F81L, T135N, H168Y, Q220H, N231D, and N260D, and wherein the amino acid position is relative to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, the engineered DLL4 ECD comprises a mutation selected from the group consisting of: (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (v) G2S, T52N, F81L, T135N, and H168Y; (vi) G2S, T52N, F81L, R66S, T135N, and H168Y; (vii) G2S, E14H, T52N, F81L, R66T, P80L, T135N, H168Y and N231D; and (viii) G2S, T52N, R66T, P80L, F81L, T135N, H168Y, Q220H and N260D.

In some embodiments, the second binding moiety comprises a DLL4 ECD or a variant thereof. In some embodiments, the second binding moiety comprises a DLL4 ECD, and optionally wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the second binding moiety comprises a variant DLL4 ECD, and optionally wherein the variant DLL4 ECD comprises the MNNL domain, DSL domain, and/or EGF1-5 domain of DLL4. In some embodiments, the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267, or a sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 126-129 and 260-267. In some embodiments, the variant DLL4 ECD comprises no more than any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions relative to SEQ ID NOs: 126-129 and 260-267.

In some embodiments, the present invention also provides a protein construct comprising any of the engineered DLL4 ECDs described herein (e.g., any of SEQ ID NOs: 126-129 and 260-267). In some embodiments, the protein construct is a bifunctional protein construct, such as any of the bifunctional protein constructs comprising an engineered DLL4 ECD described herein. In some embodiments, the protein construct further comprises a binding moiety that specifically binds to a muscle-specific molecule. In some embodiments, the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or in the basal lamina. In some embodiments, the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlein, and M-calcium adhesion protein (CDH15).

In some embodiments, the second binding moiety comprises a DLL1 ECD or a variant thereof. In some embodiments, the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD of DLL1 comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the second binding moiety comprises a variant DLL1 ECD, and optionally wherein the variant DLL1 ECD comprises a sequence having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to SEQ ID NO: 130. In some embodiments, the variant DLL1 ECD comprises no more than any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions relative to SEQ ID NO: 130.

In some embodiments, the second binding moiety comprises a DLL3 ECD or a variant thereof. In some embodiments, the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the second binding moiety comprises a variant DLL3 ECD, and optionally wherein the variant DLL3 ECD comprises a sequence having at least about 90% sequence identity (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to SEQ ID NO: 131. In some embodiments, the variant DLL3 ECD comprises no more than any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions relative to SEQ ID NO: 131.

In some embodiments, the second binding moiety comprises Jag1 ECD or a variant thereof. In some embodiments, the second binding moiety comprises Jag1 ECD, and wherein the Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, the second binding moiety comprises a variant Jag1 ECD, and wherein the variant Jag1 ECD comprises the MNNL domain, DSL domain, and EGF1-6 domain of Jag1, or a sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the sequence of the MNNL domain, DSL domain, and EGF1-6 domain of Jag1. In some embodiments, the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134.

In some embodiments, the second binding moiety comprises Jag2 ECD or a variant thereof. In some embodiments, the second binding moiety comprises Jag2 ECD, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the second binding moiety comprises a variant of Jag2 ECD, and optionally wherein the Jag2 ECD variant comprises a sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 135. In some embodiments, the Jag2 ECD variant comprises no more than any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions relative to SEQ ID NO: 135.

In some embodiments, the non-antibody portion further comprises an aptamer, a SOMAmer, a DARpin, or other engineered binding molecules or scaffolds. In some embodiments, the nick activation portion is conjugated to the 5' or 3' end of the aptamer or SOMAmer by chemical cross-linking. Anti-nick antibody portion

In some embodiments, the second binding moiety is an anti-notch antibody moiety that activates the notch receptor. Anti-notch antibodies capable of activating notch receptors (e.g., notch 1, notch 2, notch 3, and notch 4) are known in the art and include, for example, Asano et al., J Immunol. , 180(5):2796-804 (2008); Hoare et al., Nat Cell Biol. , 18(9):979-92 (2016); Falk et al., Methods ., 58(1):69-78 (2012); Thermo Fisher Scientific catalog number MA5-45604; Gerhardt et al., Genes Dev. , 28(6):576-93 (2014); Adamia et al., Blood. , 123(18):2816-25 (2014); Machuca-Parra et al., J Exp Med ., 214(8):2271-2282 (2017); Xu et al., Structure. , 23(7):1227-35 (2015); and Li et al., J Biol Chem. , 283(12):8046-54 (2008). In some embodiments, the anti-notch antibody portion is selected from the group consisting of: a full-length antibody, a Fab, a Fab', a F(ab') ₂ , a scFv, and a sdAb. C. Linker

The first binding moiety, second binding moiety, and other components of the bifunctional protein constructs described herein can be linked via one or more linkers, optionally present. The domains within the first binding moiety can be linked via one or more linkers, optionally present. The domains within the second binding moiety can be linked via one or more linkers, optionally present.

In some embodiments, the linker is a peptide linker. The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody can be used as a linker. See, for example, WO 1996/034103.

The peptide linker can have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more amino acids in length. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or fewer amino acids in length. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

In some embodiments, the peptide linker does not contain any polymerization activity. Characteristics of peptide linkers that do not promote secondary structure are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). In some embodiments, the peptide linker does not promote the formation of any secondary structure. Connection of the domains to each other can be provided, for example, by genetic engineering. Methods for preparing fused and operably linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 1999/054440; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. 1989 and 1994; or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).

The peptide linker can be a stable linker that is not cleavable by proteases, particularly matrix metalloproteinases (MMPs).

In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, where n is an integer of at least one; glycine-serine polymers (including, for example, (GS)n, where n is an integer of at least one); glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. In some embodiments, the linker is a GS linker.

In some embodiments, the peptide linker comprises the amino acid sequence of LE (SEQ ID NO: 222) or the amino acid sequence of any one of SEQ ID NOs: 207-221, 337, and 338. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 211, 212, 337, and 338. III. Methods for Preparing Binding Moieties and Bifunctional Protein Constructs

Also provided are methods of making any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECDs, and protein constructs comprising the DLL4 ECDs described herein) and bifunctional protein constructs described herein.

The bifunctional protein constructs and binding moieties described herein (e.g., antibody moieties, engineered DLL4 ECDs, and protein constructs comprising the DLL4 ECDs described herein) can be prepared by introducing nucleic acids into host cells, allowing protein expression, and purifying the protein from host cell extracts or supernatants. Methods for preparing protein constructs, constructing nucleic acids or vectors encoding them, and purifying protein constructs are known in the art. See also US20220204602 and Examples 1 and 2 described herein for exemplary methods.

Proteins comprising antibody moieties can be obtained using methods known in the art, such as by immunizing non-human mammals and obtaining hybridomas therefrom, or by cloning antibody libraries and performing subsequence selection using molecular biology techniques known in the art, or by using phage display. Also provided are nucleic acid constructs, vectors, and host cells for preparing any of the first binding moieties, second binding moieties, or bifunctional protein constructs described herein.

The polypeptide portion of the bifunctional protein constructs or binding moieties described herein (e.g., antibody moieties, engineered DLL4 ECDs, and protein constructs comprising DLL4 ECDs described herein) can be prepared by any of the protein expression and purification methods known in the art. The DNA sequence encoding the polypeptide portion of the bifunctional protein constructs or binding moieties (e.g., antibody moieties, engineered DLL4 ECDs, and protein constructs comprising DLL4 ECDs described herein) can be completely synthetic. After obtaining this sequence, it is cloned into a suitable expression vector and then transfected into a suitable host cell. The transfected host cells are cultured, and the supernatant is collected and purified to obtain the polypeptide portion of the bifunctional protein constructs or binding moieties described herein (e.g., antibody moieties, engineered DLL4 ECDs, and protein constructs comprising DLL4 ECDs described herein). The antibody moieties described herein can also be obtained by conventional immunization methods, such as by immunizing mammals (e.g., mice, llamas) and obtaining the antibody moiety from the serum; or from hybridomas, such as by fusing B cells from the lymph nodes and/or spleen of immunized animals with myeloma cells. Purification can then be performed.

These expression vectors may contain a variety of elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected, as appropriate, by one skilled in the art. For example, the promoter sequence may be selected to promote transcription of the polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, the T3 promoter, the SP6 promoter, the β-actin promoter, the EF1a promoter, the CMV promoter, and the SV40 promoter. The enhancer sequence may be selected to enhance transcription of the nucleic acid. The selectable marker can be selected to allow selection of host cells containing the inserted vector from host cells that do not contain the vector, for example, a selectable marker can be a gene that confers antibiotic resistance. The signal sequence can be selected to allow the expressed polypeptide to be exported from the host cell.

In some embodiments, two or more polypeptides of a bifunctional protein construct or binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD as described herein) are encoded by a single vector. In some embodiments, two or more polypeptides of a bifunctional protein construct or binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD as described herein) are encoded by two or more vectors. In some embodiments, two or more polypeptides of a functional protein construct or binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD as described herein) can be used for expression or cloning of isolated nucleic acids. Suitable host cells include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. The expression of binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising the DLL4 ECD described herein) in prokaryotic cells (e.g., E. coli) is well established in the art. For a review, see, for example, Pluckthun, A. BioTechnology 9: 545-551 (1991). Those skilled in the art can also use expression in cultured eukaryotic cells as an option for producing antibodies or antigen-binding fragments thereof. For recent reviews, see, for example, Ref, ME (1993) Curr. Opinion Biotech. 4: 573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6: 553-560. Higher eukaryotic cells, particularly those derived from multicellular organisms, can be used to express glycosylated polypeptides. Suitable higher eukaryotic cells include, but are not limited to, invertebrate and insect cells, as well as vertebrate cells. In some embodiments, the host cell is an Escherichia coli cell. In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell or a HEK293 cell.

Vectors can be introduced into host cells using any suitable method known in the art, including but not limited to DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, microbombardment, receptor-mediated gene delivery, and delivery mediated by polylysine, histones, chitosan, and peptides. Standard methods for transfecting and transforming cells to express vectors of interest are well known in the art. In some embodiments, the host cell contains two or more vectors, each encoding a polypeptide of any of the protein constructs or targeting moieties described herein. In some embodiments, the host cell comprises a single vector comprising isolated nucleic acids encoding two or more polypeptides of any of the bifunctional protein constructs or binding moieties described herein (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD described herein). In some embodiments, the two or more vectors are introduced into the host cell at the same time. In some embodiments, the two or more vectors are introduced into the host cell sequentially.

In some embodiments, the present application provides methods for preparing a polypeptide portion of any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD described herein) or bifunctional protein constructs described herein, comprising: i) expressing a polypeptide portion of any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD described herein) or bifunctional protein constructs described herein in a manner suitable for expressing a polypeptide portion of any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD described herein) described herein; In some embodiments, the method further comprises introducing one or more isolated nucleic acids or one or more vectors into the host cell. The isolated host cells are cultured under conditions that allow expression of the isolated nucleic acid inserted into the vector. Suitable conditions for expression of the polynucleotide may include, but are not limited to, a suitable culture medium, a suitable host cell density in the culture medium, the presence of essential nutrients, the presence of supplementary factors, a suitable temperature and humidity, and the absence of microbial contaminants. Where appropriate, one of ordinary skill in the art can select conditions suitable for the purpose of expression. In some embodiments, the preparation method further comprises purifying any of the obtained binding moieties described herein (e.g., antibody moieties, engineered DLL4 ECDs, and protein constructs comprising DLL4 ECDs described herein) or the polypeptide portions of any of the bifunctional protein constructs.

In some embodiments, polypeptides expressed by host cells can assemble together (e.g., to form a polypeptide complex, such as a dimer) and produce any of the polypeptide portions of any of the binding moieties described herein (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD described herein) or bifunctional protein constructs. In some embodiments, the polypeptide complex can be formed inside the host cell. For example, the polypeptide complex can be formed inside the host cell with the help of relevant enzymes and/or cofactors. In some embodiments, the polypeptide complex can be secreted from the cell. In some embodiments, individual polypeptides can be secreted from the host cell and then form a polypeptide complex outside the host cell.

In some embodiments, two or more polypeptides of any of the binding moieties described herein (e.g., antibody moieties, engineered DLL4 ECDs, and protein constructs comprising DLL4 ECDs described herein) or bifunctional protein constructs can be expressed separately and allowed to form (e.g., dimerize) a protein complex under suitable conditions. For example, a first polypeptide and a second polypeptide can be combined in a suitable buffer and the first protein monomer can be allowed to dimerize with the second protein monomer through suitable interactions, such as hydrophobic interactions. In some embodiments, the first polypeptide and the second polypeptide can be combined in a suitable buffer containing an enzyme and/or cofactor that promotes dimerization of the first polypeptide with the second polypeptide. In some embodiments, the first polypeptide and the second polypeptide can be combined in a suitable medium and allowed to react with each other in the presence of a suitable reagent and/or catalyst.

The expressed polypeptide and/or polypeptide complex can be collected using any suitable method. The polypeptide and/or polypeptide complex can be expressed intracellularly, in the periplasmic space, or secreted extracellularly into the culture medium. If the polypeptide and/or polypeptide complex is expressed intracellularly, the host cells containing the polypeptide and/or polypeptide complex can be lysed and the polypeptide and/or polypeptide complex can be isolated from the lysate by centrifugation or superfiltration to remove unwanted debris. If the polypeptide and/or polypeptide complex is secreted into the periplasmic space of E. coli, the cell paste can be thawed in the presence of reagents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes, and cellular debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)). If the polypeptide and/or polypeptide complex is secreted into the culture medium, the supernatant of the cell culture can be collected and concentrated using commercially available protein concentration filters, such as Amincon or Millipore Pellicon superfiltration devices. Protease inhibitors and/or antibiotics can be included in the collection and concentration steps to inhibit protein degradation and/or the growth of contaminating microorganisms.

The expressed polypeptide and/or polypeptide complex can be further purified by suitable methods, such as, but not limited to, affinity chromatography, hydroxyapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion exchange column, ethanol precipitation, reversed-phase HPLC, silica chromatography, chromatography on heparin agarose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatography focusing, SDS-PAGE, and ammonium sulfate precipitation (for reviews, see Bonner, P. L., Protein purification, published by Taylor & Francis. 2007; Janson, J. C. et al., Protein purification: principles, high resolution methods and applications, Wiley-VCH, 1998).

In some embodiments, polypeptides and/or polypeptide complexes can be purified by affinity chromatography. In some embodiments, protein A chromatography or protein A/G (a fusion protein of protein A and protein G) chromatography can be used to purify polypeptides and/or polypeptide complexes comprising components derived from antibody CH2 and/or CH3 domains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)); Zettlit, KA, Antibody Engineering, Part V, 531-535, 2010). In some embodiments, protein G chromatography can be used to purify polypeptides and/or polypeptide complexes comprising IgG γ3 heavy chains (Guss et al., EMBO J. 5:1567-1575 (1986)). In some embodiments, protein L chromatography can be used to purify polypeptides and/or polypeptide complexes containing kappa light chains (Sudhir, P., Antigen Engineering Protocols, Chapter 26, Humana Press, 1995; Nilson, BHK et al., J. Biol. Chem., 267, 2234-2239 (1992)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices can also be used. Mechanically stable matrices (such as controlled pore glass or poly(styrenedivinyl)benzene) allow for faster flow rates and shorter processing times than those achievable using agarose. When either of the bifunctional protein constructs contains an additional CH3 domain, bakerbond ABX resin (JT Baker, Phillipsburg, NJ) can be used for purification. In some embodiments, purification is performed using MabSelect SuRe. In some embodiments, the method further comprises analyzing the purified protein product, such as by SDS-PAGE and/or SEC-HPLC. Nucleic Acids and Vectors

In one aspect, provided herein are one or more nucleic acids (e.g., isolated nucleic acids) encoding one or more polypeptides of the bifunctional protein constructs or various binding moieties described herein. The one or more nucleic acids can be RNA (e.g., mRNA) or DNA. In some embodiments, the one or more nucleic acids are one or more mRNAs and the one or more mRNAs are formulated into liposomes or lipid nanoparticles. Further provided are vectors comprising any of the nucleic acids (e.g., isolated nucleic acids) described herein, such as viral vectors (e.g., AAV vectors or lentiviral vectors).

Also encompassed herein are isolated host cells comprising or expressing, for example, any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD described herein) or bifunctional protein constructs (e.g., one or more polypeptide components thereof) described herein. Also provided are isolated host cells comprising one or more nucleic acids encoding any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD described herein) or bifunctional protein constructs (e.g., one or more polypeptide components thereof) described herein. Also provided are isolated host cells comprising one or more vectors comprising a vector encoding any of the binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD described herein) or bifunctional protein constructs (e.g., one or more polypeptide components thereof) described herein. In some embodiments, one vector comprises one or more isolated nucleic acids encoding a binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD as described herein) or a bifunctional protein construct (e.g., one or more polypeptide components thereof). In some embodiments, one vector comprises one or more isolated nucleic acids encoding a binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD as described herein) or a bifunctional protein construct. In some embodiments, one vector comprises two or more isolated nucleic acids encoding a binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD as described herein) or two or more polypeptide components of a bifunctional protein construct. In some embodiments, two or more vectors comprise two or more isolated nucleic acids encoding a binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD as described herein) or two or more polypeptide components of a bifunctional protein construct. In some embodiments, two or more isolated nucleic acids encoding a binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD as described herein) or two or more polypeptide components of a bifunctional protein construct are linked to the same promoter on the same vector (e.g., via an IRES, or a nucleic acid encoding a self-cleaving 2A peptide such as P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A, or a protein construct comprising a DLL4 ECD as described herein). 2A), the same promoter on different vectors (e.g., both TetOn), different promoters on the same vector, or different promoters on different vectors (e.g., one inducible and one constitutive). In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell or a HEK293 cell.

This application also encompasses variants of these nucleic acid sequences. For example, such variants include nucleotide sequences hybridized under at least moderately stringent hybridization conditions with nucleic acid sequences encoding bifunctional protein constructs or various binding moieties described herein (e.g., antibody moieties, engineered DLL4 ECDs, and protein constructs comprising DLL4 ECDs described herein). In some embodiments, variants of the nucleic acid sequences comprise degenerate nucleotide codons. Determining the different nucleotides comprising the degenerate codons can be accomplished by one skilled in the art.

This application also provides a vector into which the nucleic acid of this application is inserted.

Nucleic acids can be cloned into a variety of vector types. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Alternatively, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, suitable restriction endonuclease sites, and one or more selectable markers (see, for example, WO 01/96584; WO 01/29058; and U.S. Patent No. 6,326,193). In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

In some embodiments, the nucleic acid encoding the binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD as described herein) or bifunctional protein construct comprises sequences or features in addition to the coding sequence encoding the binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD as described herein) or bifunctional protein construct. For example, the nucleic acid can be an mRNA (or a DNA sequence encoding the mRNA) comprising features encoded by the mRNA that improve targeting of the binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD as described herein) or bifunctional protein construct and increase the stability of the mRNA.

In some embodiments, the nucleic acid encoding one or more polypeptide chains of the bifunctional protein construct comprises a nucleic acid sequence encoding a signal peptide. A signal peptide is a short peptide that may be present at the N-terminus or C-terminus of a newly synthesized protein and that can be used to properly translocate the protein. In some embodiments, the signal peptide facilitates the translocation of the polypeptide chain of the bifunctional protein construct encoded by the nucleic acid. In some embodiments, the signal peptide translocates the polypeptide chain of the bifunctional protein construct to the cell membrane. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 253 or 336. IV. Pharmaceutical Compositions, Kits, and Articles of Manufacture

The present invention also provides pharmaceutical compositions comprising i) any of the bifunctional protein constructs described herein, any of the engineered DLL4 ECD and DLL4 ECD-containing protein constructs described herein, any of the isolated nucleic acids (DNA or RNA) encoding the bifunctional protein constructs, any of the isolated nucleic acids (DNA or RNA) encoding the engineered DLL4 ECD and DLL4 ECD-containing protein constructs described herein, or any of the vectors (e.g., viral vectors) comprising these isolated nucleic acids; and ii) optionally, a pharmaceutically acceptable carrier.

Bifunctional protein constructs described herein; nucleic acids encoding bifunctional protein constructs described herein; vectors (e.g., viral vectors) comprising nucleic acids encoding bifunctional protein constructs, engineered DLL4 ECD, and protein constructs comprising DLL4 ECD described herein; nucleic acids encoding engineered DLL4 ECD and protein constructs comprising DLL4 ECD described herein; suitable formulations (e.g., pharmaceutical compositions) of vectors (e.g., viral vectors) comprising nucleic acids encoding engineered DLL4 ECD and protein constructs comprising DLL4 ECD described herein can be prepared by combining a bifunctional protein construct (or nucleic acid encoding a bifunctional protein construct, or vector (e.g., viral vector) encoding a bifunctional protein construct) of desired purity, an engineered DLL4 ECD (or a vector (e.g., viral vector) encoding an engineered DLL4 ECD, and a protein construct comprising DLL4 ECD described herein. The preparations are obtained by mixing a nucleic acid encoding an engineered DLL4 ECD, or a vector (e.g., a viral vector) comprising a nucleic acid encoding an engineered DLL4 ECD, or a protein construct comprising a DLL4 ECD described herein (or a nucleic acid encoding a protein construct comprising a DLL4 ECD described herein, or a vector (e.g., a viral vector) comprising a nucleic acid encoding a protein construct comprising a DLL4 ECD described herein), optionally with a pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)).

