P62011579wo 1 Bispecific antigen binding proteins Field of the invention The present invention relates to the field of medicine, in particular to the fields of oncology. Specifically, the invention relates to bispecific antigen binding proteins that induce endocytosis and lysosomal degradation of PD-L1 when brought into contact with a cell expressing PD-L1 and RNF128. The invention further relates to the use of such bispecific antigen binding proteins in the treatment of cancer. Background of the invention Cells communicate with their environment by the activity of plasma membrane-embedded receptors that capture external chemical signals and initiate an intracellular signaling cascade to drive a cellular response. Receptor availability at the cell surface is a critical determinant of signal specificity and sensitivity and misregulation of these events is frequently linked to the development or progression of a disease, such as, but not limited to, cancer, auto-immune diseases, neurological disorders and inflammatory disorders, as well as therapy resistance. For example, mutational activation or overexpression of receptors is a major and widely recognized cancer-promoting mechanism in multiple tissues (e.g. EGFR, ERBB2, PDGFR, TGF^R, IGFR1, GHR, FZD, LRP6). The dependency of cancer cells on aberrant receptor activity has instigated the development of various neutralizing antibodies and small molecule inhibitors. Successful neutralization of receptor activity however requires the generation of potent binders that reach sufficient plasma concentrations to display high efficacy without inducing toxicity, which may prove difficult in case of non-covalent interactors. Furthermore, compensatory receptor stabilization or upregulation is a major pathway of resistance. Posttranslational modification of the cytosolic regions of membrane-bound receptors with ubiquitin drives their rapid removal from the cell surface via induced endocytosis. The internalised receptors may subsequently be subjected to lysosomal degradation. In healthy stem cells, high levels of Wnt signalling drive the expression of two homologous membrane-bound ubiquitin ligases, RNF43 and ZNRF3, that are known to mediate ubiquitination and removal of Frizzled (FZD), the receptors for Wnt, from the cell surface (Koo et al, Nature 2012, 488(7413):665-9). This negative feedback loop thus serves to regulate the sensitivity of stem cells to Wnt by controlling the effective number of Frizzled (FZD) receptors on the cell surface. The activity of RNF43/ZNRF3 towards FZD is neutralized in the stem cell niche by the secreted protein R-spondin (RSPO) that forms a complex with LGR4/5 receptors as well as RNF43/ZNRF3 (Hao et al, Nature 2012, 485(7397):195-200). Next, this trimeric RSPO-LGR4/5-RNF43/ZNRF3 complex undergoes removal from the cell surface, leading to stabilization of FZD receptor expression and increased levels of Wnt signaling. Wnt signaling is frequently misregulated in cancer. Such cancers display increased expression of Wnt target genes, including RNF43 and ZNRF3.
P62011579wo 2 E3 ubiquitin ligases recruit an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin to a protein substrate and assists or directly catalyses the transfer of ubiquitin to the protein substrate. Ubiquitination of receptors mediated by transmembrane ubiquitin E3 ligases is known to result in endocytosis and subsequent breakdown of the ubiquitinated substrate. It is known in the art that such breakdown preferably takes place in the lysosome. Lysosomal degradation requires ligation of monoubiquitin, multiubiquitin, Lys11-, Lys29-, Lys48- or Lys63-linked poly-ubiquitin chains to membrane-bound receptors. This is in contrast to the activity of cytosolic ubiquitin ligases, which mainly employ the proteasomal degradation pathway, i.e. by the coupling of Lys11-, Lys29- or Lys48-linked poly-ubiquitin chains to cytosolic target proteins. Hence, transmembrane E3 ubiquitin ligases may interact with different members of the E2 enzyme family to selectively target membrane-bound substrates. The ubiquitinated substrate will be internalised and may subsequently be degraded, preferably via lysosomal degradation. There is