In some embodiments, the pharmaceutical composition further comprises additional ingredients. Additional ingredients include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, buffers; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro ed., Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference. Additional excipients include agents that can act as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the walls of the container (e.g., surfactants such as polysorbates (e.g., polysorbate 80) or poloxamers). The excipients vary depending on whether the pharmaceutical composition contains a protein, a nucleic acid, or a vector (e.g., a viral vector). One skilled in the art can determine the appropriate formulation for use in a pharmaceutical composition.

For example, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; chloride); phenol, butyl alcohol, or benzyl alcohol; alkyl parahydroxybenzoates such as methyl parahydroxybenzoate or propyl parahydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartame, Amide, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). In some embodiments, lyophilized formulations are provided.

Pharmaceutical compositions for intravenous administration must be sterile. This requirement is easily achieved, for example, by filtration through a sterile filter membrane. Pharmaceutical compositions herein are generally placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle.

The pharmaceutical compositions may be prepared by any method known or later developed in the art of pharmacology. Preparation methods include, but are not limited to, combining the active ingredient with a carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single dose or multi-dose unit. The pharmaceutical composition may be prepared, packaged or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersants, wetting agents or suspending agents. Such sterile injectable formulations may be prepared using a parenterally acceptable non-toxic diluent or solvent such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and nonvolatile oils such as synthetic monoglycerides or diglycerides. Other useful parenteral formulations include those containing the active ingredient in microcrystalline form, in a liposomal formulation, or as a component of a biodegradable polymer system. Compositions for sustained-release or implantable administration may include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion-exchange resins, sparingly soluble polymers, or sparingly soluble salts.

The pharmaceutical compositions useful in the methods of the present invention can be prepared, packaged, or sold in formulations suitable for parenteral, intraperitoneal, intramuscular, intradermal, subcutaneous, or intravenous administration. Other contemplated formulations include nanoparticles, liposomal preparations, resealed red blood cells containing the active ingredient, and immunologically based formulations.

The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. A unit dose is a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject or a convenient fraction of such a dose, such as one-half or one-third of such a dose.

Also provided are kits comprising binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD as described herein) or bifunctional protein constructs (or nucleic acids encoding binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD as described herein) or bifunctional protein constructs as described herein, or vectors comprising nucleic acids encoding binding moieties (e.g., antibody moieties, engineered DLL4 ECD, and protein constructs comprising a DLL4 ECD as described herein) or bifunctional protein constructs as described herein). Such kits can be used in any of the treatment methods described herein.

The kits described in this application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The kits may optionally include additional components such as a buffer solution and instructional information.

Thus, this application also provides an article of manufacture. The article of manufacture may comprise a container and a label or instructions on or associated with the container. Suitable containers include vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like. Generally, the container holds the composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). V. Methods of Treatment

Also provided herein are methods for treating muscle-related diseases in an individual (e.g., a human) comprising administering to the individual an effective amount of any of the bifunctional protein constructs or nucleic acids or vectors encoding nucleic acids (e.g., viral vectors) described herein (or pharmaceutical compositions thereof). In some embodiments, the muscle-related disease is selected from the group consisting of Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), familial hypertrophic cardiomyopathy, distal myopathy of Ryan type, myofibrillar myopathy, and muscular dystrophy. A list of muscular dystrophy disorders with associated gene defects that can be treated by the claimed bifunctional protein constructs is presented in Table 9. Table 9 : Muscular dystrophy disorders with associated gene defects Muscular dystrophy Genes involved Genetic Mode Duchenne Dystrophin, X-linked Hidden Baker type Dystrophin, X-linked Hidden Emery-Dreyfus muscular dystrophy (EDMD) Emerin, X-linked Hidden Lamin A laminin C Congenital muscular dystrophy CMD with cardiomyopathy Titin Hidden CMD including desmin SEPN1 Hidden CMD with integrin α7 mutation Integrin-α7 gene Hidden CMD with familial junctional epidermolysis bullosa Plectin Hidden CMD with muscle hypertrophy; also known as MDC1C Fukutin-related protein (FKRP) Hidden MDC1D Large Gene Hidden CMD with myasthenia gravis DOK7 Hidden CMD with (early) spinal stiffness SEPN1 Hidden CMD with spinal stiffness and abnormalities of nuclear lamin A/C Lamin A/C dominant CMD with spinal stiffness and selenoprotein deficiency SBP2 Fukuyama CMD Fukuyama protein (fukutin) Hidden Platelet-deficient CMD; also known as MDC1A Laminin-211 Hidden Santavuori muscle-eye-brain disease POMGnT1 Hidden Ullrich CMD COLGA1, COL6A2, or COL6A3 latent or explicit Walker-Warburg syndrome Hidden MDDGA B3GNT1 Hidden MDDGA1 POMT1 Hidden MDDGA2 POMT2 Hidden MDDGA 3-6 Overlap with other CMDs Hidden MDDGA7 ISPD Hidden MDDGA8 GTDC2 Hidden MDDGA10 TMEM5 Hidden MDDGA11 B3GALNT2 Hidden MDDGA12 SGK196 Hidden Facioscapulohumeral dystrophy (FSH, FSHD) DUX4 repeat 4q35 Limb-girdle muscular dystrophy (LGMD ) LGMD1 LGMD1A Myotilin dominant LGMD1B LMNA dominant LGMD1C CAV3 dominant LGMD1D DNAJB6 dominant LGMD1E DES dominant LGMD1F TNP03 dominant LGMD1G HNRNPDL dominant LGMD1H unknown LGMD2A CAPN3 Hidden LGMD2B DYSF Hidden LGMD2C SGCG Hidden LGMD2D SGCA Hidden LGMD2E SGCB Hidden LGMD2F SGCD Hidden LGMD2G TCAP Hidden LGMD2H TRIM32 Hidden LGMD2I FKRP Hidden LGMD2J TTN Hidden LGMD2K POMT1 Hidden LGMD2L ANO5 Hidden LGMD2M FKTN Hidden LGMD2N POMT2 Hidden LGMD2O POMGnT1 Hidden LGMD2P DAG1 Hidden LGMD2Q PLEC1 Hidden LGMD2R DES Hidden LGMD2S TRAPPC11 Hidden LGMD2T GMPPB Hidden LGMD2U ISPD Hidden LGMD2V GAA Hidden LGMD2W LIMS2 Hidden LGMD2X BVES Hidden LGMD2Y TOR1A1P1 Hidden Myotonic dystrophy DM1 DMPK CTG repeat expansion > 50 DM2 ZNF9 expansion Oculopharyngeal muscular dystrophy (OPMD) PABPN1 dominant Congenital myopathy Myotubular myopathy Myotubularin X chain, hidden Myopathy Various genetic defects latent/explicit Distal myopathy Gowers-Laing distal myopathy MYH7 Miyoshi distal myopathy Dysferlin Nonaka distal myopathy GNE Hereditary inclusion body myositis type 1 (HIBM1) Various genetic defects Charcot - Marie Tooth Type 1a PMP22 dominant Type 1b MPZ Hidden X-type Connexin 32/GJB Hidden

In some embodiments, the muscle-related disease is a muscular dystrophy. In some embodiments, the muscular dystrophy comprises one or more of the following: Duchenne muscular dystrophy (DMD), Beck muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital myopathy, Charcot-Marie-Duchesne (CMT) disease, and Emory-Dreyfus muscular dystrophy (EDMD). In some embodiments, the type of limb-girdle muscular dystrophy is selected from the group consisting of LGMD2A, LGMD2B, LGMD2C, LGMD2D, LGMD2E, LGMD2F, LGMD2G, LGMD2H, LGMD2I, LGMD2J, LGMD2K, LGMD2L, LGMD2M, LGMD2N, LGMD2O, LGMD2P, LGMD2Q, LGMD2R, LGMD2S, LGMD2T, LGMD2U, LGMD2V, LGMD2W, LGMD2X, and LGMD2Y. In some embodiments, the type of myotonic dystrophy is selected from the group consisting of DM1 and DM2. In some embodiments, the type of Charcot-Marie-Dupuytren's disease is selected from the group consisting of type 1a, type 1b, and type X. In some embodiments, the muscle disease is sarcopenia.

In some embodiments, a method of renewing satellite cells in muscle tissue is provided, comprising contacting the muscle tissue with an effective amount of any of the bifunctional protein constructs or binding moieties described herein (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD described herein), or a vector (e.g., a viral vector) comprising a nucleic acid encoding a bifunctional protein construct or binding moiety described herein (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD described herein). In some embodiments, the method allows myoblasts to differentiate in the muscle tissue. In some embodiments, a method of repairing and/or regenerating muscle tissue is provided, comprising contacting the muscle tissue with an effective amount of any of the bifunctional protein constructs or binding moieties described herein (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD described herein), or a vector (e.g., a viral vector) comprising a nucleic acid encoding a bifunctional protein construct or binding moiety described herein (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD described herein). In some embodiments, the muscle tissue is damaged muscle tissue. In some embodiments, the muscle tissue is from an individual (e.g., a human) suffering from a muscle-related disease such as Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), familial hypertrophic cardiomyopathy, distal Ryan myopathy, myofibrillar myopathy, and muscular dystrophy.

In some embodiments, the methods of treating muscle-related diseases described herein can achieve one or more of the following: (i) increase or maintain muscle strength compared to a pre-existing state or compared to a state that would occur without treatment; (ii) delay the progression of muscle wasting; (iii) reduce muscle loss; (iv) increase or maintain muscle strength compared to a pre-existing state or compared to a state that would occur without treatment; Improve or maintain motor skills as compared to the state that would occur in the absence of treatment; (v) repair or maintain muscle cells or tissue; (vi) increase regeneration of muscle cells or tissue as compared to the pre-existing state or as compared to the state that would occur in the absence of treatment; (vii) enhance satellite cell turnover; and/or (viii) increase myoblast differentiation. In some embodiments, muscle strength is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to a pre-existing state or compared to the state that would occur in the absence of treatment. In some embodiments, motor skills are improved by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to a pre-existing state or compared to the state that would occur in the absence of treatment. In some embodiments, regeneration of muscle cells or tissue is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to a pre-existing state or compared to the state that would occur in the absence of treatment. In some embodiments, satellite cell turnover is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to a pre-existing state or compared to the state that would occur in the absence of treatment. In some embodiments, myoblast differentiation is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to a pre-existing state or compared to the state that would occur in the absence of treatment.

In some embodiments, the bifunctional protein construct or binding moiety (e.g., an antibody moiety, an engineered DLL4 ECD, and a protein construct comprising a DLL4 ECD described herein), or a nucleic acid or vector encoding the same (e.g., a viral vector), or a pharmaceutical composition thereof is administered to the subject once. In some embodiments, the bifunctional protein construct or a nucleic acid or vector encoding the same (e.g., a viral vector), or a pharmaceutical composition thereof is administered to the subject two or more times, e.g., weekly, biweekly, every three weeks, monthly, every two months, every three months, every six months, and the like.

In some embodiments, a bifunctional protein construct, or a nucleic acid or vector (e.g., a viral vector) is delivered to the tissue of interest by, for example, intramuscular injection, while at other times, delivery is performed intravenously, subcutaneously, transdermally, transmucosally, intraperitoneally, or by other delivery methods. Such delivery can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be delivered herein can vary widely depending on a variety of factors, such as the choice of vector, the target cell, organism, or tissue, the general condition of the individual to be treated, the degree of transformation/modification sought, the route of administration, the mode of administration, the type of transformation/modification sought, etc.

The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the individual being treated and, further, on the intended route of administration of the composition. Examples

The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention. They are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. The following examples are intended merely to illustrate the present application and, therefore, should not be construed as limiting the present application in any way.

The scope of this disclosure is not limited by the specific embodiments described, which are intended as single illustrations of individual aspects of this disclosure, and any compositions or methods that are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. For example, due to codon redundancy, changes can be made to the underlying DNA sequence without affecting the protein sequence. In addition, due to biological functional equivalence considerations, changes can be made to the protein structure without affecting the type or amount of biological effect. Therefore, it is expected that this disclosure covers modifications and variations of this disclosure as long as they come within the scope of the attached patent claims and their equivalents.

All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Example 1 : Generation of an exemplary bifunctional fusion protein ( construct muDLL4v - Fc - muLAMA2LG4-5 ) comprising a mouse DLL4 variant containing C2 - DSL and EGF1-5 , mIgG1 Fc , and mouse laminin - 211α2 LG4-5 domains

The DLL4v-Fc-LAMA2 LG4-5 construct (SEQ ID NO: 242) was generated using mouse DLL4 (Accession No. AAF76428.1) containing C2-DSL-EGF1-5 (aa 28-401) with mutations from the rat DLL4 EL12 variant (Luca et al., 2015, Science 47(6224):847-53). The mouse DLL4 variant containing C2-DSL and EGF1-5 was fused to the N-terminus of a mouse IgG1 Fc engineered to remove effector function. The C-terminus of the Fc was fused to a (GGGGS) ₃ linker (SEQ ID NO: 211) and the mouse laminin-2α (Accession No. NP_032507.2) LG domains 4-5 (aa 2870-3118) (SEQ ID NO: 250). Assemble the complete DNA sequence, codon-optimized for expression in CHO or HEK293 cells. Clone the DNA into the pcDNA3.x vector (Thermo Fisher Scientific). Transform the pcDNA3.x vector containing the cloned construct into E. coli (DH5α competent cells), pick a single colony, verify the DNA sequence, propagate the colony by overnight culture, and obtain the plasmid DNA using a commercially available plasmid purification kit. After generating a stable cell pool, transiently or stably transfect the pcDNA3.x plasmid DNA into CHO or HEK293 cells for expression. Purify the recombinant protein from the cell culture supernatant using a 25 ml MabSelect™ SuRe™ column (Cytiva). The protein was eluted from the column with 0.1 M glycine (pH 3.0) and immediately neutralized with 20% by volume of 1 M Tris (pH 8.0). The eluted sample was then buffer-exchanged into 1× PBS and analyzed for monodispersity using analytical size exclusion chromatography (SEC). When the monodispersity of the recombinant protein was less than 93%, a second purification step was performed using preparative SEC in 1× PBS. The purity and integrity of the purified protein were analyzed using analytical HPLC-SEC (Figure 3B) and SDS-PAGE (Figure 3A). HPLC-SEC (Figure 3B) and SDS-PAGE (Figure 3A) analysis showed that the DLL4v-Fc-LAMA2 LG4-5 construct had the predicted molecular weight. Purified proteins with a monodispersity greater than 93% were used for functional studies.

A bifunctional protein with the wild-type DLL4 sequence (construct DLL4wt-Fc-LAMA2 LG4-5) was similarly expressed (Figures 4A and 4B). SDS-PAGE (Figure 4A) and HPLC-SEC (Figure 4B) analysis showed that the DLL4wt-Fc-LAMA2 LG4-5 construct (SEQ ID NO: 241) had the predicted molecular weight. Example 2 : Generation of an exemplary bifunctional fusion protein ( construct muDLL4v - Fc - LAMA2scFv ) comprising a mouse DLL4 variant containing C2 - DSL and ( EGF1-5 ) , mIgG1 Fc , and anti -laminin - 2α

The DLL4v-Fc-LAMA2 scFv construct was generated using mouse DLL4 (Accession No. AAF76428.1) containing C2-DSL-EGF1-5 (aa 28-401) with mutations from the rat DLL4 EL12 variant (Luca et al., 2015, Science 47(6224):847-53). The mouse DLL4 variant containing C2-DSL and EGF1-5 was fused to the N-terminus of a mouse IgG1 Fc engineered to remove effector function. The C-terminus of the Fc was fused to a (GGGGS)3 linker and anti-mouse laminin-2α (SEQ ID NO:251). The complete DNA sequence was assembled, codon-optimized for expression in CHO or HEK293 cells, and cloned into the pcDNA3.x vector (Thermo Fisher Scientific). pcDNA3.x containing the selected constructs was transformed into E. coli (DH5α competent cells). Single colonies were picked, DNA sequences verified, and colonies were propagated overnight. Plasmid DNA was harvested using a commercially available plasmid purification kit. After generating stable cell pools, the pcDNA3.x plasmid DNA was transiently or stably transfected into CHO or HEK293 cells for expression. Recombinant protein was purified from the cell culture supernatant using a 25 ml MabSelect™ SuRe™ column (Cytiva). Protein was eluted from the column with 0.1 M glycine (pH 3.0) and immediately neutralized with 20% by volume of 1 M Tris (pH 8.0). The eluted sample was then buffer-exchanged into 1× PBS and analyzed for monodispersity using analytical size exclusion chromatography (SEC). When the monodispersity of the recombinant protein was less than 93%, a second purification step was performed using preparative SEC in 1× PBS. The purity and integrity of the purified protein were analyzed using analytical HPLC-SEC (Figure 5B) and SDS-PAGE (Figure 5A). SDS-PAGE (Figure 5A) and HPLC-SEC (Figure 5B) analyses demonstrated that the DLL4v-Fc-LAMA2scFv construct had the predicted molecular weight. Purified proteins with a monodispersity greater than 93% were used for functional studies.

The sequences of exemplary bifunctional proteins comprising a muscle targeting portion and a Notch activation portion used in preclinical testing are listed in Table 5. Example 3 : Expression of human LG4-5 domains

Human LG5 (aa 2870-3118, accession number: NP_000417.3) was cloned into pcDNA3.x with an N-terminal signal peptide (SEQ ID NO: 252) and a His6 tag. The recombinant protein was expressed in HEK293 cells as described above and purified from the cell culture supernatant on a Ni-NTA nickel chelate resin column (ThermoFisher Scientific) using the manufacturer's protocol. The lysate containing the recombinant protein was buffer-exchanged into PBS in a Slide-A-Lyzer dialysis cassette with a molecular weight cutoff (MWCO) of 10 kDa (ThermoFisher Scientific). Example 4 : Demonstration of binding of the mouse bifunctional construct to the target

Binding of the muDLL4v-Fc-LAMA2 scFv, muDLL4wt-Fc-LAMA2 LG4-5, and muDLL4v-Fc-LAMA2 LG4-5 constructs to Notch was confirmed using a sandwich ELISA with mouse recombinant Notch 1 protein (Sino Biologicals). A 96-well ELISA plate was coated with 100 μL/well of 2 μg/mL recombinant Notch 1 protein at 4°C. After 16 hours, the wells were washed five times with 200 μL/well of 0.05% PBST, blocked with 200 μL/well of 1% BSA for 1 hour at 25°C, and washed five times with 200 μL/well of 0.05% PBST. The wells were serially diluted three times with 100 μL of 10 μL/ml dual-functional protein per well for 1 hour at 25°C, washed five times with 200 μL of 0.05% PBST per well, and incubated with 100 μL of goat anti-mouse IgG-HRP (1:10,000) per well for 1 hour at 25°C. After washing five times with 200 μL of 0.05% PBST per well, 100 μL of TMB was added per well at 25°C in the absence of light for 10 minutes, 50 μL of 1 M HCl was added per well, and the OD at 450 nm was read on a plate reader. As shown in Figure 6, DLL4v-Fc-LAMA2 LG4-5 and DLL4v-Fc-LAMA2scFv have higher binding affinity to nick 1 than DLL4wt -Fc-LAMA2 LG4-5. Example 5 : Binding of recombinant bifunctional proteins containing mouse laminin - 2 LG4-5 to matrix glycans

Following published methods (Michele et al., 2002, Nature. 418(6896):417-22), stromal glycans on α-myotoxin were purified from rabbit muscle homogenate using WGA agarose beads. Binding of the DLL4v-Fc-LG4-5 construct to stromal glycans was analyzed using a Biacore T200 (Cytiva). The DLL4v-Fc-LG4-5 construct was captured on an anti-Fc chip at a concentration of 10 μg/ml and signal subtraction was performed on the same anti-Fc chip using TBST buffer as a reference. Stromal glycan samples were injected to confirm binding. The sensor map clearly indicated binding of the DLL4v-Fc-LG4-5 construct to matrix glycans; however, due to the uncertainty of matrix glycan concentrations, binding kinetics were not calculated (Figure 7A). As shown in Figure 7B, binding of the DLL4v-Fc-LG4-5 construct to matrix glycans (on α-myosin) was also confirmed by Western blotting after laminin overlay analysis (Michele et al . , 2002, Nature. 418(6896):417-22). Example 6 : Confirmation of Binding of Mouse DLL4v - Fc - LAMA2 scFv to Recombinant Human LG4-5 by Biacore

Binding of the muLL4v-Fc-LAMA2scFv construct was tested as described above. The DLL4v-Fc-LAMA2scFv construct was captured on an anti-Fc chip at a concentration of 10 μg/ml and used for signal subtraction on the same anti-Fc chip using TBST buffer as a reference. Binding was confirmed using single-cycle kinetics with injections of recombinant LG5 at concentrations of 0.16 nM, 0.8 nM, 4 nM, 20 nM, and 100 nM. Binding kinetics were generated using a 1:1 binding model. As shown in Figure 8, the binding signal increased with increasing concentrations of recombinant LG5. Kinetic analysis indicated a binding affinity of KD of 12 nM (ka = 1.65E+5 1/Ms, kd = 0.002 1/s, KD = 12 nM) . Example 7 : Evaluation of the efficacy of the recombinant construct in the D2.mdx model

Animals . This study used male mice of the DBA/2J strain (The Jackson Laboratory, colony #000671) in the control group and mdx mice of the DBA/2J background (D2.mdx; The Jackson Laboratory, colony #013141) in the experimental group. Mice were housed in cages of 3–5 mice each with free access to food (NIH-31 open formulation diet; Envigo #7917) and water, and provided with ample housing. Mice were maintained on a 12-h light/dark cycle. All animal research protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Tufts University and were conducted accordingly. Male mice of the DBA/2J and D2.mdx backgrounds, 5-6 weeks of age, were purchased from the Jackson Laboratory. After approximately one week of acclimation, the mice were trained for a forelimb grip strength test. Based on forelimb grip strength, D2.mdx mice were randomly assigned to control or treatment groups.

Administration of Test Articles . D2.mdx male mice, approximately 7-8 weeks of age, were injected intraperitoneally (ip) with 5 mg/kg of muDLL4v-Fc-LAMA2scFv (Figure 9A). Control D2.mdx male mice of similar age were injected with 20 mg/kg of an isotype control antibody against trinitrophenol (TNP). D2.mdx mice in both control and treated groups were injected once every three weeks. Starting with the second dose, mice were pretreated with 5 mg/kg diphenhydramine (ip) 10 minutes prior to administration of the isotype control or DLL4v-Fc-LAMA2scFv to prevent potential allergic reactions.

Forelimb grip strength test . Forelimb grip strength was assessed using a grid connected to an isometric force transducer (Columbus Instruments, Columbus, OH, USA) according to a previously published protocol (Aartsma-Rus and van Putten, 2014, J Vis Exp. (85):51303) and TREAT-NMD standard operating protocol DMD_M.2.2.001. Briefly, mice were grasped by the tail and had their forelimbs grasp a wire connected to the force transducer on the handgrip dynamometer. The mouse was pulled backward by the tail until it released the wire. The maximum force required to release the mouse from the mesh surface was recorded using the transducer. Five replicates were performed on the same mouse, each with a 30-second interval, and the maximum grip force was recorded. All groups were tested in the morning of each month.

As shown in Figure 9B, DBA/2J wild-type mice showed a decrease in grip strength over time. Although grip strength was comparable between D2.mdx control and treated groups at 13 weeks, D2.mdx mice injected with the isotype control exhibited progressively reduced forelimb grip strength compared to D2.mdx mice injected with muDLL4v-Fc-LAMA2scFv (Figure 9B). By 35 weeks, although both control and treated mice had lower overall forelimb grip strength than DBA/2J wild-type mice, mice injected with DLL4v-Fc-LAMA2scFv had 65% greater forelimb grip strength than mice injected with the isotype control (Figure 9B). In addition, the body weight of the control and treated groups reached a stable level and was generally less than that of DBA/2J wild-type mice (Figure 9C), indicating that despite the low body weight, mice given the DLL4v-Fc-LAMA2scFv construct were able to maintain muscle function. In summary, the data demonstrate that the use of the DLL4v- Fc -LAMA2scFv construct has long-term benefits in maintaining or improving muscle function. Example 8 : Production of a bifunctional fusion protein comprising human DLL4 variants containing C2 - DSL and EGF1-5 , hIgG1 Fc , and anti- LAMA2 scFv

Parental human DLL4v (SEQ ID NO: 260) was generated using human DLL4 containing C2-DSL-EGF1-5, which contains G2S, F81L, and H168Y mutations at the N-terminus of EGF5 relative to wild-type DLL4 (SEQ ID NO: 126). Parental human DLL4v was fused to the N-terminus of a human IgG1 Fc engineered to remove effector function. The C-terminus of the Fc was fused to a (GGGGS)3 linker and an anti-human LAMA2 scFv (SEQ ID NO: 268). Because mutations in the rat DLL4 EL12 variant (US2019/0248894; Luca et al., 2015, Science 47(6224):847-53; WO2020/223610; and WO2021/007573) cause low yield and high aggregation in human DLL4, mutations were introduced to overcome poor chemistry, manufacturing, and control (CMC) developability. The N-glycosylation sites from the huD4v_G2S_F81L_H168Y-FcAAG-LG21DS parental construct were reintroduced into positions T52 and T135 of human DLL4v (SEQ ID NO: 260), and new mutations identified by yeast display screening were incorporated to identify DLL4 mutations that confer higher affinity for the nick receptor. Based on the parental human DLL4v (SEQ ID NO: 260), the following seven human DLL4 variants were generated: DLL4v11 (SEQ ID NO: 261), DLL4v12 (SEQ ID NO: 262), DLL4v13 (SEQ ID NO: 263), DLL4v14 (SEQ ID NO: 264), DLL4v22 (SEQ ID NO: 265), DLL4v23 (SEQ ID NO: 266), and DLL4v24 (SEQ ID NO: 267). These seven human DLL4 variants were further fused to an anti-LAMA2 scFv (SEQ ID NO: 268) to create bifunctional constructs (e.g., SEQ ID NOs: 270-276). The N-glycosylation site and yeast display mutations are summarized in Table 10 . Relative to wild-type DLL4 (SEQ ID NO: 126), bold amino acids indicate amino acid mutations and non-bold amino acids are native amino acids. Table 10 : Human DLL4 mutations Amino acid position 14 52 66 80 135 220 231 260 huDLL4v11 E N R P N Q N N huDLL4v12 E N S P N Q N N huDLL4v13 H N T L N Q D N huDLL4v14 E N T L N H N D huDLL4v22 E T S P T Q N N huDLL4v23 H T T L T Q D N huDLL4v24 E T T L T H N D