still a strong need in the art to effectively target and inhibit activity of membrane- bound receptors, especially membrane-bound receptors that are involved in the development or progression of a disease. There is in particular a strong need in the art to effectively target and inhibit the activity of membrane-bound receptors that are involved in the development or progression of e.g. cancer, auto-immune diseases, neurological disorders, rare diseases and inflammatory disorders, as well as therapy resistance. Summary of the invention In a first aspect, the invention relates to a bispecific antigen binding protein comprising: i) a first an immunoglobulin single variable domain (ISVD) that specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128); and, ii) a second ISVD that specifically binds an extracellular portion of a tumor associated antigen (TAA). In one embodiment, in the bispecific antigen binding protein: i) the first ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s)
P62011579wo 3 with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9; and, ii) the second ISVD specifically binds a TAA is selected from the group consisting of: PD- L1, PD1, PSCA, L1-CAM, EpCAM, Her2 (ErbB2/Neu), Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, Glycoprotein NMB, CanAg, CD22 (Siglec2), CD33 (Siglec3), CD79, CD123, CD138, CD171, CTLA-4 (CD152), PSMA (prostate specific membrane antigen), BCMA, TROP2, STEAP1, CD52, CD56, CD80, CD70, E-selectin, EphB2, EPHA4, Melanotransferrin, Mud 6, TMEFF2, Killer lg-Like Receptor, Killer lg-Like Receptor 3DL2 (KIR3DL2), B7.1, B7.2, B7-H3, B7-H4, B7-H6, IL-6 receptor, IL-1 accessory Protein, MAGE, MART-1/Melan-A, gp100, MICA, MICB, colorectal associated antigen (CRC)-C017-1A/GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1, nectin-4, a UL16-binding protein (ULBP), a RAET1 protein, carcinoembryonic antigen (CEA), CEACAM5, etv6, , prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-A3, a GAGE-tumor antigen, anti-Müllerian hormone Type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, NTRKR1 (EC 2.7.10.1), SLAMF7, TRAILR1, TRAILR2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC1, MUC1-C, VEGF, VEGFR2, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR/ERBB1), HER-3/ERBB3, HER-4/ERBB4, a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, cMET, CA125, integrin receptors, a5β3 integrins, a5β1 integrins, αllbβ3-integrins, PDGF alpha receptor, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, IL-1 accessory protein, CSF1R, α-fetoprotein, mesothelin (MSLN), Isoform 2 of Claudin-18 (Claudin 18.2, CLDN18), folate receptor alpha (FRα, FOLR1), tissue factor (TF, CD142), P-cadherin, E- cadherin, , Plexin-A1, TNFRSF10B, AXL, EDNRB, OLR1, ADAM12, PLAUR, CCR4, CCR6, p120ctn, PRAME, NY-ESO-1, cdc27, CDCP1, , fodrin, Connexin 37, Ig-idiotype, p15, gp75, a GM2 ganglioside, a GD2 ganglioside, a human papillomavirus protein, imp- 1, P1A, SCP-1 CT-7, c-erbB-2, FcRL5/FcRH5, Flt3, muc16, muc17, mmp9, FAP, Lewis- Y, EGFRvIII, GPC3, GPRC5D, gpA33, 5T4, SSTR2, CD73, CD25, CD45, and CD133. In a further aspect, the invention provides for a bispecific antigen binding protein comprising:
P62011579wo 4 i) a first an immunoglobulin single variable domain (ISVD that specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128), wherein the first ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9; and, ii) a second ISVD that specifically binds an extracellular portion of PD-L1, wherein the second ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12; e) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has 2 or 1 amino acid difference(s)
P62011579wo 5 with the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 15; and, f) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 18. In one embodiment, in the bispecific antigen binding protein: i) the first ISVD comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 19; b) the amino acid sequence as set forth in SEQ ID NO: 20; and c) the amino acid sequence as set forth in SEQ ID NO: 21; and, ii) ) the second ISVD comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: d) the amino acid sequence as set forth in SEQ ID NO: 22; e) the amino acid sequence as set forth in SEQ ID NO: 23; and f) the amino acid sequence as set forth in SEQ ID NO: 24. In one embodiment, at least one of the first and second ISVD is humanized. In one embodiment, at least one of i) the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
P62011579wo 6 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 114; b) the amino acid sequence as set forth in SEQ ID NO: 115; c) the amino acid sequence as set forth in SEQ ID NO: 116; and d) the amino acid sequence as set forth in SEQ ID NO: 117; and ii) the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: e) the amino acid sequence as set forth in SEQ ID NO: 108; and f) the amino acid sequence as set forth in SEQ ID NO: 110. In one embodiment, at least one of i) the first humanized ISVD comprises an amino acid sequence having at least one of SEQ ID NO: 114, 115, 116 and 117, and ii) the second humanized ISVD comprises an amino acid sequence having at least one of SEQ ID NO: 108 and 110. In one embodiment, the bispecific antigen binding protein is a bivalent bispecific antibody comprising: i) a first polypeptide comprising a first human Fc fragment and the first ISVD; and, ii) a second polypeptide comprising a second human Fc fragment and the second ISVD, wherein preferably, the antibody contains no antibody light chains. In one embodiment, each of the Fc fragments comprises a CH2 domain and a CH3 domain. In one embodiment, the first Fc fragment and the second Fc fragment comprise different amino acid sequence comprising complementary knob-into-hole modifications promoting association of the first and the second polypeptide chains of the Fc region to form a heterodimeric Fc region. In one embodiment bispecific antigen binding protein has at least one biological activity selected from: a) the bispecific antigen binding protein induces endocytosis and lysosomal degradation of PD-L1 when brought into contact with a cell expressing PD-L1 and RNF128; and, b) the bispecific antigen binding protein induces at least one of secretion of a cytokine and killing of tumor cells expressing PD-L1 and RNF128, when added to a coculture of the tumor cells and human PBMCs, wherein the secreted cytokine includes at least one of IL-2 and interferon γ. In a further aspect, the invention provides for a pharmaceutical composition comprising a bispecific antigen binding protein as described herein and a pharmaceutically acceptable carrier. In yet a further aspect, the invention provides for a bispecific antigen binding protein as described herein, or a pharmaceutical composition according as described herein for use as a medicament. In one embodiment the bispecific antigen binding protein as described herein, or a
P62011579wo 7 pharmaceutical composition according as described herein for use in the treatment of a cancer, preferably a cancer comprising tumor cells expressing PD-L1. In yet a further aspect, the invention provides for a nucleic acid molecule comprising one or more nucleotide sequences encoding at least one polypeptide chain of a bispecific antigen binding protein as described herein, wherein preferably the one or more nucleotide sequences are operably linked to regulatory sequences for expression of the one or more polypeptide chains in a host cell. In yet a further aspect, the invention provides for a host cell comprising the nucleic acid molecule as described herein. In yet a further aspect, the invention provides for a method for producing a bispecific antigen binding protein as described herein, the method comprising culturing a host cell as described herein such that the one or more nucleotide sequences are expressed, and the bispecific antigen binding protein is produced. In one embodiment, the method further comprises the steps of: recovery of the bispecific antigen binding protein, and, optionally, formulation of the bispecific antigen binding protein with a pharmaceutically acceptable carrier. In yet a further aspect, the invention provides for an RNF128 binding protein comprising an immunoglobulin single variable domain (ISVD) that specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128), wherein the ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9.