Assemble the complete DNA sequence, codon-optimized for expression in CHO or HEK293 cells. Clone the DNA into the pcDNA3.x vector (Thermo Fisher Scientific). Transform the pcDNA3.x vector containing the cloned construct into E. coli (DH5α competent cells), pick a single colony, verify the DNA sequence, propagate the colony by overnight culture, and obtain the plasmid DNA using a commercially available plasmid purification kit. After generating a stable cell pool, transiently or stably transfect the pcDNA3.x plasmid DNA into CHO or HEK293 cells for expression. Purify the recombinant protein from the cell culture supernatant using a 25 ml MabSelect™ SuRe™ column (Cytiva). The protein was eluted from the column with 0.1 M glycine (pH 3.0) and immediately neutralized with 20% by volume of 1 M Tris (pH 8.0). The eluted sample was then buffer-exchanged into 1× PBS and analyzed for monodispersity using analytical size exclusion chromatography (SEC). When the monodispersity of the recombinant protein was less than 93%, a second purification step was performed using preparative SEC in 1× PBS. The purity and integrity of the purified protein were analyzed using analytical HPLC-SEC, which showed that the initial fractions of v11, v12, v13, and v14 contained 92%, 92%, 78%, and 84% monomeric protein, respectively (Figures 10A and 10B). Furthermore, from 30 ml of starting material, v11 and v12 achieved high yields, while v13 and v14 achieved moderate yields ( Figure 10B ). Thus, constructs with N-glycosylation sites (v11, v12, v13, and v14) demonstrated superior protein expression, stability, and high recovery compared to constructs v22, v23, and v24. As shown in Figures 11A and 11B , when v11, v12, v13, and v14 were tested for their binding affinity to the murine Notch 1 receptor in an ELISA assay, these constructs exhibited comparable binding affinities to the huD4v_G2S_F81L_H168Y-Fc(AAG)-LG21scFvDS parent construct (SEQ ID NO:269) at higher fusion protein concentrations and superior binding affinities to those of the mouse surrogate molecule (muD4v; SEQ ID NO:349). These data demonstrate that v11, v12, v13, and v14 possess excellent CMC developability. Sequence Listing SEQ ID NO: 1 anti- LAMA2 LG03 HC-CDR1SYTMS SEQ ID NO: 2 anti- LAMA2 LG03 HC-CDR2SISSGGGNTYYPDSVKG SEQ ID NO: 3 anti- LAMA2 LG03 HC-CDR3FDYGSSLDS SEQ ID NO: 4 anti- LAMA2 LG03 LC-CDR1RASQSISNNLH SEQ ID NO: 5 anti- LAMA2 LG03 LC-CDR2YASQSIS SEQ ID NO: 6 anti- LAMA2 LG03 LC-CDR3QQSKNWPRT SEQ ID NO: 7 anti- LAMA2 LG21 Parents /huVH7/huVH10 HC-CDR1SYTMS SEQ ID NO: 8 Anti-LAMA2 LG21 Parents /huVH7/huVH10 HC-CDR2TISSSGDNTYYPDSVKG SEQ ID NO: 9 anti- LAMA2 LG21 Parents /huVH7/huVH10 HC-CDR3FDYGSSLDS SEQ ID NO: 10 anti- LAMA2 LG21 Parents /huVL3/huVL7 LC-CDR1RASQSISNYLH SEQ ID NO: 11 anti- LAMA2 LG21 Parents /huVL3/huVL7 LC-CDR2YASQSIS SEQ ID NO: 12 anti- LAMA2 LG21 Parents /huVL3/huVL7 LC-CDR3QQSKSWPRT SEQ ID NO: 13 anti- LAMA2 LG21 huVH1 HC-CDR1RYTMS SEQ ID NO: 14 anti- LAMA2 LG21 huVH1 HC-CDR2TISSSGDNTYYPDSVKG SEQ ID NO: 15 anti- LAMA2 LG21 huVH1 HC-CDR3FDYGSSLDS SEQ ID NO: 16 anti- LAMA2 LG21 huVH2 HC-CDR1SYTMS SEQ ID NO: 17 anti- LAMA2 LG21 huVH2 HC-CDR2TIESSGDNTYYPDSVKG SEQ ID NO: 18 anti- LAMA2 LG21 huVH2 HC-CDR3FDYGSSLDS SEQ ID NO: 19 anti- LAMA2 LG21 huVH3 HC-CDR1RYTMS SEQ ID NO: 20 anti- LAMA2 LG21 huVH3 HC-CDR2TIESSGQITYYPDSVKG SEQ ID NO: 21 anti- LAMA2 LG21 huVH3 HC-CDR3FDYGSSLDS SEQ ID NO: 22 anti- LAMA2 LG21 huVH4 HC-CDR1RYTMS SEQ ID NO: 23 anti- LAMA2 LG21 huVH4 HC-CDR2TISSSGQETYYPDSVKG SEQ ID NO: 24 anti- LAMA2 LG21 huVH4 HC-CDR3FDYGSSLDS SEQ ID NO: 25 anti- LAMA2 LG21 huVH5 HC-CDR1SYTMS SEQ ID NO: 26 anti- LAMA2 LG21 huVH5 HC-CDR2TISSSGSNTYYPDSVKG SEQ ID NO: 27 anti- LAMA2 LG21 huVH5 HC-CDR3FDYGSSLDS SEQ ID NO: 28 anti- LAMA2 LG21 huVH6 HC-CDR1SYTMS SEQ ID NO: 29 anti- LAMA2 LG21 huVH6 HC-CDR2TISNSGDNTYYPDSVKG SEQ ID NO: 30 anti- LAMA2 LG21 huVH6 HC-CDR3FDYGSSLDS SEQ ID NO: 31 anti- LAMA2 LG21 huVH8 HC-CDR1FDYGSSLDS SEQ ID NO: 32 anti- LAMA2 LG21 huVH8 HC-CDR2FDYGSSLDS SEQ ID NO: 33 anti- LAMA2 LG21 huVH8 HC-CDR3FDYGSSLDS SEQ ID NO: 34 anti- LAMA2 LG21 huVH9 HC-CDR1SYTMS SEQ ID NO: 35 anti- LAMA2 LG21 huVH9 HC-CDR2TISSSGDITYYPDSVKG SEQ ID NO: 36 anti- LAMA2 LG21 huVH9 HC-CDR3FDYGSSLDL SEQ ID NO: 37 anti- LAMA2 LG21 huVL1 LC-CDR1RASQSISNYLH SEQ ID NO: 38 anti- LAMA2 LG21 huVL1 LC-CDR2YASQSIS SEQ ID NO: 39 anti- LAMA2 LG21 huVL1 LC-CDR3QQSRSWPRT SEQ ID NO: 40 anti- LAMA2 LG21 huVL2 LC-CDR1RASQSISNYLH SEQ ID NO: 41 anti- LAMA2 LG21 huVL2 LC-CDR2YASQSIS SEQ ID NO: 42 anti- LAMA2 LG21 huVL2 LC-CDR3QQSKYWPRT SEQ ID NO: 43 anti- LAMA2 LG21 huVL4/huVL5 LC-CDR1RASQSISNYLH SEQ ID NO: 44 anti- LAMA2 LG21 huVL4/huVL5 LC-CDR2YASQSIS SEQ ID NO: 45 anti- LAMA2 LG21 huVL4/huVL5 LC-CDR3QQSKSFPRT SEQ ID NO: 46 anti- LAMA2 LG21 huVL6/huVL8 LC-CDR1RASQSISNYLH SEQ ID NO: 47 anti- LAMA2 LG21 huVL6/huVL8 LC-CDR2YASQSIS SEQ ID NO: 48 anti- LAMA2 LG21 huVL6/huVL8 LC-CDR3QQSKSDPRT SEQ ID NO: 49 anti- LAMA2 LG03 VH (CDR Sequence underlined )DVMLVESGGDLVKPGGSLKLSCAASGFTFS SYTMSWVRQTPEKRLEWVA SISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 50 anti- LAMA2 LG03 VL (CDR Sequence underlined )DIVLTQSPATLSVTPGDSVSLSC RASQSISNNLHWYQQKSHESPRLLIK YASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFC QQSKNWPRTFGGGTKLEIK SEQ ID NO: 51 anti- LAMA2 LG03 scFv (CDR Sequence underlined )DIVLTQSPATLSVTPGDSVSLSC RASQSISNNLHWYQQKSHESPRLLIK YASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFC QQSKNWPRTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSDVMLVESGGDLVKPGGSLKLSCAASGFTFS SYTMSWVRQTPEKRLEWVA SISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 52 anti- LAMA2 LG03 VH-CH1 (CDR Sequence underlined )DVMLVESGGDLVKPGGSLKLSCAASGFTFS SYTMSWVRQTPEKRLEWVA SISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCAR FDYGSSLDSWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC SEQ ID NO: 53 anti- LAMA2 LG03 VL-CL (CDR Sequence underlined )DIVLTQSPATLSVTPGDSVSLSC RASQSISNNLHWYQQKSHESPRLLIK YASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFC QQSKNWPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 54 anti- LAMA2 LG21 huVHpar (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMSWVRQAPGKGLEWVA TISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 55 anti- LAMA2 LG21 huVH1 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYTMSWVRQAPGKGLEWVA TISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 56 anti- LAMA2 LG21 huVH2 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMSWVRQAPGKGLEWVA TIESSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 57 anti- LAMA2 LG21 huVH3 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYTMSWVRQAPGKGLEWVA TIESSGQITYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 58 anti- LAMA2 LG21 huVH4 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYTMSWVRQAPGKGLEWVA TISSSGQETYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 59 anti- LAMA2 LG21 huVH5 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMSWVRQAPGKGLEWVA TISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 60 anti- LAMA2 LG21 huVH6 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMSWVRQAPGKGLEWVA TISNSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 61 anti- LAMA2 LG21 huVH7 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFPFS SYTMSWVRQAPGKGLEWVA TISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 62 anti- LAMA2 LG21 huVH8 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMSWVRQAPGKGLEWVA TISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDLWGQGTTLTVSS SEQ ID NO: 63 anti- LAMA2 LG21 huVH9 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYTMSWVRQAPGKGLEWVA TISSSGDITYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDLWGQGTTLTVSS SEQ ID NO: 64 anti- LAMA2 LG21 huVH10 (CDR Sequence underlined )EVQLVESGGGLVQPGGSLRLSCAASGFEFS SYTMSWVRQAPGKGLEWVA TISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCAR FDYGSSLDSWGQGTTLTVSS SEQ ID NO: 65 anti- LAMA2 LG21 huVLpar (CDR Sequence underlined )EIVLTQSPDFLSVTPKEKVTLSC RASQSISNYLHWYQQKSDQSPKLLIK YASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFC QQSKSWPRTFGGGTKLEIK SEQ ID NO: 66 anti- LAMA2 LG21 huVL1 (CDR Sequence underlined )EIVLTQSPDFLSVTPGEKVILTC RASQSISNYLHWYQQKSDQSPKLLIK YASQSISGVPSRFSGSGSGTDFTLTISSVEAEDVATYFC QQSRSWPRTFGGGTKLEIK SEQ ID NO: 67 anti- LAMA2 LG21 huVL2 (CDR Sequence underlined )EIVLTQSPDFLSVTPGEKVILTC RASQSISNYLHWYQQKSDQSPKLLIK YASQSISGVPSRFSGSGSGTDFTLTISSVEAEDVATYFC QQSKYWPRTFGGGTKLEIK SEQ ID NO: 68 anti- LAMA2 LG21 huVL3 (CDR Sequence underlined )EIVLTQSPDFLSVTPGEKVTLSC RASQSISNYLHWYQQKSDQSPKLLIK YASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFC QQSKSWPRTFGGGTKLEIK SEQ ID NO: 69 anti- LAMA2 LG21 huVL4 (CDR Sequence underlined )EIVLTQSPDFLSVTPGEKVTLSC RASQSISNYLHWYQQKSDQSPKLLIK YASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFC QQSKSFPRTFGGGTKLEIK SEQ ID NO: 70 anti- LAMA2 LG21 huVL5 (CDR Sequence underlined )EIVLTQSPDFLSVTPGEKVTLSC RASQSISNYLHWYQQKSDQSPKLLIK YASQSISGIPSRFSGSGSGTDFTLTINSVEAEDAATYFC QQSKSFPRTFGGGTKLEIK SEQ ID NO: 71 anti- LAMA2 LG21 huVL6 (CDR Sequence underlined )EIVLTQSPDFLSVTPGEKVTLSC RASQSISNYLHWYQQKSDQSPKLLIK YASQSISGIPSRFSGSGSGTDFTLTINSVEAEDAATYFC QQSKSDPRTFGGGTKLEIK SEQ ID NO: 72 anti- LAMA2 LG21 huVL7 (CDR Sequence underlined )EIVLTQSPDFLSVTPKEKVTLSC RASQSISNYLHWYQQKSDQPPKLLIK YASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFC QQSKSWPRTFGGGTKLEIK SEQ ID NO: 73 anti- LAMA2 LG21 huVL8 (CDR Sequence underlined )EIVLTQSPDFLSVTPGEKVTLSC RASQSISNYLHWYQQKSDQSPKLLIK YASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFC QQSKSDPRTFGGGTKLEIK SEQ ID NO: 74 anti- ADG41/huVH1/huVH2 HC-CDR1GYTMN SEQ ID NO: 75 anti- ADG41/huVH1/huVH2 HC-CDR2LINPYNGGTSYNQKFKG SEQ ID NO: 76 anti- ADG41/huVH1/huVH2 HC-CDR3GTMATGFAY SEQ ID NO: 77 anti- ADG41 LC-CDR1SASSSVSSIYLY SEQ ID NO: 78 anti- ADG41 LC-CDR2STSNLAS SEQ ID NO: 79 anti- ADG41 LC-CDR3HQWSSYPLT SEQ ID NO: 80 anti- ADG41 huVH3/huVH4 HC-CDR1GYTMN SEQ ID NO: 81 anti- ADG41 huVH3/huVH4 HC-CDR2LINPYNGGTSYAQKFQG SEQ ID NO: 82 anti- ADG41 huVH3/huVH4 HC-CDR3GTMATGFAY SEQ ID NO: 83 anti- ADG41 huVL1/huVL2 LC-CDR1RASSSVSSIYLY SEQ ID NO: 84 anti- ADG41 huVL1/huVL2 LC-CDR2STSNLAS SEQ ID NO: 85 anti- ADG41 huVL1/huVL2 LC-CDR3HQWSSYPLT SEQ ID NO: 86 anti- ADG41 huVL3/huVL4 LC-CDR1RASSSVSSIYLY SEQ ID NO: 87 anti- ADG41 huVL3/huVL4 LC-CDR2STSNRAT SEQ ID NO: 88 anti- ADG41 huVL3/huVL4 LC-CDR3HQWSSYPLT SEQ ID NO: 89 anti- ADG41 VH (CDR Sequence underlined )EVQLQQSGPELVKPGASMKISCKASGYSFT GYTMNWVKQSHGKNLEWIG LINPYNGGTSYNQKFKGKATLTVDKSSTTAYMELLSLLTSEDSAVYYCAS GTMATGFAYWGQGTLVTVSA SEQ ID NO: 90 anti- ADG41 huVH1 (CDR Sequence underlined )EVQLVQSGPEVVKPGASMKISCKASGYSFT GYTMNWVRQAPGQGLEWIG LINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCAS GTMATGFAYWGQGTLVTVSS SEQ ID NO: 91 anti- ADG41 huVH2 (CDR Sequence underlined )EVQLVQSGAEVKKPGASMKVSCKASGYSFT GYTMNWVRQAPGQGLEWMG LINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCAS GTMATGFAYWGQGTLVTVSS SEQ ID NO: 92 anti- ADG41 huVH3 (CDR Sequence underlined )QVQLVQSGAEVKKPGASMKVSCKASGYSFT GYTMNWVRQAPGQGLEWMG LINPYNGGTSYAQKFQGRVTLTVDKSTTTAYMELSSLTSEDTAVYYCAS GTMATGFAYWGQGTLVTVSS SEQ ID NO: 93 anti- ADG41 huVH4 (CDR Sequence underlined )QVQLVQSGAEVKKPGASVKVSCKASGYSFT GYTMNWVRQAPGQGLEWMG LINPYNGGTSYAQKFQGRVTMTVDKSTTTAYMELSSLTSEDTAVYYCAS GTMATGFAYWGQGTLVTVSS SEQ ID NO: 94 anti- ADG41 VL (CDR Sequence underlined )QIVLTQSPAIMSASPGEKVTLTC SASSSVSSIYLYWYQQKPGSSPKLWIY STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFC HQWSSYPLTFGAGTKLELK SEQ ID NO: 95 anti- ADG41 huVL1 (CDR Sequence underlined )QIVLTQSPGTLSLSPGERATLSC RASSSVSSIYLYWYQQKPGQSPRLWIY STSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFC HQWSSYPLTFGQGTKLEIK SEQ ID NO: 96 anti- ADG41 huVL2 (CDR Sequence underlined )QIVLTQSPGTLSLSPGERATLSC RASSSVSSIYLYWYQQKPGQSPRLWIY STSNLASGVPARFSGSGSGTDYTLTISSLEAEDAASYYC HQWSSYPLTFGQGTKLEIK SEQ ID NO: 97 anti- ADG41 huVL3 (CDR Sequence underlined )EIVLTQSPGTLSLSPGERATLSC RASSSVSSIYLYWYQQKPGQAPRLWIY STSNRATGVPDRFSGSGSGTDYTLTISSLEPEDAAVYYC HQWSSYPLTFGQGTKLEIK SEQ ID NO: 98 anti- ADG41 huVL4 (CDR Sequence underlined )EIVLTQSPGTLSLSPGERATLSC RASSSVSSIYLYWYQQKPGQAPRLWIY STSNRATGIPDRFSGSGSGTDFTLTISRLEPEDAAVYYC HQWSSYPLTFGQGTKLEIK SEQ ID NO: 99 anti- ADG41 scFv (CDR Sequence underlined )QIVLTQSPAIMSASPGEKVTLTC SASSSVSSIYLYWYQQKPGSSPKLWIY STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFC HQWSSYPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASMKISCKASGYSFT GYTMNWVKQSHGKNLEWIG LINPYNGGTSYNQKFKGKATLTVDKSSTTAYMELLSLLTSEDSAVYYCAS GTMATGFAYWGQGTLVTVSA SEQ ID NO: 100 anti- ADG41 VH-CH1 (CDR Sequence underlined )EVQLQQSGPELVKPGASMKISCKASGYSFT GYTMNWVKQSHGKNLEWIG LINPYNGGTSYNQKFKGKATLTVDKSSTTAYMELLSLLTSEDSAVYYCAS GTMATGFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC SEQ ID NO: 101 anti- ADG41 VL-CL (CDR Sequence underlined )QIVLTQSPAIMSASPGEKVTLTC SASSSVSSIYLYWYQQKPGSSPKLWIY STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFC HQWSSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 102 anti- CDH15 HC-CDR1SYDMS SEQ ID NO: 103 anti- CDH15 HC-CDR2SINRGGSYTFYPDSVKG SEQ ID NO: 104 anti- CDH15 HC-CDR3GDDGYFFSWFAY SEQ ID NO: 105 anti- CDH15 LC-CDR1RSSQSIVHSSGNTYLE SEQ ID NO: 106 anti- CDH15 LC-CDR2KVSNRFS SEQ ID NO: 107 anti- CDH15 LC-CDR3FQGSHVPFT SEQ ID NO: 108 anti- CDH15 VH (CDR Sequence underlined )EVKLVESGGGLVKPGGALRLSCAASGFGFS SYDMSWVRQTSEKRLEWVA SINRGGSYTFYPDSVKGRFTISRDNARNTLYLQMNSLRSEDSALYYCAR GDDGYFFSWFAYWGQGTLVTVSA SEQ ID NO: 109 anti- CDH15 VL (CDR Sequence underlined )DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSSGNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYC FQGSHVPFTFGSGTKLEIK SEQ ID NO: 110 anti- CDH15 scFv (CDR Sequence underlined )DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSSGNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYC FQGSHVPFTFGSGTKLEIKGGGGSGGGGSGGGGSGGGGSEVKLVESGGGLVKPGGALRLSCAASGFGFS SYDMSWVRQTSEKRLEWVA SINRGGSYTFYPDSVKGRFTISRDNARNTLYLQMNSLRSEDSALYYCAR GDDGYFFSWFAYWGQGTLVTVSA SEQ ID NO: 111 anti- CDH15 VH-CH1 (CDR Sequence underlined )EVKLVESGGGLVKPGGALRLSCAASGFGFS SYDMSWVRQTSEKRLEWVA SINRGGSYTFYPDSVKGRFTISRDNARNTLYLQMNSLRSEDSALYYCAR GDDGYFFSWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC SEQ ID NO: 112 anti- CDH15 VL-CL (CDR Sequence underlined )DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSSGNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYC FQGSHVPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 113 Human LAMA2 LG domainSVSSGGDCIRTYKPEIKKGSYNNIVVNVKTAVADNLLFYLGSAKFIDFLAIEMRKGKVSFLWDVGSGVGRVEYPDLTIDDSYWYRIVASRTGRNGTISVRALDGPKASIVPSTHHSTSPPGY TILDVDANAMLFVGGLTGKLKKADAVRVITFTGCMGETYFDNKPIGLWNFREKEGDCKGCTVSPQVEDSEGTIQFDGEGYALVSRPIRWYPNISTVMFKFRTFSSSALLMYLATRRDLRDFMS VELTDGHIKVSYDLGSGMASVVSNQNHNDGKWKSFTLSRIQKQANISIVDIDTNQEENIATSSGNNFGLDLKADDKIYFGGLPTLRNLSMKARPEVNLKKYSGCLKDIEISRTPYNILSSP DYVGVTKGCSLENVYTVSFPKPGFVELSPVPIDVGTEINLSFSTKNESGIILLGSGGTPAPPRRKRRQTGQAYYAILLNRGRLEVHLSTGARTMRKIVIRPEPNLFHDGREHSVHVERTRGI FTVQVDENRRYMQNLTVEQPIEVKKLFVGGAPPEFQPSPLRNIPPFEGCIWNLVINSVPMDFARPVSFKNADIGRCAHQKLREDEDGAAPAAEIVIQPEPVPTPAFPTPTPVLTHGPCAAESE PALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADI LDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFH VDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 114 Human LAMA1 LG domainYNTLTLNVKTQEPDNLLFYLGSSTASDFLAVEMRRRGRVAFLWDLGSGSTRLEFPDFPIDDNRWHSIHVARFGNIGSLSVKEMSSNQKSPTKTSKSPGTANVLDVNNSTLMFVGGLGGQIKKSPAVKVTHFKGCLGEAFLNGKSIGLWNYIEREGKCRGCFGSSQNEDPSFHFDGSGYSVVEKSLPATVTQIIMLFNTFSPNGL LLYLGSYGTKDFLSIELFRGRVKVMTDLGSGPITLLTDRRYNNNGTWYKIAFQRNRKQGVLAVIDAYNTSNKETKQGETPGASSDLNRLDKDPIYVGGLPRSRVVRRGVTTKSFVGCIKNLEISRSTFDLLRNSYGVRKGCLLEPIRSVSFLKGGYIELPPKSLSPESEWLVTFATTNSSGIILAALGGDVEKRGDREEAHVPFF SVMLIGGNIEVHVNPGDGTGLRKALLHAPTGTCSDGQAHSISLVRNRRIITVQLDENNPVEMKLGTLVESRTINVSNLYVGGIPEGEGTSLLTMRRSFHGCIKNLIFNLELLDFNSAVGHEQVDLDTCWLSERPKLAPDAEDSKLLPEPRAFPEQCVVDAALEYVPGAHQFGLTQNSHFILPFNQSAVRKKLSVELSIRTFASS GLIYYMAHQNQADYAVLQLHGGRLHFMFDLGKGRTKVSHPALLSDGKWHTVKTDYVKRKGFITVDGRESPMVTVVGDGTMLDVEGLFYLGGLPSQYQARKI GNITHSIPACIGDVTVNSKQLDKDSPVSAFTVNRCYAVAQEGTYFDGSGYAALVKEGYKVQSDVNITLEFRTSSQNGVLLGISTAKVDAIGLELVDGKVGAS SEQ ID NO: 115 Human LAMA3 LG domainLQTVIKEDLPRKAKTLSSNSDKLLNEAKMTQKKLKQEVSPALNNLQQTLNIVTVQKEVIDTNLTTLRDGLHGIQRGDIDAMISSAKSMVRKANDITDEVLDGLNPIQTDVERIKDTYGRTQNEDFKKALTDADNSVNKLTN KLPDLWRKIESINQQLLPLGNISDNMDRIRELIQQARDAASKVAVPMRFNGKSGVEVRLPNDLEDLKGYTSLSLFLQRPNSRENGGTENMFVMYLGNKDASRDYIGMAVVDGQLTCVYNLGDREAELQVDQILTKSETKEA VMDRVKFQRIYQFARLNYTKGATSSKPETPGVYDMDGRNSNTLLNLDPENVVFYVGGYPPDFKLPSRLSFPPYKGCIELDDLNENVLSLYNFKKTFNLNTTEVEPCRRRKEESDKNYFEGTGYARVPTQPHAPIPTFGQTI QTTVDRGLLFFAENGDRFISLNIEDGKLMVRYKLNSELPKERGVGDAINNGRDHSIQIKIGKLQKRMWINVDVQNTIIDGEVFDFSTYYLGGIPIAIRERFNISTPAFRGCMKNLKKTSGVVRLNDTVGVTKKCSEDWKLVR SASFSRGGQLSFTDLGLPPTDHLQASFGFQTFQPSGILLDHQTWTRNLQVTLEDGYIELSTSDSGGPIFKSPQTYMDGLLHYVSVISDNSGLRLLIDDQLLRNSKRLKHISSSRQSLRLGGSNFEGCISNVFVQRLSLSPE VLDLTSNSLKRDVSLGGCSLNKPPFLMLLKGSTRFNKTKTFRINQLLQDTPVASPRSVKVWQDACSPLPKTQANHGALQFGDIPTSHLLFKLPQELLKPRSQFAVDMQTTSSRGLVFHTGTKNSFMALYLSKGRLVFALGTD GKKLRIKSKEKCNDGKWHTVVFGHDGEKGRLVVDGLRAREGSLPGNSTISIRAPVYLGSPPSGKPKSLPTNSFVGCLKNFQLDSKPLYTPSSSFGVSSCLGGPLEKGIYFSEEGGHVVLAHSVLLGPEFKLVFSIRPRSLT GILIHIGSQPGKHLCVYLEAGKVTASMDSGAGGTSTSVTPKQSLCDGQWHSVAVTIKQHILHLELDTDSSYTAGQIPFPPASTQEPLHLGGAPANLTTLRIPVWKSFFGCLRNIHVNHIPVPVTEALEVQGPVSLNGCPDQ SEQ ID NO: 116 Human LAMA4 LG domainFTSLSLYMKPPVKRPELTETADQFILYLGSKNAKKEYMGLAIKNDNLVYVYNLGTKDVEIPLDSKPVSSWPAYFSIVKIERVGKHGKVFLTVPSLSSTAEEKFIKKGEFSGDDSLLDLDP EDTVFYVGGVPSNFKLPTSLNLPGFVGCLELATLNNDVISLYNFKHIYNMDPSTSVPCARDKLAFTQSRAASYFFDGSGYAVVRDITRRGKFGQVTRFDIEVRTPADNGLILLMVNGSMFF RLEMRNGYLHVFYDFGFSSGRVHLEDTLKKAQINDAKYHEISIIYHNDKKMILVVDRRHVKSMDNEKMKIPFTDIYIGGAPPEILQSRALRAHLPLDINFRGCMKGFQFQKKDFNLLEQTE TLGVGYGCPEDSLISRAYFNGQSFIASIQKISFFDGFEGGFNFRTLQPNGLLFYYASGSDVFSISLDNGTVIMDVKGIKVQSVDKQYNDGLSHFVISSVSPTRYELIVDKSRVGSKNPTK GKIEQTQASEKKFYFGGSPISAQYANFTGCISNAYFTRVDRDVEVEDFQRYTEKVHTSLYECPIESSPLFLLHKKGKNLSKPKASQNKKGGKSKDAPSWDPVALKLPERNTPRNSHCHLSN SPRAIEHAYQYGGTANSRQEFEHLKGDFGAKSQFSIRLRTRSSHGMIFYVSDQEENDFMTLFLAHGRLVYMFNVGHKKLKIRSQEKYNDGLWHDVIFIRERSSGRLVIDGLRVLEESLPPT EATWKIKGPIYLGGVAPGKAVKNVQINSIYSFSGCLSNLQLNGASITSASQTFSVTPCFEGPMETGTYFSTEGGYVVLDESFNIGLKFEIAFEVRPRSSSGTLVHGHSVNGEYLNVHMKN GQVIVKVNNGIRDFSTSVTPKQSLCDGRWHRITVIRDSNVVQLDVDSEVNHVVGPLNPKPIDHREPVFVGGVPESLLTPRLAPSKPFTGCIRHFVIDGHPVSFSKAALVSGAVSINSCPAA SEQ ID NO: 117 Human LAMA5 LG domainYTALKFYLQGPEPEPGQGTEDRFVMYMGSRQATGDYMGVSLRDKKVHWVYQLGEAGPAVLSIDEDIGEQFAAVSLDRTLQFGHMSVTVERQMIQETKGDTVAPGAEGLLNLRPDDFV FYVGGYPSTFTPPPLLRFPGYRGCIEMDTLNEEVVSLYNFERTFQLDTAVDRPCARSKSTGDPWLTDGSYLDGTGFARISFDSQISTTKRFEQELRLVSYSGVLFFLKQQSQFLCLA VQEGSLVLLYDFGAGLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVVRVERATVYSVEQDNDLELADAYYLGGVPPDQLPPSLRRLFPTGGSVRGCVKGIKALGKYVDLKRLNTT GVSAGCTADLLVGRAMTFHGHGFLRLALSNVAPLTGNVYSGFGFHSAQDSALLYYRASPDGLCQVSLQQGRVSLQLLRTEVKTQAGFADGAPHYVAFYSNATGVWLYVDDQLQQMKPH RGPPPELQPQPEGPPRLLLGGLPESGTIYNFSGCISNVFVQRLLGPQRVFDLQQNLGSVNVSTGCAPALQAQTPGLGPRGLQATARKASRRSRQPARHPACMLPPHLRTTRDSYQFG GSLSSHLEFVGILARHRNWPSLSMHVLPRSSRGLLLFTARLRPGSPSLALFLSNGHFVAQMEGLGTRLRAQSRQRSRPGRWHKVSVRWEKNRILLVTDGARAWSQEGPHRQHQGAEH PQPHTLFVGGLPASSHSSKLPVTVGFSGCVKRLRLHGRPLGAPTRMAGVTPCILGPLEAGLFFPGSGGVITLDLPGATLPDVGLELEVRPLAVTGLIFHLGQARTPPYLQLQVTEKQ VLLRADDGAGEFSTSVTRPSVLCDGQWHRLAVMKSGNVLRLEVDAQSNHTVGPLLAAAAGAPAPLYLGGLPEPMAVQPWPPAYCGCMRRRLAVNRSPVAMTRSVEVHGAVGASGCPAA SEQ ID NO: 118 mice LAMA2 domain 4-5 「 LG4-5 "VPTPAFPFPAPTMVHGPCVAESEPALLTGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGFPYFSYDLGSGDTSTMIPTKINDGQWHKIKIVRVKQEGILYVDDASSQTISPKKADILDVVGILYVGGLPINYTTRRIGPVTYSLDGCVRNLHMEQAPVDLDQPTS SFHVGTCFANAESGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTRPTGVLLGVSSQKMDGMGIEMIDEKLMFHVDNGAGRFTAIYDAGIPGHMCNGQ WHKVTAKKIKNRLELVVDGNQVDAQSPNSASTSADTNDPVFVGGFPGGLNQFGLTTNIRFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPTT SEQ ID NO: 119 Human LAMA2 domain 4-5 「 LG4-5 "IQPEPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQP TSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 120 Human pearl LG domainVKAFAHLQVPERVVPYFTQTPYSFLPLPTIKDAYRKFEIKITFRPDSADGMLLYNGQKRVPGSPTNLANRQPDFISFGLVGGRPEFRFDAGSGMATIRHPTPLALGHFHTVTLLRSLTQGSLIVGDLAPVNGTSQGKFQGLDLNEELYLGGYPDYGAIPKAGLSSGFIGCVRELRIQGEEIVFHD LNLTAHGISHCPTCRDPCQNGGQCHDSESSSYVCVCPAGFTGSRCEHSQALHCHPEACGPDATCVNRPDGRGYTCCRCHLGRSGLRCEEGVTVTTPSLSGAGSYLALPALTNTHHELRLDVEFKPLAPDGVLLFSGGKSGPVEDFVSLAMVGGHLEFRYELGSGLAVLRSAEPLALGRWHRVSAER LNKDGSLRVNGGRPVLRSSPGKSQGLNLHTLLYLGGVEPSVPLSPATNMSAHFRGCVGEVSVNGKRLDLTYSFLGSQGIGQCYDSSPCERQPCQHGATCMPAGEYEFQCLCRDGFKGDLCEHEENPCQLREPCLHGGTCQGTRCLCLPGFSGPRCQQGSGHGIAESDWHLEGSGGNDAPGQYGAY FHDDGFLAFPGHVFSRSLPEVPETIELEVRTSTASGLLLWQGVEVGEAGQGKDFISLGLQDGHLVFRYQLGSGEARLVSEDPINDGEWHRVTALREGRRGSIQVDGEELVSGRSPGPNVAVNAKGSVYIGGAPDVATLTGGRFSSGITGCVKNLVLHSARPGAPPPQPLDLQHRAQAGANTRPPCS SEQ ID NO: 121 Human aggrecan LG domainTLRLALEFRALEPQGLLLYNGNARGKDFLALALLDGRVQLRFDTGSGPAVLTSAVPVEPGQWHRLELSRHWRRGTLSVDGETPVLGESPSGTTDGLNLDTDLFVGGVPEDQAAVALERTFVGAGLRGCIRLLDVNNQRLELGIGPGAATRGSGVGECGDHPCLPNPCHGG APCQNLEAGRFHCQCPPGRVGPTCADEKSPCQPNPCHGAAPCRVLPEGGAQCECPLGREGTFCQTASGQDGSGPFLADFNGFSHLELRGLHTFARDLGEKMALEVVFLARGPSGLLLYNGQKTDGKGDFVSLALRDRRLEFRYDLGKGAAVIRSREPVTLGAWTRVSLE RNGRKGALRVGDGPRVLGESPKSRKVPHTVLNLKEPLYVGGAPDFSKLARAAAVSSGFDGAIQLVSLGGRQLLTPEHVLRQVDVTSFAGHPCTRASGHPCLNGASCVPREAAYVCLCPGGFSGPHCEKGLVEKSAGDVDTLAFDGRTFVEYLNAVTESELANEIPVPET LDSGALHSEKALQSNHFELSLRTEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVVGRHPLHLLEDAVTKPELRPCPTP SEQ ID NO: 122 Human agrin laminin coiled-coil binding domainCPERALERREEEANVVLTGTVEEILNVDPVQHTYSCKVRVWRYLKGKDLVARESLLDGGNKVVISGFGDPLICDNQVSTGDTRIFFVNPAPPYLWPAHKNELMLNSSLMRITLRN LEEVEFCVEDKPGTHFTPVPPTPPDACRGMLCGFGAVCEPNAEGPGRASCVCKKSPCPSVVAPVCGSDASTYSNECELQRAQCSQQRRIRLLSRGPCGSRDPCSNVTCSFGSTCAR SEQ ID NO: 123 Human nestin - 1 Laminin γ binding domainCANNRHQCSVHAECRDYATGFCCSCVAGYTNGGRQCVAEGSPQRVNGKVKGRIFVGSSQVPIVFENTDLHSYVVMNHGRSYTAISTIPETVGYSLLPLAPVGGIIGW MFAVEQDGFKNGFSITGGEFTRQAEVTFVGHPGNLVIKQRFSGIDEHGHLTIDTELEGRVPQIPFGSSVHIEPYTELYHYSTSVITSSSTREYTVTEPERDGASPSR IYTYQWRQTITFQECVHDDSRPALPSTQQLSVDSVFVLYNQEEKILRYALSNSIGPVREGSPDALQNPCYIGTHGCDTNAACRPGPRTQFTCECSIGFRGDGRTCYD IDECSEQPSVCGSHTICNNHPGTFRCECVEGYQFSDEGTCVAVVDQRPINYCETGLHNCDIPQRAQCIYTGGSSYTCSCLPGFSGDGQACQDVDECQPSRCHPDAFCY NTPGSFTCQCKPGYQGDGFRCVPGEVEKTRCQHEREHILGAAGATDPQRPIPGLFVPECDAHGHYAPTQCHGSTGYCWCVDRDGREVEGTRTRPGMTPPCLSTVAP PIHQGPAVPTAVIPLPPGTHLLFAQTGKIERLPLEGNTMRKTEAKAFLHVPAKVIIGLAFDCVDKMVYWTDITEPSIGRASLHGGEPTTIIRQDLGSPEGIAVDHLGR NIFWTDSNLDRIEVAKLDGTQRRVLFETDLVNPRGIVTDSVRGNLYWTDWNRDNPKIETSYMDGTNRRILVQDDLGLPNGLTFDAFSSQLCWVDAGTNRAECLNPSQ PSRRKALEGLQYPFAVTSYGKNLYFTDWKMNSVVALDLAISKETDAFQPHKQTRLYGITTALSQCPQGHNYCSVNNGGCTHLCLATPGSRTCRCPDNTLGVDCIEQK SEQ ID NO: 124 Human nestin - 2 Laminin γ binding domainCEHNHRQCSRHAFCTDYATGFCCHCQSKFYGNGKHCLPEGAPHRVNGKVSGHLHVGHTPVHFTDVDLHAYIVGNDGRAYTAISHIPQPAAQALLPLTPIGGLFGWLFALEK PGSENGFSLAGAAFTHDMEVTFYPGEETVRITQTAEGLDPENYLSIKTNIQGQVPYVSANFTAHISPYKELYHYSDSTVTSTSSRDYSLTFGAINQTWSYRIHQNITYQVC RHAPRHPSFPTTQQLNVDRVFALYNDEERVLRFAVTNQIGPVKEDSDPTPGNPCYDGSHCMCDTTARCHPGTGVDYTCECASGYQGDGRNCVDENECATGFHRCGPNSVCIN LPGSYRCECRSGYEFADDRHTCILITPPANPCEDGSHTCAPAGQARCVHHGGSTFSCACLPGYAGDGHQCTDVDECSENRCHPAATCYNTPGSFSCRCQPGYYGDGFQCIP DSTSSLTPCEQQQRHAQAQYAYPGARFHIPQCDEQGNFLPLQCHGSTGFCWCVDPDGHEVPGTQTPPGSTPPHCGPSPEPTQRPPTICERWRENLLEHYGGTPRDDQYVPQ CDDLGHFIPLQCHGKSDFCWCVDKDGREVQGTRSQPGTTPACIPTVAPPMVRPTPRPDVTPPSVGTFLLYTQGQQIGYLPLNGTRLQKDAAKTLLSLHGSIIVGIDYDCRE RMVYWTDVAGRTISRAGLELGAEPETIVNSGLISPEGLAIDHIRRTMYWTDSVLDKIESALLDGSERKVLFYTDLVNPRAIAVDPIRGNLYWTDWNREAPKIETSSLDGEN RRILINTDIGLPNGLTFDPFSKLLCWADAGTKKLECTLPDGTGRRVIQNNLKYPFSIVSYADHFYHTDWRRDGVVSVNKHSGQFTDEYLPEQRSHLYGITAVYPYCPTGRK SEQ ID NO: 125 DLL4wt 27-529 ECDSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVG QNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGG LFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKV DRCTSPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPSFPW SEQ ID NO: 126 exist EGF5 ECD Of N End DLL4wtSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 127 exist EGF5 ECD Of N End DLL4v1 (G2S 、 F81L 、 I117F 、 H168Y 、 L180P)SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 128 exist EGF5 ECD Of N End DLL4max (G2S 、 F81L 、 I117F 、 H168Y 、 L180P 、 N231P 、 T245L 、 F254Y 、 S275R 、 Q279P)SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATTCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 129 DLL4v1_derisk ECD (G2S 、 F81L 、 H168Y 、 L180P)SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 130 DLL1wt 18-545 ECDQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLAT QRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWG GLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSP CSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPW SEQ ID NO: 131 DLL3wt 27-492 ECDAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSL LARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLS PRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYL SEQ ID NO: 132 Jag1wt 31-1067 ECDASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSG MINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWG GQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAK PCVNAKSCKNLIASYYCDCLPGWMGQNCDININDCLGQCQNDASCRDLVNGYRCICPPGYAGDHCERDIDECASNPCLNGGHCQNEINRFQCLCPTGFSGNLCQLDIDYCEPNPCQNGAQCYNRASDYFC KCPEDYEGKNCSHLKDHCRTTPCEVIDSCTVAMASNDTPEGVRYISSNVCGPHGKCKSQSGGKFTCDCNKGFTGTYCHENINDCESNPCRNGGTCIDGVNSYKCICSDGWEGAYCETNINDCSQNPCHN GGTCRDLVNDFYCDCKNGWKGKTCHSRDSQCDEATCNNGGTCYDEGDAFKCMCPGGWEGTTCNIARNSSCLPNPCHNGGTCVVNGESFTCVCKEGWEGPICAQNTNDCSPHPCYNSGTCVDGDNWYRCEC APGFAGPDCRININECQSSPCAFGATCVDEINGYRCVCPPGHSGAKCQEVSGRPCITMGSVIPDGAKWDDDCNTCQCLNGRIACSKVWCGPRPCLLHKGHSECPSGQSCIPILDDQCFVHPCTGVGECR SSSLQPVKTKCTSDSYYQDNCANITFTFNKEMMSPGLTTEHICSELRNLNILKNVSAEYSIYIACEPSPSANNEIHVAISAEDIRDDGNPIKEITDKIIDLVSKRDGNSSLIAAVAEVRVQRRPLKNRTD SEQ ID NO: 133 exist EGF6 ECD Of N End Jag1wtASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTL LVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCS PKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSD PCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCD SEQ ID NO: 134 exist EGF6 ECD Of N End Jag1v1ALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTL LVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCS PKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSD PCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCD SEQ ID NO: 135 Jag2wt 27-1080 ECDMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAAGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTP NEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVE PWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQ LDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYN LEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCLPDPCHS RGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAP GFAGPDCRINIDECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDSCKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGE CGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGTTVGAICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSST SEQ ID NO: 136 DLL4wt-LG4-5SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPIC LSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSIQP EPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPT SSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 137 DLL4v-LG4-5SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPIC LSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSIQP EPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPT SSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 138 DLL4max-LG4-5SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPIC LSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSIQP EPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPT SSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 139 DLL4deimmune (DLL4di)-LG4-5SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPIC LSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSIQP EPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPT SSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 140 DLL1wt-LG4-5QVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDD LATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQG RYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAM TCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCE HAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWGGGGSGGGGSGGGGSIQPEPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRN SHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVG GLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGA GRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 141 DLL3wt-LG4-5AGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTG PLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARC EFPVHPDGASALPAAPPGLRPGDPQRYLGGGGSGGGGGSGGGGSIQPEPVPTPAFPTPPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVR TEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTY SIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYD AGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 142 Jag1wt-LG4-5ASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQG CSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHAC LSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMG QNCDGGGGSGGGGSGGGGSIQPEPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHAD FATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHM AEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPG HLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 143 Jag1v-LG4-5ALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQG CSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHAC LSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMG QNCDGGGGSGGGGSGGGGSIQPEPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHAD FATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHM AEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPG HLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 144 Jag2wt-LG4-5MGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNK FCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASN PCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPR EPCPGGACRVIDGCGSDAGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCLPDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPN PCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRP PCEAWGECGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGTTVGAICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTGGGGSGGGGSGGGGSIQPEPVPTPAFPTPTPVLTHGPCAAESEP ALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQ RGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 145 LG21 scFvEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 146 DLL4wt-LG21scFvSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDN CSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKD QEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDA ATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 147 DLL4v-LG21 scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDN CSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKD QEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDA ATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 148 DLL4max-LG21scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDN CSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKD QEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDA ATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 149 DLL4di-LG21scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDN CSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKD QEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDA ATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 150 DLL1wt-LG21scFvQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCN PGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYC SSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSP KLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 151 DLL3wt-LG21scFvAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRP CAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPR CEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPS RFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 152 Jag1wt-LG21scFvASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNK FCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWT GPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFT LSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 153 Jag1v-LG21scFvALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNK FCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWT GPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFT LSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 154 Jag2wt-LG21scFvMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAH LELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTS NPCANGGSCHEVPSGFECHPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLA DGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCLPD PCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVD EINGYRCSCPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGTTVGAICSGIRSLPATRAVARDR LLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSIN SVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 155 ADG41 scFvQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGG GGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 156 DLL4wt-ADG41scFvSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDN CSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKD QEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAED AASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 157 DLL4v-ADG41scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDN CSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKD QEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAED AASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 158 DLL4max-ADG41scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDN CSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKD QEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAED AASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 159 DLL4di-ADG41scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDN CSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKD QEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAED AASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 160 DLL1wt-ADG41scFvQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCN PGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYC SSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSP RLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 161 DLL3wt-ADG41scFvAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRP CAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPR CEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVP ARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 162 Jag1wt-ADG41scFvASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNK FCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWT GPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDY SLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 163 Jag1v-ADG41scFvALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNK FCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWT GPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDY SLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 164 Jag2wt-ADG41scFvMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAH LELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTS NPCANGGSCHEVPSGFECHPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLA DGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCLPD PCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVDE INGYRCSCPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGTTVGAICSGIRSLPATRAVARDRL LVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTIS SMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 165 LG21-VH-CH1EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQ GTTLTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 166 LG21-VL-CLEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 167 DLL4wt-LG21-FabSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSD NYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELLSEC DSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFS GSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 168 DLL4v-LG21-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSD NYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSEC DSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFS GSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 169 DLL4max-LG21-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSD NYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSEC DSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFS GSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 170 DLL4di-LG21-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSD NYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSEC DSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFS GSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 171 DLL1wt-LG21-FabQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGE RGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVG YSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRA SQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 172 DLL3wt-LG21-FabAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRC GPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHA LRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKS DQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 173 Jag1wt-LG21-FabASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYY GFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKET SLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYAS QSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 174 Jag1v-LG21-FabALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYY GFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKET SLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYAS QSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 175 Jag2wt-LG21-FabMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGH VAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKA EHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPC HSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCCFCPSGWEG ELCDTNPNDCLPDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINI DECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGTTVG AICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQS ISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 176 ADG41-VH-CH1EVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID NO: 177 LG21-VL-CLQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 178 DLL4wt-ADG41-FabSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSD NYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELLSEC DSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFS GSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 179 DLL4v-ADG41-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSD NYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSEC DSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFS GSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 180 DLL4max-ADG41-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSD NYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSEC DSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFS GSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 181 DLL4di-ADG41-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSD NYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSEC DSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFS GSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 182 DLL1wt-ADG41-FabQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGE RGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVG YSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRA SSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 183 DLL3wt-ADG41-FabAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRC GPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHA LRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKP GQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 184 Jag1wt-ADG41-FabASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYY GFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKET SLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTS NLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 185 Jag1v-ADG41-FabALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYY GFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKET SLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTS NLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 186 Jag2wt-ADG41-FabMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGH VAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKA EHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPC HSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCCFCPSGWEG ELCDTNPNDCLPDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINI DECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGTTVG AICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSN LASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 187 Human wild type IgG1DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 188 Human IgG1 Fc LALA + H435ADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK SEQ ID NO: 189 DLL4wt-Fc-LG21-FabSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDL RPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYC SKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQS PDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 190 DLL4v-Fc-LG21-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDL RPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYC SKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQS PDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 191 DLL4max-Fc-LG21-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDL RPEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYC SKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATTCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQS PDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 192 DLL4di-Fc-LG21-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDL RPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYC SKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQS PDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 193 DLL1wt-Fc-LG21-FabQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENP ERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGC LHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPD GGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECAR GYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQS KSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 194 DLL3wt-Fc-LG21-FabAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSS CLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGD PQRYLDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG KGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFG GGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 195 Jag1wt-Fc-LG21-FabASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAW DSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLP GDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLG FECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSG GGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 196 Jag1v-Fc-LG21-FabALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAW DSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLP GDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLG FECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSG GGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 197 Jag2wt-Fc-LG21-FabMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARAAGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPR NDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQ VDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSD AGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCLPDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGW EGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDCSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNC ARLTLHFNRDHVPQGTTVGAICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTDKTHTCPPCPAPEAAGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRA SQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 198 DLL4wt-Fc-ADG41-FabSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLR PEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCS KPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSP GTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 199 DLL4v-Fc-ADG41-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLR PEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCS KPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSP GTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 200 DLL4max-Fc-ADG41-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLR PEALPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCS KPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSP GTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 201 DLL4di-Fc-ADG41-FabSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLR PEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCS KPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSP GTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 202 DLL1wt-Fc-ADG41-FabQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENP ERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGC LHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPD GGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARG YGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQW SSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 203 DLL3wt-Fc-ADG41-FabAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWS LLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSC LSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDP QRYLDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK GGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 204 Jag1wt-Fc-ADG41-FabASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAW DSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPG DCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGF ECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGG GGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSILYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 205 Jag1v-Fc-ADG41-FabALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAW DSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPG DCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGF ECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGG GGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSILYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 206 Jag2wt-Fc-ADG41-FabMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARAAGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRN DFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQV DGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDA GPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCLPDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWE GRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDCSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNCA RLTLHFNRDHVPQGTTVGAICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRAS SSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 207 connector(GS) _n(n is an integer at least 1)SEQ ID NO: 208 connector(GGGS) _n(n is an integer at least 1)SEQ ID NO: 209 connector(GGGGS) _n(n is an integer at least 1)SEQ ID NO: 210 connector(GSGGS) _n(n is an integer at least 1)SEQ ID NO: 211 connectorGGGGSGGGGSGGGGS SEQ ID NO: 212 connectorGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 213 connectorGGGGS SEQ ID NO: 214 connectorGGGGGGGG SEQ ID NO: 215 connectorGGGGGG SEQ ID NO: 216 connectorEAAAKEAAAKEAAAK SEQ ID NO: 217 connector(EAAAK) _n(n is an integer at least 1)SEQ ID NO: 218 connectorAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA SEQ ID NO: 219 connectorAEAAAKEAAAKA SEQ ID NO: 220 connectorPAPAP SEQ ID NO: 221 connector(AP) _n(n is an integer at least 5) SEQ ID NO: 222 connectorLE SEQ ID NO: 223 DLL4wt-Fc-LG21scFvSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 224 DLL4v-Fc-LG21scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 225 DLL4max-Fc-LG21scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 226 DLL4di-Fc-LG21scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 227 DLL1wt-Fc-LG21scFvQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERL ISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQ QPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPV GYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPE LPPGPAVVDLTEKLEGQGGPFPWDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGG GSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGG GGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 228 DLL3wt-Fc-LG21scFvAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLA RVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGP SSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIV LTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 229 Jag1wt-Fc-LG21scFvASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSS NDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQY GWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWT GPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTP KEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLV ESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 230 Jag1v-Fc-LG21scFvALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSS NDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQY GWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWT GPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTP KEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLV ESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 231 Jag2wt-Fc-LG21scFvMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFF GHYTCDQYGNKACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECI CPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAA SGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCLPDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCN PLPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDCSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGT TVGAICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLI KYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 232 DLL4wt-Fc-ADG41scFvSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASS SVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEV VKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 233 DLL4v-Fc-ADG41scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASS SVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEV VKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 234 DLL4max-Fc-ADG41scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASS SVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEV VKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 235 DLL4di-Fc-ADG41scFvSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASS SVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPEV VKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 236 DLL1wt-Fc-ADG41scFvQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERL ISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQ PWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVG YSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPEL PPGPAVVDLTEKLEGQGGPFPWDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGG SGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGG GGSGGGGSEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 237 DLL3wt-Fc-ADG41scFvAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLA RVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGP SSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAG FAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVL TQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGG SEVQLVQSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 238 Jag1wt-Fc-ADG41scFvASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSS NDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQY GWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWTG PTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPG ERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLV QSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 239 Jag1v-Fc-ADG41scFvALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSS NDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQY GWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWTG PTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPG ERATLSCRASSSVSSIYLYWYQQKPGQSPRLWIYSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLV QSGPEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 240 Jag2wt-Fc-ADG41scFvMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFF GHYTCDQYGNKACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECI CPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAA SGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCLPDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCNP LPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRSCWSRGTPFPHGSSWVEDCNSCRCLDGRRDCSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGTT VGAICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSQIVLTQSPGTLSLSPGERATLSCRASSSVSSIYLYWYQQKPGQSPRLWI YSTSNLASGVPARFSGSGSGTDYSLTISSMEAEDAASYFCHQWSSYPLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGPGEVVKPGASMKISCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNGGTSYNQKFKGRATLTVDKSTTTAYMELSSLTSEDTAVYYCASGTMATGFAYWGQGTLVTVSS SEQ ID NO: 241 DLL4wt-Fc-LG4-5SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVG QNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGG LFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSIQPEPVPTPAFPTPT PVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRT ISPKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEK LMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 242 DLL4v-Fc-LG4-5SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVG QNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGG LFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSIQPEPVPTPAFPTPT PVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRT ISPKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEK LMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 243 DLL4max-Fc-LG4-5SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSLAVG QNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTPWQCLCDEGWGG LYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSIQPEPVPTPAFPTPT PVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRT ISPKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEK LMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 244 DLL4di-Fc-LG4-5SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVG QNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGG LFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSIQPEPVPTPAFPTPT PVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRT ISPKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEK LMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 245 DLL1wt-Fc-LG4-5QVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQD LHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTG QGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCA NGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSIQPEPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGS GDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGV LLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 246 DLL3wt-Fc-LG4-5AGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQ RAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCP RGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLLCDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCA HGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSL SPGKGGGGSGGGGSGGGGSIQPEPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQW HKIKIMRSKQEGILYVDGASNRTISPKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQK MDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 247 Jag1wt-Fc-LG4-5ASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMI NPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLC DKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAK SCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGG SIQPEPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEG ILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGI EMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 248 Jag1v-Fc-LG4-5ALGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTGDECNTYFKVCLKEYQSRVRAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMI NPSRQWQTLKQNTGVAHFEYRIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLC DKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAK SCKNLIASYYCDCLPGWMGQNCDDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGG SIQPEPVPTPAFPTPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEG ILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGI EMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 249 Jag2wt-Fc-LG4-5MGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVV IPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMG KECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGR NCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCD CIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELERDECASSPCHSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFR CFCPSGWEGELCDTNPNDCLPDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGT CVGSGASFSCICRDGWEGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQEVIGFGRS CWSRGTPFPHGSSWVEDCNSCRCLDGRRDSKVWCGWKPCLLAGQPEALSAQCPLGQRCLEKAPGQCLRPPCEAWGECGAEEPPSTPCLPRSGHLDNNCARLTLHFNRDHVPQGTTVGAICSGIRSLPATRAVARDRLLVLLCDRASSGASAVEVAVSFSPARDLPDSSLIQGAAHAIVAAITQRGNSSLLLAVTEVKVETVVTGGSSTDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN AYTQKSLSLSPGKGGGGSGGGGSGGGGSIQPEPVPTPAFPTPPTPVLTHGPCAAESEPALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYM ARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVR NLHMEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGV PGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 250 mice DLL4wt-Fc-LG4-5SGIFQLRLQEFVNQRGMLANGQSCEPGCRTFFRICLKHFQATFSEGPCTFGNVSTPVLGTNSFVVRDKNSGSGRNPLQLPFNFTWPGTFSLNIQAWHTPGDDLRPETSPGNSLISQIIIQGSLAV GKIWRTDEQNDTLTRLSYSYRVICSDNYYGESCSRLCKKRDDHFGHYECQPDGSLSCLPGWTGKYCDQPICLSGCHEQNGYCSKPDECICRPGWQGRLCNECIPHNGCRHGTCSIPWQCACDEGWG GLFCDQDLNYCTHHSPCKNGSTCSNSGPKGYTCTCLPGYTGEHCELGLSKCASNPCRNGGSCKDQENSYHCLCPPGYYGQHCEHSTLTCADSPCFNGGSCRERNQGSSYACECPPNFTGSNCEVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVY TIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSGGGGSGGGGSVPTPAFPFPAPT MVHGPCVAESEPALLTGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGFPYFSYDLGSGDTSTMIPTKINDGQWHKIKIVRVKQEGILYVDDASSQTI SPKADILDVVGILYVGGLPINYTTRRIGPVTYSLDGCVRNLHMEQAPVDLDQPTSSFHVGTCFANAESGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTRPTGVLLGVSSQKMDGMGIEMIDEK LMFHVDNGAGRFTAIYDAGIPGHMCNGQWHKVTAKKIKNRLELVVDGNQVDAQSPNSASTSADTNDPVFVGGFPGGLNQFGLTTNIRFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPTT SEQ ID NO: 251 mice DLL4v1-Fc-LG4-5SSIFQLRLQEFVNQRGMLANGQSCEPGCRTFFRICLKHFQATFSEGPCTFGNVSTPVLGTNSFVVRDKNSGSGRNPLQLPLNFTWPGTFSLIIQAWHTPGDDLRPETSPGNSLISQFIIQGSLAV GKIWRTDEQNDTLTRLSYSYRVICSDNYYGESCSRLCKKRDDYFGHYECQPDGSPSCLPGWTGEYCDQPICLSGCHEQNGYCSKPDECICRPGWQGRLCNECIPHNGCRHGTCSIPWQCACDEGWG GLFCDQDLNYCTHHSPCKNGSTCSNSGPKGYTCTCLPGYTGEHCELGLSKCASNPCRNGGSCKDQENSYHCLCPPGYYGQHCEHSTLTCADSPCFNGGSCRERNQGSSYACECPPNFTGSNCEVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVY TIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSGGGGSGGGGSVPTPAFPFPAPT MVHGPCVAESEPALLTGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGFPYFSYDLGSGDTSTMIPTKINDGQWHKIKIVRVKQEGILYVDDASSQTI SPKADILDVVGILYVGGLPINYTTRRIGPVTYSLDGCVRNLHMEQAPVDLDQPTSSFHVGTCFANAESGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTRPTGVLLGVSSQKMDGMGIEMIDEK LMFHVDNGAGRFTAIYDAGIPGHMCNGQWHKVTAKKIKNRLELVVDGNQVDAQSPNSASTSADTNDPVFVGGFPGGLNQFGLTTNIRFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPTT SEQ ID NO: 252 mice DLL4v1-Fc-LG03scFvSSIFQLRLQEFVNQRGMLANGQSCEPGCRTFFRICLKHFQATFSEGPCTFGNVSTPVLGTNSFVVRDKNSGSGRNPLQLPLNFTWPGTFSLIIQAWHTPGDDLRPET SPGNSLISQFIIQGSLAVGKIWRTDEQNDTLTRLSYSYRVICSDNYYGESCSRLCKKRDDYFGHYECQPDGSPSCLPGWTGEYCDQPICLSGCHEQNGYCSKPDECIC RPGWQGRLCNECIPHNGCRHGTCSIPWQCACDEGWGGLFCDQDLNYCTHHSPCKNGSTCSNSGPKGYTCTCLPGYTGEHCELGLSKCASNPCRNGGSCKDQENSYHCL CPPGYYGQHCEHSTLTCADSPCFNGGSCRERNQGSSYACECPPNFTGSNCEVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSW FVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWN GQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGGGGSGGGGSGGGGSDIVLTQSPATLSVTPGDSVSLSCRASQSIS NNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSKNWPRTTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSDVMLVESGGD LVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVASISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 253 signal peptideMHSSALLCCLVLLTGVRA SEQ ID NO: 254 mice FcICTVPEVSSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 255 Human IgG1 Fc Second Unit , in X ₂ Department Asp or GluDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSX ₂LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 256 Fc Human IgG1 Fc + LALA , in X ₃ Department Arg 、 His or LysDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLX ₃SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 257 Human IgG1 Fc + LALA , in X ₄ Department Asp or GluDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSX ₄LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 258 Human IgG1 Fc + LALA + H435A , in X ₅ Department Arg 、 His or LysDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLX ₅SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK SEQ ID NO: 259 Human IgG1 Fc + LALA + H435A , in X ₆ Department Asp or GluDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSX ₆LTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK SEQ ID NO: 260 Human DLL4v_G2S_F81L_H168YSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 261 Human DLL4v11_G2S_T52N_F81L_T135N_H168YSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGNVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQNSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 262 Human DLL4v12_G2S_R66S_F81L_H168YSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVSDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 263 Human DLL4v13_G2S_E14H_T52N_R66T_P76L_ F81L_T135N_H168Y_N231DSSVFQLQLQEFINHRGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGNVSTPVLGTNSFAVTDDSSGGGRNLLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQNSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHDGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 264 Human DLL4v14_G2S_T52N_R66T_P76L_F81L_ T135N_H168Y_Q220H_N260DSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGNVSTPVLGTNSFAVTDDSSGGGRNLLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQNSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWHGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLDYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 265 Human DLL4v22_G2S_ R66S_F81L_H168YSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVSDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 266 Human DLL4v23_G2S_E14H_R66T_P76L_ F81L_H168Y_N231DSSVFQLQLQEFINHRGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVTDDSSGGGRNLLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHDGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 267 Human DLL4v24_G2S_R66T_P76L_F81L_H168Y _Q220H_N260DSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVTDDSSGGGRNLLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWT GEYCQQPICLSGCHEQNGYCSKPAECLCRPGWHGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLDYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE SEQ ID NO: 268 anti- LAMA2_D178S scFvEIVLTQSPDFLSVTPKEKVTLSCRASQSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 269 huD4v_G2S_F81L_H168Y-Fc(AAG)-LG21scFvDSSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 270 huD4v11_G2S_T52N_F81L_T135N_H168Y-Fc(AAG)-LG21scFvDSSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGNVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQNSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 271 huD4v12_G2S_R66S_F81L_H168Y-Fc(AAG)-LG21scFvDSSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGNVSTPVLGTNSFAVSDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQNSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 272 huD4v13_G2S_E14H_T52N_R66T_P76L_F81L_T135N_H168Y_N231D-Fc(AAG)-LG21scFvDSSSVFQLQLQEFINHRGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGNVSTPVLGTNSFAVTDDSSGGGRNLLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQNSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHDGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 273 huD4v14_G2S_T52N_R66T_P76L_F81L_T135N_H168Y_Q220H_N260D-Fc(AAG)-LG21scFvDSSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGNVSTPVLGTNSFAVTDDSSGGGRNLLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQNSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWHGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLDYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 274 huD4v22_G2S_ R66S_F81L_H168Y-Fc(AAG)-LG21scFvDSSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVSDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 275 huD4v23_G2S_E14H_R66T_P76L_F81L_H168Y_N231D-FcAAG-LG21DSSSVFQLQLQEFINHRGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVTDDSSGGGRNLLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHDGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 276 huD4v24_G2S_R66T_P76L_F81L_H168Y_Q220H_N260D-FcAAG-LG21DSSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVTDDSSGGGRNLLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWHGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLDYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGSNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS SEQ ID NO: 277 Human IgG1 Fc LALA + P329GDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 278 Jagged1 MNNL (C2)KVCGASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQ SEQ ID NO: 279 Jagged1 DSLWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGREC SEQ ID NO: 280 Jagged1 EGF Repeated sequence 1-3NRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCE SEQ ID NO: 281 Jagged1 EGF Repeated sequence 4-6IAEHACLSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCD SEQ ID NO: 282 Jagged1 EGF Repeated sequence 7-16ININDCLGQCQNDASCRDLVNGYRCICPPGYAGDHCERDIDECASNPCLDGGHCQNEINRFQCLCPTGFSGNLCQLDIDYCEPNPCQNGAQCYNRASDYFCKCPEDYEGKNCSHLKDHCRTTPCEVIDSCTVAMASNDTPEGVRYISSNVCGPHGKCKSQSGGKFTCDCNKGFTGTYCHENINDCESNPCRNGGTCIDGVNSYK CICSDGWEGAYCETNINDCSQNPCHNGGTCRDLVNDFYCDCKNGWKGKTCHSRDSQCDEATCNNGGTCYDEGDAFKCMCPGGWEGTTCNIARNSSCLPNPCHNGGTCVVNGESFTCVCKEGWEGPICAQNTNDCSPHPCYNSGTCVDGDNWYRCECAPGFAGPDCRININECQSSPCAFGATCVDEINGYRCVCPPGHSGAKCQ SEQ ID NO: 283 Jagged2 MNNL (C2)VQAARPMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDR SEQ ID NO: 284 Jagged2 DSLWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVH SEQ ID NO: 285 Jagged2 EGF Repeated sequence 1-3GSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCA SEQ ID NO: 286 Jagged2 EGF Repeated sequence 4-6LDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCEL SEQ ID NO: 287 Jagged2 EGF Repeated sequence 7-15LERDECASSPCHSGGLCEDLADGFHCHCPQGFSGPLCEVDVDLCEPSPCRNGARCYNLEGDYYCACPDDFGGKNCSVPREPCPGGACRVIDGCGSDAGPGMPGTAASGVCGPHGRCVSQPGGNFSCICDSGFTGTYCHENIDDCLGQPCRNGGTCIDEVDAFRCFCPSGWEGELCDTNPNDCL PDPCHSRGRCYDLVNDFYCACDDGWKGKTCHSREFQCDAYTCSNGGTCYDSGDTFRCACPPGWKGSTCAVAKNSSCLPNPCVNGGTCVGSGASFSCICRDGWEGRTCTHNTNDCNPLPCYNGGICVDGVNWFRCECAPGFAGPDCRINIDECQSSPCAYGATCVDEINGYRCSCPPGRAGPRCQ SEQ ID NO: 288 DLL1 MNNL (C2)SGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEE SEQ ID NO: 289 DLL1 DSLWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEP SEQ ID NO: 290 DLL1 EGF Repeated sequence 1-3ICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCE SEQ ID NO: 291 DLL1 EGF Repeated sequence 4-6LGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCD SEQ ID NO: 292 DLL1 EGF Repeated sequence 7-8DNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQ SEQ ID NO: 293 DLL4 MNNL (C2)SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQN SEQ ID NO: 294 DLL4 DSLWLLDEQTSTLTLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYC SEQ ID NO: 295 DLL4 EGF Repeated sequence 1-3QQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCE SEQ ID NO: 296 DLL4 EGF Repeated sequence 4-6LELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCE SEQ ID NO: 297 DLL4 EGF Repeated sequence 7-8LHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCE SEQ ID NO: 298 DLL3 MNNL (C2) and DSLSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLED SEQ ID NO: 299 DLL3 EGF Repeated sequence 1-3ECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCE SEQ ID NO: 300 DLL3 EGF Repeated sequence 4-6KRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCE SEQ ID NO: 301 Human laminin - α - 2 LG domain 1 - 5SVSSGGDCIRTYKPEIKKGSYNNIVVNVKTAVADNLLFYLGSAKFIDFLAIEMRKGKVSFLWDVGSGVGRVEYPDLTIDDSYWYRIVASRTGRNGTISVRALDGPKASIVPSTHHSTSPPGY TILDVDANAMLFVGGLTGKLKKADAVRVITFTGCMGETYFDNKPIGLWNFREKEGDCKGCTVSPQVEDSEGTIQFDGEGYALVSRPIRWYPNISTVMFKFRTFSSSALLMYLATRRDLRDFMS VELTDGHIKVSYDLGSGMASVVSNQNHNDGKWKSFTLSRIQKQANISIVDIDTNQEENIATSSGNNFGLDLKADDKIYFGGLPTLRNLSMKARPEVNLKKYSGCLKDIEISRTPYNILSSP DYVGVTKGCSLENVYTVSFPKPGFVELSPVPIDVGTEINLSFSTKNESGIILLGSGGTPAPPRRKRRQTGQAYYAILLNRGRLEVHLSTGARTMRKIVIRPEPNLFHDGREHSVHVERTRGI FTVQVDENRRYMQNLTVEQPIEVKKLFVGGAPPEFQPSPLRNIPPFEGCIWNLVINSVPMDFARPVSFKNADIGRCAHQKLREDEDGAAPAAEIVIQPEPVPTPAFPTPTPVLTHGPCAAESE PALLIGSKQFGLSRNSHIAIAFDDTKVKNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADI LDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFH VDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 302 Human laminin - α - 1 LG domain 1 - 5YNTLTLNVKTQEPDNLLFYLGSSTASDFLAVEMRRRGRVAFLWDLGSGSTRLEFPDFPIDDNRWHSIHVARFGNIGSLSVKEMSSNQKSPTKTSKSPGTANVLDVNNSTLMFVGGLGGQIKKSPAVKVTHFKGCLGEAFLNGKSIGLWNYIEREGKCRGCFGSSQNEDPSFHFDGSGYSVVEKSLPATVTQIIMLFNTFSPNGL LLYLGSYGTKDFLSIELFRGRVKVMTDLGSGPITLLTDRRYNNNGTWYKIAFQRNRKQGVLAVIDAYNTSNKETKQGETPGASSDLNRLDKDPIYVGGLPRSRVVRRGVTTKSFVGCIKNLEISRSTFDLLRNSYGVRKGCLLEPIRSVSFLKGGYIELPPKSLSPESEWLVTFATTNSSGIILAALGGDVEKRGDREEAHVPFF SVMLIGGNIEVHVNPGDGTGLRKALLHAPTGTCSDGQAHSISLVRNRRIITVQLDENNPVEMKLGTLVESRTINVSNLYVGGIPEGEGTSLLTMRRSFHGCIKNLIFNLELLDFNSAVGHEQVDLDTCWLSERPKLAPDAEDSKLLPEPRAFPEQCVVDAALEYVPGAHQFGLTQNSHFILPFNQSAVRKKLSVELSIRTFASS GLIYYMAHQNQADYAVLQLHGGRLHFMFDLGKGRTKVSHPALLSDGKWHTVKTDYVKRKGFITVDGRESPMVTVVGDGTMLDVEGLFYLGGLPSQYQARKI GNITHSIPACIGDVTVNSKQLDKDSPVSAFTVNRCYAVAQEGTYFDGSGYAALVKEGYKVQSDVNITLEFRTSSQNGVLLGISTAKVDAIGLELVDGKVGAS SEQ ID NO: 303 Human laminin - α - 3 LG domain 1 - 5LQTVIKEDLPRKAKTLSSNSDKLLNEAKMTQKKLKQEVSPALNNLQQTLNIVTVQKEVIDTNLTTLRDGLHGIQRGDIDAMISSAKSMVRKANDITDEVLDGLNPIQTDVERIKDTYGRTQNEDFKKALTDADNSVNKLTN KLPDLWRKIESINQQLLPLGNISDNMDRIRELIQQARDAASKVAVPMRFNGKSGVEVRLPNDLEDLKGYTSLSLFLQRPNSRENGGTENMFVMYLGNKDASRDYIGMAVVDGQLTCVYNLGDREAELQVDQILTKSETKEA VMDRVKFQRIYQFARLNYTKGATSSKPETPGVYDMDGRNSNTLLNLDPENVVFYVGGYPPDFKLPSRLSFPPYKGCIELDDLNENVLSLYNFKKTFNLNTTEVEPCRRRKEESDKNYFEGTGYARVPTQPHAPIPTFGQTI QTTVDRGLLFFAENGDRFISLNIEDGKLMVRYKLNSELPKERGVGDAINNGRDHSIQIKIGKLQKRMWINVDVQNTIIDGEVFDFSTYYLGGIPIAIRERFNISTPAFRGCMKNLKKTSGVVRLNDTVGVTKKCSEDWKLVR SASFSRGGQLSFTDLGLPPTDHLQASFGFQTFQPSGILLDHQTWTRNLQVTLEDGYIELSTSDSGGPIFKSPQTYMDGLLHYVSVISDNSGLRLLIDDQLLRNSKRLKHISSSRQSLRLGGSNFEGCISNVFVQRLSLSPE VLDLTSNSLKRDVSLGGCSLNKPPFLMLLKGSTRFNKTKTFRINQLLQDTPVASPRSVKVWQDACSPLPKTQANHGALQFGDIPTSHLLFKLPQELLKPRSQFAVDMQTTSSRGLVFHTGTKNSFMALYLSKGRLVFALGTD GKKLRIKSKEKCNDGKWHTVVFGHDGEKGRLVVDGLRAREGSLPGNSTISIRAPVYLGSPPSGKPKSLPTNSFVGCLKNFQLDSKPLYTPSSSFGVSSCLGGPLEKGIYFSEEGGHVVLAHSVLLGPEFKLVFSIRPRSLT GILIHIGSQPGKHLCVYLEAGKVTASMDSGAGGTSTSVTPKQSLCDGQWHSVAVTIKQHILHLELDTDSSYTAGQIPFPPASTQEPLHLGGAPANLTTLRIPVWKSFFGCLRNIHVNHIPVPVTEALEVQGPVSLNGCPDQ SEQ ID NO: 304 Human laminin - α - 4 LG domain 1 - 5FTSLSLYMKPPVKRPELTETADQFILYLGSKNAKKEYMGLAIKNDNLVYVYNLGTKDVEIPLDSKPVSSWPAYFSIVKIERVGKHGKVFLTVPSLSSTAEEKFIKKGEFSGDDSLLDLDP EDTVFYVGGVPSNFKLPTSLNLPGFVGCLELATLNNDVISLYNFKHIYNMDPSTSVPCARDKLAFTQSRAASYFFDGSGYAVVRDITRRGKFGQVTRFDIEVRTPADNGLILLMVNGSMFF RLEMRNGYLHVFYDFGFSSGRVHLEDTLKKAQINDAKYHEISIIYHNDKKMILVVDRRHVKSMDNEKMKIPFTDIYIGGAPPEILQSRALRAHLPLDINFRGCMKGFQFQKKDFNLLEQTE TLGVGYGCPEDSLISRAYFNGQSFIASIQKISFFDGFEGGFNFRTLQPNGLLFYYASGSDVFSISLDNGTVIMDVKGIKVQSVDKQYNDGLSHFVISSVSPTRYELIVDKSRVGSKNPTK GKIEQTQASEKKFYFGGSPISAQYANFTGCISNAYFTRVDRDVEVEDFQRYTEKVHTSLYECPIESSPLFLLHKKGKNLSKPKASQNKKGGKSKDAPSWDPVALKLPERNTPRNSHCHLSN SPRAIEHAYQYGGTANSRQEFEHLKGDFGAKSQFSIRLRTRSSHGMIFYVSDQEENDFMTLFLAHGRLVYMFNVGHKKLKIRSQEKYNDGLWHDVIFIRERSSGRLVIDGLRVLEESLPPT EATWKIKGPIYLGGVAPGKAVKNVQINSIYSFSGCLSNLQLNGASITSASQTFSVTPCFEGPMETGTYFSTEGGYVVLDESFNIGLKFEIAFEVRPRSSSGTLVHGHSVNGEYLNVHMKN GQVIVKVNNGIRDFSTSVTPKQSLCDGRWHRITVIRDSNVVQLDVDSEVNHVVGPLNPKPIDHREPVFVGGVPESLLTPRLAPSKPFTGCIRHFVIDGHPVSFSKAALVSGAVSINSCPAA SEQ ID NO: 305 Human laminin - α - 5 LG domain 1 - 5YTALKFYLQGPEPEPGQGTEDRFVMYMGSRQATGDYMGVSLRDKKVHWVYQLGEAGPAVLSIDEDIGEQFAAVSLDRTLQFGHMSVTVERQMIQETKGDTVAPGAEGLLNLRPDDFV FYVGGYPSTFTPPPLLRFPGYRGCIEMDTLNEEVVSLYNFERTFQLDTAVDRPCARSKSTGDPWLTDGSYLDGTGFARISFDSQISTTKRFEQELRLVSYSGVLFFLKQQSQFLCLA VQEGSLVLLYDFGAGLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVVRVERATVYSVEQDNDLELADAYYLGGVPPDQLPPSLRRLFPTGGSVRGCVKGIKALGKYVDLKRLNTT GVSAGCTADLLVGRAMTFHGHGFLRLALSNVAPLTGNVYSGFGFHSAQDSALLYYRASPDGLCQVSLQQGRVSLQLLRTEVKTQAGFADGAPHYVAFYSNATGVWLYVDDQLQQMKPH RGPPPELQPQPEGPPRLLLGGLPESGTIYNFSGCISNVFVQRLLGPQRVFDLQQNLGSVNVSTGCAPALQAQTPGLGPRGLQATARKASRRSRQPARHPACMLPPHLRTTRDSYQFG GSLSSHLEFVGILARHRNWPSLSMHVLPRSSRGLLLFTARLRPGSPSLALFLSNGHFVAQMEGLGTRLRAQSRQRSRPGRWHKVSVRWEKNRILLVTDGARAWSQEGPHRQHQGAEH PQPHTLFVGGLPASSHSSKLPVTVGFSGCVKRLRLHGRPLGAPTRMAGVTPCILGPLEAGLFFPGSGGVITLDLPGATLPDVGLELEVRPLAVTGLIFHLGQARTPPYLQLQVTEKQ VLLRADDGAGEFSTSVTRPSVLCDGQWHRLAVMKSGNVLRLEVDAQSNHTVGPLLAAAAGAPAPLYLGGLPEPMAVQPWPPAYCGCMRRRLAVNRSPVAMTRSVEVHGAVGASGCPAA SEQ ID NO: 306 Human pearl LG domain 1 - 5VKAFAHLQVPERVVPYFTQTPYSFLPLPTIKDAYRKFEIKITFRPDSADGMLLYNGQKRVPGSPTNLANRQPDFISFGLVGGRPEFRFDAGSGMATIRHPTPLALGHFHTVTLLRSLTQGSLIVGDLAPVNGTSQGKFQGLDLNEELYLGGYPDYGAIPKAGLSSGFIGCVRELRIQGEEIVFHD LNLTAHGISHCPTCRDPCQNGGQCHDSESSSYVCVCPAGFTGSRCEHSQALHCHPEACGPDATCVNRPDGRGYTCCRCHLGRSGLRCEEGVTVTTPSLSGAGSYLALPALTNTHHELRLDVEFKPLAPDGVLLFSGGKSGPVEDFVSLAMVGGHLEFRYELGSGLAVLRSAEPLALGRWHRVSAER LNKDGSLRVNGGRPVLRSSPGKSQGLNLHTLLYLGGVEPSVPLSPATNMSAHFRGCVGEVSVNGKRLDLTYSFLGSQGIGQCYDSSPCERQPCQHGATCMPAGEYEFQCLCRDGFKGDLCEHEENPCQLREPCLHGGTCQGTRCLCLPGFSGPRCQQGSGHGIAESDWHLEGSGGNDAPGQYGAY FHDDGFLAFPGHVFSRSLPEVPETIELEVRTSTASGLLLWQGVEVGEAGQGKDFISLGLQDGHLVFRYQLGSGEARLVSEDPINDGEWHRVTALREGRRGSIQVDGEELVSGRSPGPNVAVNAKGSVYIGGAPDVATLTGGRFSSGITGCVKNLVLHSARPGAPPPQPLDLQHRAQAGANTRPPCS SEQ ID NO: 307 Human aggrecan LG domain 1 - 5TLRLALEFRALEPQGLLLYNGNARGKDFLALALLDGRVQLRFDTGSGPAVLTSAVPVEPGQWHRLELSRHWRRGTLSVDGETPVLGESPSGTTDGLNLDTDLFVGGVPEDQAAVALERTFVGAGLRGCIRLLDVNNQRLELGIGPGAATRGSGVGECGDHPCLPNPCHGG APCQNLEAGRFHCQCPPGRVGPTCADEKSPCQPNPCHGAAPCRVLPEGGAQCECPLGREGTFCQTASGQDGSGPFLADFNGFSHLELRGLHTFARDLGEKMALEVVFLARGPSGLLLYNGQKTDGKGDFVSLALRDRRLEFRYDLGKGAAVIRSREPVTLGAWTRVSLE RNGRKGALRVGDGPRVLGESPKSRKVPHTVLNLKEPLYVGGAPDFSKLARAAAVSSGFDGAIQLVSLGGRQLLTPEHVLRQVDVTSFAGHPCTRASGHPCLNGASCVPREAAYVCLCPGGFSGPHCEKGLVEKSAGDVDTLAFDGRTFVEYLNAVTESELANEIPVPET LDSGALHSEKALQSNHFELSLRTEATQGLVLWSGKATERADYVALAIVDGHLQLSYNLGSQPVVLRSTVPVNTNRWLRVVAHREQREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLPELPVGPALPKAYGTGFVGCLRDVVVGRHPLHLLEDAVTKPELRPCPTP 5 SEQ ID NO: 308 Human agrin laminin - spiral Binding domainCPERALERREEEANVVLTGTVEEILNVDPVQHTYSCKVRVWRYLKGKDLVARESLLDGGNKVVISGFGDPLICDNQVSTGDTRIFFVNPAPPYLWPAHKNELMLNSSLMRITLRN LEEVEFCVEDKPGTHFTPVPPTPPDACRGMLCGFGAVCEPNAEGPGRASCVCKKSPCPSVVAPVCGSDASTYSNECELQRAQCSQQRRIRLLSRGPCGSRDPCSNVTCSFGSTCAR SEQ ID NO: 309 Human nestin - 1 Laminin - γ ear binding domainCANNRHQCSVHAECRDYATGFCCSCVAGYTNGGRQCVAEGSPQRVNGKVKGRIFVGSSQVPIVFENTDLHSYVVMNHGRSYTAISTIPETVGYSLLPLAPVGGIIGW MFAVEQDGFKNGFSITGGEFTRQAEVTFVGHPGNLVIKQRFSGIDEHGHLTIDTELEGRVPQIPFGSSVHIEPYTELYHYSTSVITSSSTREYTVTEPERDGASPSR IYTYQWRQTITFQECVHDDSRPALPSTQQLSVDSVFVLYNQEEKILRYALSNSIGPVREGSPDALQNPCYIGTHGCDTNAACRPGPRTQFTCECSIGFRGDGRTCYD IDECSEQPSVCGSHTICNNHPGTFRCECVEGYQFSDEGTCVAVVDQRPINYCETGLHNCDIPQRAQCIYTGGSSYTCSCLPGFSGDGQACQDVDECQPSRCHPDAFCY NTPGSFTCQCKPGYQGDGFRCVPGEVEKTRCQHEREHILGAAGATDPQRPIPGLFVPECDAHGHYAPTQCHGSTGYCWCVDRDGREVEGTRTRPGMTPPCLSTVAP PIHQGPAVPTAVIPLPPGTHLLFAQTGKIERLPLEGNTMRKTEAKAFLHVPAKVIIGLAFDCVDKMVYWTDITEPSIGRASLHGGEPTTIIRQDLGSPEGIAVDHLGR NIFWTDSNLDRIEVAKLDGTQRRVLFETDLVNPRGIVTDSVRGNLYWTDWNRDNPKIETSYMDGTNRRILVQDDLGLPNGLTFDAFSSQLCWVDAGTNRAECLNPSQ PSRRKALEGLQYPFAVTSYGKNLYFTDWKMNSVVALDLAISKETDAFQPHKQTRLYGITTALSQCPQGHNYCSVNNGGCTHLCLATPGSRTCRCPDNTLGVDCIEQK SEQ ID NO: 310 Human nestin - 2 Laminin - γ ear binding domainCEHNHRQCSRHAFCTDYATGFCCHCQSKFYGNGKHCLPEGAPHRVNGKVSGHLHVGHTPVHFTDVDLHAYIVGNDGRAYTAISHIPQPAAQALLPLTPIGGLFGWLFALEK PGSENGFSLAGAAFTHDMEVTFYPGEETVRITQTAEGLDPENYLSIKTNIQGQVPYVSANFTAHISPYKELYHYSDSTVTSTSSRDYSLTFGAINQTWSYRIHQNITYQVC RHAPRHPSFPTTQQLNVDRVFALYNDEERVLRFAVTNQIGPVKEDSDPTPGNPCYDGSHCMCDTTARCHPGTGVDYTCECASGYQGDGRNCVDENECATGFHRCGPNSVCIN LPGSYRCECRSGYEFADDRHTCILITPPANPCEDGSHTCAPAGQARCVHHGGSTFSCACLPGYAGDGHQCTDVDECSENRCHPAATCYNTPGSFSCRCQPGYYGDGFQCIP DSTSSLTPCEQQQRHAQAQYAYPGARFHIPQCDEQGNFLPLQCHGSTGFCWCVDPDGHEVPGTQTPPGSTPPHCGPSPEPTQRPPTICERWRENLLEHYGGTPRDDQYVPQ CDDLGHFIPLQCHGKSDFCWCVDKDGREVQGTRSQPGTTPACIPTVAPPMVRPTPRPDVTPPSVGTFLLYTQGQQIGYLPLNGTRLQKDAAKTLLSLHGSIIVGIDYDCRE RMVYWTDVAGRTISRAGLELGAEPETIVNSGLISPEGLAIDHIRRTMYWTDSVLDKIESALLDGSERKVLFYTDLVNPRAIAVDPIRGNLYWTDWNREAPKIETSSLDGEN RRILINTDIGLPNGLTFDPFSKLLCWADAGTKKLECTLPDGTGRRVIQNNLKYPFSIVSYADHFYHTDWRRDGVVSVNKHSGQFTDEYLPEQRSHLYGITAVYPYCPTGRK SEQ ID NO: 311 signal peptideMGWSCIILFLVATATGVHS SEQ ID NO: 312 hIgG1 Heavy Chain CH1-Fc- FestivalASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 313 hIgG1 Heavy chain hinge area -Fc- holeEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: 314 LC κRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 315 DLL4(EGF1-6)-hlgG1-Fc holeSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRL CKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHC LCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: 316 DLL1(EGF1-6)-hlgG1-Fc holeSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYY GEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLE NSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: 317 Jagged1(EGF1-6)-hlgG1-Fc holeKVCGASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDD YYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGRECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKE TSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: 318 Jagged2(EGF1-6)-hlgG1-Fc holeVQAARPMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVR CDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPS GWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: 319 DLL4(EGF1-6)-GS connector - Laminin -a2 LG4-5SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEY CQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCT SNPCANGGQCLNRGPSRMCRCRPGFTGTYCEGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHV GTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 320 DLL4(EGF1-6)-HSA- Laminin -a2 LG4-5SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHF GHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLS CADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCEGGGGSGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEM ADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDR ADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHHDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSGGGSGGGS NRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAV GGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 321 DLL4(EGF1-6)-hlgG1-Fc- Laminin -a2 LG4-5SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLA VGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDE GWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSN CEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPK KADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLM FHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 322 DLL1(EGF1-6)- Laminin -a2 LG4-5SGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCN PGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKI DYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSF HVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 323 DLL1(EGF1-6)-HSA- Laminin -a2 LG4-5SGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVF CRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKI CELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDGGGGSGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRE TYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLEC ADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTL VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSGGGSGG GSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKA VGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 324 DLL1(EGF1-6)-hlgG1-Fc- Laminin -a2 LG4-5SGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISR LATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQP WQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVG YSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTIS PKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKL MFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 325 Jagged1(EGF1-6)- Laminin -a2 LG4-5KVCGASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKT CMEGWMGRECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLD ANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSF HVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 326 Jagged1(EGF1-6)-HSA- Laminin -a2 LG4-5KVCGASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGC NKFCRPRDDFFGHYACDQNGNKTCMEGWMGRECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWT GPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDGGGGSGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLR ETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPT LVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSGGGSG GGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAK AVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 327 Jagged1(EGF1-6)-hlgG1-Fc- Laminin -a2 LG4-5KVCGASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSI IEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGRECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICN EPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPP QWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTI SPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEK LMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 328 Jagged2(EGF1-6)- Laminin -a2 LG4-5VQAARPMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYG NKACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLN AFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHAD FATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHM AEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPG HLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 329 Jagged2(EGF1-6)-HSA- Laminin -a2 LG4-5VQAARPMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVR CDENYYSATCNKFCRPRNDFFGHYTCDQYGNACMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPS GWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELGGGGSGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEV TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFP KAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEY KFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAAS QAALGLGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYF DGTGFAKAVGGFKVGLDLLVEFEFFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 330 Jagged-2(EGF1-6)-hlgG1-Fc- Laminin -a2 LG4-5VQAARPMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWD NDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNKAMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVHG SCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVG ATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQE GILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGI EMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 331 DLL4(EGF1-6)-hlgG4-Fc- Laminin -a2 LG4-5SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLA VGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDE GWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSN CEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLXGKEYKCKVSXKGLPSSIEKTISXAXGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSXWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKK ADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLM FHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 332 DLL1(EGF1-6)-hlgG4-Fc- Laminin -a2 LG4-5SGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISR LATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQP WQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVG YSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLXGKEYKCKVSXKGLPSSIEKTISXAXGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSXWQEGNVF SCSVMHEALHNHYTQKSLSLSLGKGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISP KKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKL MFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 333 Jagged1(EGF1-6)-hlgG4-Fc- Laminin -a2 LG4-5KVCGASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEAWDSSNDTVQPDSI IEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGRECNRAICRQGCSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGIC NEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCP PQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLXGKEYKCKVSXKGLPSSIEKTISXAXGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSXWQEGNV FSCSVMHEALHNHYTQKSLSLSLGKKGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTIS PKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKL MFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 334 Jagged2(EGF1-6)-hlgG4-Fc- Laminin -a2 LG4-5VQAARPMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWPRSFTLIVEAWDWD NDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNKAMDGWMGKECKEAVCKQGCNLLHGGCTVPGECRCSYGWQGRFCDECVPYPGCVH GSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWSGPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWV GATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCELESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLXGKEYKCKVSXKGLPSSIEKTISXAXGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSXWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGSGGGSGGGSNRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQE GILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAVGGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGI EMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 335 Hinge area -hIgG4-Fc S228PESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLXGKEYKCKVSXKGLPSSI EKTISXAXGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSXWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 336 signal peptideMAAASRSASGWALLLLVALWQQRAAG SEQ ID NO: 337 connectorGGGSGGGSGGGS SEQ ID NO: 338 connectorGGGSGGGS SEQ ID NO: 339 DLL4 EGF1-6SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDL RPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGY CSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCE SEQ ID NO: 340 DLL1 EGF1-6SGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCD EQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSP CKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCD SEQ ID NO: 341 Jag1 EGF1-6KVCGASGQFELEILSMQNVNGELQNGNCCGGARNPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRS YTLLVEAWDSSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGRECNRAICRQG CSPKHGSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGICNEPWQCLCETNWGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLS DPCHNRGSCKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPCVNAKSCKNLIASYYCDCLPGWMGQNCD SEQ ID NO: 342 Jag2 EGF1-6VQAARPMGYFELQLSALRNVNGELLSGACCDGDGRTTRAGGCGHDECDTYVRVCLKEYQAKVTPTGPCSYGHGATPVLGGNSFYLPPAGAAGDRARARARAGGDQDPGLVVIPFQFAWP RSFTLIVEAWDWDNDTTPNEELLIERVSHAGMINPEDRWKSLHFSGHVAHLELQIRVRCDENYYSATCNKFCRPRNDFFGHYTCDQYGNKAMDGWMGKECKEAVCKQGCNLLHGGCTVP GECRCSYGWQGRFCDECVPYPGCVHGSCVEPWQCNCETNWGGLLCDKDLNYCGSHHPCTNGGTCINAEPDQYRCTCPDGYSGRNCEKAEHACTSNPCANGGSCHEVPSGFECHCPSGWS GPTCALDIDECASNPCAAGGTCVDQVDGFECICPEQWVGATCQLDANECEGKPCLNAFSCKNLIGGYYCDCIPGWKGINCHINVNDCRGQCQHGGTCKDLVNGYQCVCPRGFGGRHCEL SEQ ID NO: 343 Laminin -a2 LG4-5NRLTIELEVRTEAESGLLFYMARINHADFATVQLRNGLPYFSYDLGSGDTHTMIPTKINDGQWHKIKIMRSKQEGILYVDGASNRTISPKKADILDVVGMLYVGGLPINYTTRRIGPVTYSIDGCVRNLHMAEAPADLEQPTSSFHVGTCFANAQRGTYFDGTGFAKAV GGFKVGLDLLVEFEFRTTTTTGVLLGISSQKMDGMGIEMIDEKLMFHVDNGAGRFTAVYDAGVPGHLCDGQWHKVTANKIKHRIELTVDGNQVEAQSPNPASTSADTNDPVFVGGFPDDLKQFGLTTSIPFRGCIRSLKLTKGTGKPLEVNFAKALELRGVQPVSCPAN SEQ ID NO: 344 HSADAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEV ENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCK HPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL SEQ ID NO: 345 hIgG1 FcEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 346 hIgG4 FcESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLXGKEYKCKVSXKGLPSSIE KTISXAXGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSXWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKG SEQ ID NO: 347 FcDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 348 Human IgG1 Fc First unit , in X ₁ Department Arg 、 His or LysDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLX ₁SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 349 muD4vSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEA LPPDALISKFAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYH CLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQSPDFLSVTPKEKVTLSCRAS QSISNYLHWYQQKSDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEAEDAATYFCQQSKSWPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSSGDNTYYPDSVKGRFTISRDNSKNTLYLQMSSLRAEDTALYYCARFDYGSSLDSWGQGTTLTVSS