P62011579wo 8 In an embodiment, the immunoglobulin single variable domain comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9. In an embodiment, the ISVD comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 19; b) the amino acid sequence as set forth in SEQ ID NO: 20; and c) the amino acid sequence as set forth in SEQ ID NO: 21. In an embodiment, the ISVD comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 19. In an embodiment, the ISVD is humanized. In an embodiment the humanized ISVD comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 114; b) the amino acid sequence as set forth in SEQ ID NO: 115; c) the amino acid sequence as set forth in SEQ ID NO: 116; and d) the amino acid sequence as set forth in SEQ ID NO: 117. Description of the invention Definitions Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. “A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article “a” or “an” thus usually means “at least one”. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
P62011579wo 9 “About” and “approximately”: these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth. “And/or”: The term “and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases. “Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. Exemplary": this term means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein. As used herein "cancer'' and "cancerous", refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer is also referred to as malignant neoplasm. As used herein, "in combination with" is intended to refer to all forms of administration that provide a first drug together with a further (second, third) drug. The drugs may be administered simultaneous, separate or sequential and in any order. Drugs administered in combination have biological activity in the subject to which the drugs are delivered. As used herein "simultaneous" administration refers to administration of more than one drug at the same time, but not necessarily via the same route of administration or in the form of one combined formulation. For example, one drug may be provided orally whereas the other drug may be provided intravenously during a patient’s visit to a hospital. Separate includes the administration of the drugs in separate form and/or at separate moments in time, but again, not necessarily via the same route of administration. Sequentially indicates that the administration of a first drug is followed, immediately or in time, by the administration of the second drug. A used herein "compositions", "products" or "combinations" useful in the methods of the present disclosure include those suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral or mucosal application. The compositions, formulations, and products
P62011579wo 10 according to the disclosure invention normally comprise the drugs (alone or in combination) and one or more suitable pharmaceutically acceptable excipients. As used herein, "an effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a cancer varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. Thus, in connection with the administration of a drug which, in the context of the current disclosure, is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition. “Sequence identity” is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The terms “sequence identity” or “sequence similarity” means that two (poly)peptide or two nucleotide sequences, when optimally aligned, preferably over the entire length (of at least the shortest sequence in the comparison) and maximizing the number of matches and minimizes the number of gaps such as by the programs ClustalW (1.83), GAP or BESTFIT using default parameters, share at least a certain percentage of sequence identity as defined elsewhere herein. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning protein sequences of the invention is ClustalW (1.83) using a blosum matrix and default settings (Gap opening penalty:10; Gap extension penalty: 0.05). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments,
P62011579wo 11 the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blosum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below. Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W) Alternative conservative amino acid residue substitution classes. 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W Alternative physical and functional classifications of amino acid residues. Alcohol group-containing residues S and T Aliphatic residues I, L, V, and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T
P62011579wo 12 Flexible residues Q, T, K, S, G, P, D, E, and R The term "agent" refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. An agent can be a compound or a composition. An agent can e.g. be selected from the group consisting of: polynucleotides, polypeptides, small molecules, (bispecific) antigen binding proteins, such as antibodies and functional fragments thereof. The term "antigen-binding domain" or "antigen-binding region" refers to the portion of an antigen-binding protein that is capable of specifically binding to an antigen or epitope. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region, e.g. comprising both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding regions include single-chain Fv (scFv), single-chain antibody, Fv, single-chain Fv2 (scFv2), Fab, and Fab'. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region from a single domain antibody consisting only of heavy chains and devoid of light chains as are known e.g. from camelids, wherein the antigen-binding site is present on, and formed by, the single variable domain (also referred to as an "immunoglobulin single variable domain" or "ISVD"). Examples of such ISVDs include the single variable domains of camelid heavy chain antibodies (VHHs), also referred to as nanobodies, domain antibodies (dAbs), and single domains derived from shark antibodies (IgNAR domains). In other embodiments, an antigen-binding region comprises a non-immunoglobulin-derived domain capable of specifically binding to an antigen or epitope, such as darpins; affilins; antikalins, etc. The term "antibody" herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological and/or immunological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al.. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). An antibody can be human and/or humanized. "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. The terms "full length antibody", "intact antibody", and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG),
P62011579wo 13 or m (IgM), some of which may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An "antibody fragment" comprises a portion of a full-length antibody, e.g. the antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab’)2, F(ab)s, Fv (typically the VH and VL domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VHH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g.. Ill et al.. Protein Eng 1997;10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g. Plückthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, N.Y., pp.269-315 (1994); see also WO 93/16185; and U.S. Patent Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No.5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Various types of antibody fragments have been described or reviewed in, e.g.. Heiliger and Hudson, Nat Biotechnol 2005; 23, 1126-1136; WO2005/040219, US20050238646 and US20020161201. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. CHO, E. coli or phage), as described herein. The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, N.Y. (1988); Hammerling et al., in: "Monoclonal Antibodies and T-Cell Hybridomas," Elsevier, N.Y. (1981), pp.563-681 (both of which are incorporated herein by reference in their entireties). The term "monospecific" antibody as used herein denotes that the antibody-part of a multispecific antigen binding protein as described herein, has one or more antigen-binding sites each of which bind to the same epitope of the same antigen. The term "bispecific" means that the antibody-part of a multispecific antigen binding protein as described herein, has at least two antigen- binding sites that are able to specifically bind to at least two distinct antigenic determinants.