Figures 1A to 1M depict different configurations of bifunctional proteins comprising an anti-muscle target protein and a nicked ligand or agonist. Figure 1A depicts a monovalent nicked ligand with an anti-muscle target protein (Ab) in a "button-in-hole" format. Figures 1B and 1C depict additional formats and configurations of bifunctional proteins, where R1, R2, R3, and R4 can be an anti-muscle target protein or a nicked ligand/agonist. Figure 1D depicts a bivalent fusion protein in which the nicked ligand is fused to the N-terminus of the Fc and the anti-muscle target protein Fab is fused to the C-terminus of the Fc. Figure 1E depicts a bivalent fusion protein in which the nicked ligand is fused to the N-terminus of the Fc and the anti-muscle target protein scFv is fused to the C-terminus of the Fc. Figure 1F depicts a bivalent fusion protein in which the nicked ligand is fused to the N-terminus of the Fc and an anti-muscle target protein antibody single chain Ab (VHH or nanobody) is fused to the C-terminus of the Fc. Figure 1G depicts a bivalent fusion protein in which the nicked ligand is fused to the C-terminus of the Fc and an anti-muscle protein antibody Fab is fused to the N-terminus of the Fc. Figure 1H depicts a bivalent fusion protein in which the nicked ligand is fused to the C-terminus of the Fc and an anti-muscle protein scFv is fused to the N-terminus of the Fc. Figure 1I depicts a bivalent fusion protein in which the nicked ligand is fused to the C-terminus of the Fc and an anti-muscle protein VHH/nanobody is fused to the N-terminus of the Fc. Figure 1J depicts a bivalent fusion protein in which the nicked ligand is fused to the C-terminus of the antibody light chain. Figure 1K depicts a bivalent fusion protein in which the nicked ligand is fused to the N-terminus of the antibody light chain. Figure 1L depicts a bivalent fusion protein in which the Notch ligand is fused to the N-terminus of the antibody heavy chain. Figure 1M depicts a tetravalent fusion protein in which the Notch ligand is fused to the N-termini of both the antibody light and heavy chains.

Figures 2A to 2J depict different configurations of bifunctional proteins comprising a non-antibody portion (e.g., the LG domain of laminin, agrin, nidogen, and perlein) and a nick ligand or agonist. Figure 2A depicts a monovalent protein in which the nick ligand is fused to the N-terminus of a laminin G-like domain (LG domain) monomer, or to an scFv, sdAb, or Fab directed against a muscle-specific protein. Figure 2B depicts a monovalent protein in which the nick ligand is fused to the N-terminus of an LG domain dimer, or to an scFv, sdAb, or Fab that specifically recognizes a muscle-specific protein. Figure 2C depicts a monovalent protein in which the nick ligand is fused to the N-terminus of a tandem LG domain trimer or multimer, or to an scFv, sdAb, or Fab directed against a muscle-specific protein. Figure 2D depicts a monovalent protein consisting of, from N' to C' terminus: a nicked ligand-albumin, an anti-albumin scFv or sdAb, or a monomeric human Fc-LG domain monomer. Figure 2E depicts a monovalent protein consisting of, from N' to C' terminus: a nicked ligand-albumin, an anti-albumin scFv or sdAb, or a monomeric human Fc-LG domain dimer. Figure 2F depicts a monovalent protein consisting of, from N' to C' terminus: a nicked ligand-albumin, an anti-albumin scFv or sdAb, or a monomeric human Fc-tandem LG domain trimer or multimer. Figures 2G and 2H depict bivalent fusion proteins in which the nicked ligand is fused to the N-terminus of Fc and the LG domain is fused to the C-terminus of Fc as a monomer, dimer, or tandem multimer, respectively. Figures 2I and 2J depict bivalent fusion proteins in which the Notch ligand is fused to the C-terminus of Fc and the LG domain is fused to the N-terminus of Fc in monomeric, dimer, or tandem multimeric form, respectively.

Figure 3A depicts SDS-PAGE of reduced and non-reduced DLL4v-Fc-LG4-5 constructs. Figure 3B depicts size exclusion chromatography (SEC) curves of a protein standard (upper curve) and the DLL4v-Fc-LG4-5 construct (lower curve).

Figure 4A depicts SDS-PAGE of reduced and non-reduced DLL4wt-Fc-LG4-5 constructs. Figure 4B depicts size exclusion chromatography (SEC) curves of protein standards (upper curve) and DLL4wt-Fc-LG4-5 construct (lower curve).

Figure 5A depicts SDS-PAGE of reduced and non-reduced DLL4v-Fc-LAMA2scFv constructs. Figure 5B depicts size exclusion chromatography (SEC) curves of protein standards (upper curve) and DLL4v-Fc-LAMA2scFv construct (lower curve).

Figure 6 depicts the binding of the bifunctional proteins DLL4v-Fc-LG4-5, DLL4wt-Fc-LG4-5, and DLL4v-Fc-LAMA2scFv to Notch-1 as determined by sandwich ELISA. DLL4v-Fc-LG4-5 and DLL4v-Fc-LAMA2scFv have higher affinity (or avidity) for Notch-1 compared to the DLL4wt-Fc-LG4-5 construct.

Figure 7A depicts kinetic analysis of the binding of the DLL4v-Fc-LG4-5 construct to various concentrations of purified matrix glycans using a Biacore system. The y-axis represents absolute response units (RU), and the x-axis represents time in seconds. Arrows indicate the injection points of purified matrix glycans. Figure 7B depicts Western blot analysis of a laminin overlay assay, demonstrating binding of DLL4v-Fc-LG4-5 to matrix glycans.

Figure 8 depicts kinetic analysis of the binding of the DLL4v-Fc-LAMA2scFv construct to varying concentrations of recombinant human LG4-5 using a Biacore system, confirming binding of LAMA2scFv to LG4-5. Arrows indicate the injection site of recombinant human LG4-5.

Figure 9A depicts the study plan. Muscle function was assessed by forelimb grip strength in male D2.mdx mice, approximately 7-9 weeks of age. Mice were randomly divided into two groups (n=10/group). The control group was treated with 20 mg/kg of an isotype control antibody against the irrelevant antigen trinitrophenol (TNP) every three weeks. The experimental group was treated with 5 mg/kg of DLL4v-Fc-LAMA2scFv every three weeks. The wild-type (WT) group received no treatment. Forelimb grip strength was assessed monthly over 7 months. Figure 9B depicts forelimb grip strength in WT D2.mdx mice, D2.mdx mice treated with DLL4v-Fc-LAMA2scFv, and D2.mdx mice treated with isotype controls over 7 months. Figure 9C depicts the body weight of WT D2.mdx mice, DLL4v-Fc-LAMA2scFv-treated D2.mdx mice, and isotype control-treated D2.mdx mice over 7 months.

Figure 10A depicts size exclusion chromatography (SEC) curves of protein standards (upper curve), huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG-LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS. Figure 10B depicts a table summarizing the yield and percentage of the main peak determined by analytical size exclusion chromatography (aSEC).

Figure 11A depicts the binding affinity of huD4v11_2N-FcAAG-LG21DS, huD4v12_2N_A2-FcAAG-LG21DS, huD4v13_2N_-FcAAG-LG21DS, and huD4v14_2N_H2-FcAAG-LG21DS to the murine Notch 1 receptor, as compared to the binding affinity of huD4v_G2S_F81L_H168Y-Fc(AAG)-LG21scFvDS (parent) and mud4v constructs, as determined by ELISA. Figure 11B depicts a table summarizing the binding affinity ( _KD ) of each construct, measured in nM.

TW202525856A_113133987_SEQL.xml

Claims (57)

A bifunctional protein construct comprises a first binding moiety and a second binding moiety, wherein the first binding moiety specifically binds to a muscle-specific molecule, and wherein the second binding moiety specifically binds to a Notch receptor and activates the Notch receptor. The bifunctional protein construct of claim 1, wherein the muscle-specific molecule is a target antigen on the sarcolemma, between the sarcolemma and the basal lamina, or in the basal lamina. The bifunctional protein construct of claim 2, wherein the target antigen is selected from the group consisting of laminin, agrin, nidogen, perlecan, and M-cadherin (CDH15). The bifunctional protein construct of claim 2 or 3, wherein the target antigen is a component of the dystrophin-related glycoprotein complex (DGC). The bifunctional protein construct of claim 4, wherein the target antigen is selected from the group consisting of α-dystroglycan (α-DG), β-DG, laminin-211, perlein, collagen, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, ε-sarcoglycan, ζ-sarcoglycan, biglycan, sarcospan, and matriglycan on α-DG. The bifunctional protein construct of any one of claims 3 to 5, wherein the target antigen is laminin subunit α-2 (LAMA2) of laminin-211, CDH15, α-DG, or a matrix glycan on α-DG. The bifunctional protein construct of any one of claims 1 to 6, wherein the first binding moiety comprises an antibody portion that specifically binds to the muscle-specific molecule. The bifunctional protein construct of claim 7, wherein the antibody portion is selected from the group consisting of: full-length antibody, Fab, Fab', F(ab') ₂ , scFv and sdAb. [Claim 9a] The bifunctional protein construct of claim 7 or 8, wherein the antibody portion specifically binds to LAMA2 (anti-LAMA2 antibody portion). [Claim 9b] The bifunctional protein construct of claim 9a, wherein the anti-LAMA2 antibody portion comprises: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. [Claim 9c] The bifunctional protein construct of claim 9b, wherein the anti-LAMA2 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 50. [Claim 9d] The bifunctional protein construct of claim 9a, wherein the anti-LAMA2 antibody portion comprises: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12. [Claim 9e] The bifunctional protein construct of claim 9d, wherein the anti-LAMA2 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 65. [Claim 9f] The bifunctional protein construct of claim 7 or 8, wherein the antibody portion specifically binds to a matrix glycan on α-DG (anti-matrix glycan antibody portion). [Claim 9g] The bifunctional protein construct of claim 9f, wherein the anti-matrix antibody portion comprises: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 83, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 85. [Claim 9h] The bifunctional protein construct of claim 9g, wherein the anti-matrix antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 95. [Claim 9i] The bifunctional protein construct of claim 7 or 8, wherein the antibody portion specifically binds to CDH15 (anti-CDH15 antibody portion). [Claim 9j] The bifunctional protein construct of claim 9i, wherein the anti-CDH15 antibody portion comprises: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 102, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 103, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 105, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 107. [Claim 9k] The bifunctional protein construct of claim 9j, wherein the anti-CDH15 antibody portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 108 and a VL comprising the amino acid sequence of SEQ ID NO: 109. The bifunctional protein construct of any one of claims 1 to 6, wherein the first binding moiety comprises a non-antibody portion that specifically binds to the muscle-specific molecule. The bifunctional protein construct of claim 10, wherein the non-antibody portion comprises a protein domain selected from the group consisting of: the laminin G-like domain (LG domain) of laminin, the LG domain of agrin, the LG domain of nidogen, the LG domain of perlein, the laminin coiled-coil binding domain of agrin, and the laminin γ binding domain of nidogen. The bifunctional protein construct of claim 11, wherein the non-antibody portion comprises the LG domain of laminin. The bifunctional protein construct of claim 12, wherein the non-antibody portion comprises the LG4-5 domain of LAMA2 (LAMA2 LG4-5). The bifunctional protein construct of claim 13, wherein LAMA2 LG4-5 comprises the amino acid sequence of SEQ ID NO: 118 or 119. The bifunctional protein construct of any one of claims 1 to 14, wherein the second binding moiety comprises the extracellular domain (ECD) of a Notch ligand or a variant thereof, wherein the Notch ligand is selected from the group consisting of Delta-like 1 (DLL1), DLL3, DLL4, Jagged1 (Jag1), and Jag2. [Claim 16a] The bifunctional protein construct of claim 15, wherein the second binding moiety comprises DLL4 ECD or a variant thereof, wherein the DLL4 ECD comprises the amino acid sequence of SEQ ID NO:125, and wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs:126-129 and 260-267. [Claim 16b] The bifunctional protein construct of claim 15, wherein the second binding moiety comprises DLL1 ECD or a variant thereof, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO:130. [Claim 16c] The bifunctional protein construct of claim 15, wherein the second binding moiety comprises DLL3 ECD or a variant thereof, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO:131. [Claim 16d] The bifunctional protein construct of claim 15, wherein the second binding moiety comprises Jag1 ECD or a variant thereof, wherein the Jag1 ECD comprises the amino acid sequence of SEQ ID NO:132, and wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO:133 or 134. [Claim 16e] The bifunctional protein construct of claim 15, wherein the second binding moiety comprises Jag2 ECD or a variant thereof, and wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO:135. The bifunctional protein construct of any one of claims 1 to 16e, wherein the first binding moiety is fused to the second binding moiety via an optional linker. The bifunctional protein construct of claim 17, wherein: a) the second binding moiety comprises a variant DLL4 ECD comprising the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267; b) the second binding moiety comprises a DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130; c) the second binding moiety comprises a DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131; d) the second binding moiety comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NOs: 133 or 134; or e) the second binding moiety comprises a Jag2 ECD comprising the amino acid sequence of SEQ ID NO: 135. The bifunctional protein construct of claim 18, wherein the bifunctional protein construct comprises the amino acid sequence of any one of SEQ ID NOs: 136-144. The bifunctional protein construct of claim 18, wherein the first binding moiety comprises a Fab that specifically binds to a muscle-specific molecule. The bifunctional protein construct of claim 20, wherein the second binding moiety is fused to the N-terminus of the VL of the Fab via an optional linker. The bifunctional protein construct of any one of claims 1 to 16e, wherein the bifunctional protein construct further comprises an Fc domain, wherein the Fc domain comprises a first unit and a second unit. The bifunctional protein construct of claim 22, wherein: a) the Fc domain is derived from human IgG1 comprising the amino acid sequence of SEQ ID NO: 187; b) each subunit of the Fc domain comprises an L234A/L235A mutation (EU numbering); c) each subunit of the Fc domain comprises an H435A mutation (EU numbering); d) each subunit of the Fc domain comprises a P329G mutation (EU numbering); e) each subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 188 or 277; f) the first subunit of the Fc domain or the second subunit of the Fc domain comprises an H435R/Y436F mutation (EU numbering); and/or g) the Fc domain comprises a knob-into-hole mutation, and wherein: i) the first Fc domain unit comprises a knob mutation, and the second Fc domain unit comprises a hole mutation; or ii) the second Fc domain unit comprises a knob mutation, and the first Fc domain unit comprises a hole mutation. The bifunctional protein construct of claim 23, wherein the knob mutation is T366W (EU numbering) and the hole mutation is T366S/L368A/Y407V (EU numbering). A bifunctional protein construct according to any one of claims 22 to 24, wherein: a) the bifunctional protein construct comprises: i) a first unit that specifically binds to a second binding moiety of a first nick receptor, and ii) a second unit that specifically binds to a second binding moiety of a second nick receptor, wherein the first unit of the second binding moiety and the second unit of the second binding moiety each independently comprise an ECD of a nick ligand or a variant thereof, the nick ligand being selected from the group consisting of DLL1, DLL3, DLL4, Jag1, and Jag2; and/or b) the bifunctional protein construct comprises: i) a first unit that specifically binds to a first binding moiety of a first muscle-specific molecule, and ii) a second unit that specifically binds to a first binding moiety of a second muscle-specific molecule. The bifunctional protein construct of any one of claims 22 to 25, wherein the first unit of the first binding moiety comprises a first antibody portion that specifically binds to the first muscle-specific molecule, the second unit of the first binding moiety comprises a second antibody portion that specifically binds to the second muscle-specific molecule, and wherein the first antibody portion and the second antibody portion are each independently selected from the group consisting of: Fab, scFv, and sdAb. [Claim 27a] The bifunctional protein construct of claim 26, wherein the first antibody portion is a first Fab (Fab1) and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit specifically binding to the second binding portion of a first notch receptor - optionally a linker - a first unit of the Fc domain - optionally a linker - VH1-(H1-CH1); ii) a second polypeptide comprising, from N' to C', a second unit specifically binding to the second binding portion of a second notch receptor - optionally a linker - a second unit of the Fc domain - optionally a linker - VH2-(H2-CH1); iii) a third polypeptide comprising, from N' to C', VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C', VL2-(L2-CL); and wherein VL1-(L1-CL) forms Fab1 with VH1-(H1-CH1), and VH2-(H2-CH1) forms Fab2 with VL2-(L2-CL). [Claim 27b] The bifunctional protein construct of claim 26, wherein the first antibody portion is a first Fab (Fab1) and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit that specifically binds to the second binding portion of a first nick receptor - optionally a linker - a first unit of the Fc domain - optionally a linker - VL1-(L1-CL); ii) a second polypeptide comprising, from N' to C', a second unit that specifically binds to the second binding portion of a second nick receptor - optionally a linker - a second unit of the Fc domain - optionally a linker - VL2-(L2-CL); iii) a third polypeptide comprising, from N' to C', VH1-(H1-CH1); and iv) a fourth polypeptide comprising, from N' to C', VH2-(H2-CH1); and wherein VL1-(L1-CL) and VH1-(H1-CH1) form Fab1, and VH2-(H2-CH1) and VL2-(L2-CL) form Fab2. [Claim 27c] The bifunctional protein construct of claim 27a or 27b, wherein both Fab1 and Fab2 specifically bind to LAMA2, and wherein the anti-LAMA2 Fab1 and/or anti-LAMA2 Fab2 comprise: a first polypeptide comprising the amino acid sequence of SEQ ID NO:165 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:166. [Claim 27d] The bifunctional protein construct of any one of claims 25 to 27c, wherein: a) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, and wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267; b) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, and wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; c) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, and wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; d) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134; or e) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. The bifunctional protein construct of claim 26, wherein the first antibody portion is a first sdAb (sdAb1) and the second antibody portion is a second sdAb (sdAb2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit that specifically binds to the second binding portion of a first nick receptor - optionally a linker - a first unit of the Fc domain - optionally a linker - sdAb1; and ii) a second polypeptide comprising, from N' to C', a second unit that specifically binds to the second binding portion of a second nick receptor - optionally a linker - a second unit of the Fc domain - optionally a linker - sdAb2. The bifunctional protein construct of claim 26, wherein the first antibody portion is a first scFv (scFv1) and the second antibody portion is a second scFv (scFv2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit that specifically binds to the second binding portion of a first nick receptor - optionally a linker - a first unit of the Fc domain - optionally a linker - scFv1; and ii) a second polypeptide comprising, from N' to C', a second unit that specifically binds to the second binding portion of a second nick receptor - optionally a linker - a second unit of the Fc domain - optionally a linker - scFv2. The bifunctional protein construct of claim 29, wherein both scFv1 and scFv2 specifically bind to LAMA2, and wherein the anti-LAMA2 scFv1 and/or anti-LAMA2 scFv2 comprise the amino acid sequence of SEQ ID NO: 145 or 268. A bifunctional protein construct according to any one of claims 28 to 30, wherein: a) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD comprising the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267; b) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD comprising the amino acid sequence of SEQ ID NO: 130; c) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD comprising the amino acid sequence of SEQ ID NO: 131; d) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD comprising the amino acid sequence of SEQ ID NO: 133 or 134; or e) The first unit of the second binding moiety and/or the second unit of the second binding moiety comprises Jag2 ECD, which comprises the amino acid sequence of SEQ ID NO: 135. The bifunctional protein construct of claim 31, wherein the bifunctional protein construct comprises a first polypeptide and a second polypeptide, each comprising the amino acid sequence of any one of SEQ ID NOs: 223-231 and 269-276. A bifunctional protein construct as claimed in claim 25 or 26, wherein the first antibody portion is a first Fab (Fab1) and the second antibody portion is a second Fab (Fab2); wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit specifically binding to the second binding portion of a first notch receptor, optionally a linker, VH1-(H1-CH1)-optionally a linker-a first unit of the Fc domain; ii) a second polypeptide comprising, from N' to C', a second unit specifically binding to the second binding portion of a second notch receptor, optionally a linker, VH2-(H2-CH1)-optionally a linker-a second unit of the Fc domain; iii) a third polypeptide comprising, from N' to C', a third unit that specifically binds to the second binding portion of a third nick receptor, optionally a linker, VL1-(L1-CL); and iv) a fourth polypeptide comprising, from N' to C', a fourth unit that specifically binds to the second binding portion of a fourth nick receptor, optionally a linker, VL2-(L2-CL); and wherein VL1-(L1-CL) forms a Fab1 with VH1-(H1-CH1), and VH2-(H2-CH1) forms a Fab2 with VL2-(L2-CL). The bifunctional protein construct of any one of claims 22 to 25, wherein the first unit of the first binding moiety comprises a first non-antibody moiety that specifically binds to the first muscle-specific molecule, the second unit of the first binding moiety comprises a second non-antibody moiety that specifically binds to the second muscle-specific molecule, and wherein the first non-antibody moiety and the second non-antibody moiety are each independently selected from the group consisting of: the LG domain of laminin, the LG domain of agrin, the LG domain of nidogen, and the LG domain of perlein. The bifunctional protein construct of claim 34, wherein the first non-antibody portion and/or the second non-antibody portion comprises LAMA2 LG4-5, which comprises the amino acid sequence of SEQ ID NO: 118 or 119. The bifunctional protein construct of claim 34 or 35, wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', the first unit of the first binding moiety - optionally a linker - the first unit of the Fc domain - optionally a linker - the first unit of the second binding moiety that specifically binds to the first nicked receptor; and ii) a second polypeptide comprising, from N' to C', the second unit of the first binding moiety - optionally a linker - the second unit of the Fc domain - optionally a linker - the second unit of the second binding moiety that specifically binds to the second nicked receptor. The bifunctional protein construct of claim 34 or 35, wherein the bifunctional protein construct comprises: i) a first polypeptide comprising, from N' to C', a first unit that specifically binds to the second binding portion of a first nick receptor - optionally a linker - a first unit of the Fc domain - optionally a linker - a first unit of the first binding portion; and ii) a second polypeptide comprising, from N' to C', a second unit that specifically binds to the second binding portion of a second nick receptor - optionally a linker - a second unit of the Fc domain - optionally a linker - a second unit of the first binding portion. A bifunctional protein construct according to any one of claims 34 to 37, wherein: a) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant DLL4 ECD, wherein the variant DLL4 ECD comprises the amino acid sequence of any one of SEQ ID NOs: 126-129 and 260-267; b) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL1 ECD, wherein the DLL1 ECD comprises the amino acid sequence of SEQ ID NO: 130; c) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a DLL3 ECD, wherein the DLL3 ECD comprises the amino acid sequence of SEQ ID NO: 131; d) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a variant Jag1 ECD, wherein the variant Jag1 ECD comprises the amino acid sequence of SEQ ID NO: 133 or 134; or e) the first unit of the second binding moiety and/or the second unit of the second binding moiety comprises a Jag2 ECD, wherein the Jag2 ECD comprises the amino acid sequence of SEQ ID NO: 135. The bifunctional protein construct of claim 22, wherein: i) the first binding moiety is fused to the N-terminus of the first subunit of the Fc domain via a first linker, and the second binding moiety is fused to the N-terminus of the second subunit of the Fc domain via a second linker, or ii) the first binding moiety is fused to the N-terminus of the second subunit of the Fc domain via a first linker, and the second binding moiety is fused to the N-terminus of the first subunit of the Fc domain via a second linker, or One or more isolated nucleic acids encoding the bifunctional protein construct of any one of claims 1 to 39. One or more vectors comprising one or more isolated nucleic acids of claim 40. One or more vectors as claimed in claim 41, which are viral vectors. A host cell expressing the bifunctional protein construct of any one of claims 1 to 39, comprising one or more isolated nucleic acids of claim 40, or comprising one or more vectors of claim 41 or 42. A pharmaceutical composition comprising: i) the bifunctional protein construct of any one of claims 1 to 39, one or more isolated nucleic acids of claim 40, or one or more vectors of claim 41 or 42; and ii) a pharmaceutically acceptable excipient. A method for producing a bifunctional protein construct comprising: i) culturing a host cell comprising one or more isolated nucleic acids according to claim 40 or one or more vectors according to claim 41 or 42, or a host cell according to claim 43, under conditions suitable for expressing the bifunctional protein construct; and ii) recovering the expressed bifunctional protein construct from the cultured host cell. A method for treating a muscle-related disease in a subject, comprising administering to the subject an effective amount of the bifunctional protein construct of any one of claims 1 to 39 or the pharmaceutical composition of claim 44. The method of claim 46, wherein the muscle-related disease is selected from the group consisting of: Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), familial hypertrophic cardiomyopathy, Laing distal myopathy, myofibrillar myopathy, and muscular dystrophy. The method of claim 47, wherein the muscle-related disease is muscular dystrophy. The method of claim 48, wherein the muscular dystrophy comprises one or more of: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), congenital myopathy, Charcot-Marie-Tooth (CMT) disease, and Emery-Dreifuss muscular dystrophy (EDMD). The method of any one of claims 46 to 49, wherein the bifunctional protein construct or the pharmaceutical composition is administered intravenously, subcutaneously, or intramuscularly. An engineered DLL4 extracellular domain (ECD), wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of T52N and T135N, and wherein the amino acid positions are relative to a reference DLL4 ECD comprising the amino acid sequence of SEQ ID NO: 126. The engineered DLL4 ECD of claim 51, wherein the engineered DLL4 ECD further comprises a mutation at one or more amino acid positions selected from the group consisting of: G2, E14, R66, P80, F81, H168, Q220, N231, and N260. The engineered DLL4 ECD of claim 52, wherein the additional mutation is selected from the group consisting of G2S, E14H, R66S, R66T, P80L, F81L, H168Y, Q220H, N231D, and N260D. The engineered DLL4 ECD of any one of claims 51 to 53, wherein the engineered DLL4 ECD comprises a mutation selected from the group consisting of: (i) T52N and T135N; (ii) T52N, R66S, and T135N; (iii) E14H, T52N, R66T, P80L, T135N, and N231D; (iv) T52N, R66T, P80L, T135N, Q220H, and N260D; (v) G2S, T52N, F81L, T135N, and H168Y; (vi) G2S, T52N, F81L, R66S, T135N, and H168Y; (vii) G2S, E14H, T52N, F81L, R66T, P80L, T135N, H168Y, and N231D; and (viii) G2S, T52N, R66T, P80L, F81L, T135N, H168Y, Q220H, and N260D. The engineered DLL4 ECD of claim 54, wherein the engineered DLL4 ECD comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 261-264. A protein construct comprising the engineered DLL4 ECD of any one of claims 51 to 55. The protein construct of claim 56, wherein the protein construct further comprises a binding portion that specifically binds to a muscle-specific molecule.