P62011579wo 14 Typically, a bispecific antigen binding molecule comprises two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells. The term "valent" or "valency" as used within the current application denotes the presence of a specified number of binding sites in an antigen binding molecule. As such, the terms "bivalent", "tetravalent", and "hexavalent" denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen binding molecule. An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen. Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell expressing such an antigen. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen. Ascites fluid, which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones. Typically, an immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). Light and heavy chain variable regions contain four "framework" regions interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity-determining region" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, USA) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et
P62011579wo 15 al., supra. Phrases such as “Kabat position”, "variable domain residue numbering as in Kabat" and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. The term "framework" or "FR" residues as used herein refers to the region of an antibody variable domain exclusive of those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4). The term "constant region" as defined herein refers to an antibody-derived constant region that is encoded by one of the light or heavy chain immunoglobulin constant region genes. By "constant light chain" or "light chain constant region" as used herein is meant the region of an antibody encoded by the kappa (Ck) or lambda (Cλ) light chains. The constant light chain typically comprises a single domain, and as defined herein refers to positions 108-214 of Cκ or Cλ, wherein numbering is according to the EU index (Kabat et al., 1991, supra). The term "constant heavy chain" or "heavy chain constant region" as used herein refers to the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full length IgG antibodies, the constant heavy chain, as defined herein, refers to the N-terminus of the CH1 domain to the C- terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. As used herein, Thus, the term "Fab fragment" " or "Fab region" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. Fab may refer to this region in isolation, or this region in the context of a polypeptide, multispecific antigen binding protein or antigen-binding region, or any other embodiments as outlined herein. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab’-SH are Fab’ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.
P62011579wo 16 The term "single-chain Fv" or "scFv" as used herein refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, N.Y., pp.269-315 (1994). "Scaffold antigen-binding proteins" are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen- binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In one aspect of the invention, a scaffold antigen-binding protein is selected from the group consisting of CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-domain (Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrin repeat protein (DARPin), a variable domain of antibody light chain or heavy chain (single-domain antibody, sdAb), a variable domain of antibody heavy chain (nanobody, aVH), VNAR fragments, a fibronectin (AdNectin), a C-type lectin domain (Tetranectin); a variable domain of a new antigen receptor beta-lactamase (VNAR fragments), a human gamma-crystallin or ubiquitin (Affilin molecules); a Kunitz type domain of human protease inhibitors, microbodies such as the proteins from the knottin family, peptide aptamers and fibronectin (adnectin). Programmed death-ligand 1 (PD-L1) is a 40kDa type 1 transmembrane protein. Engagement of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1 and US20070224633. An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen. The domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see Protein Eng. Des. Sel.17, 455-462 (2004) and EP1641818A1. Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulfide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further
P62011579wo 17 details see Nature Biotechnology 23(12), 1556 - 1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). A transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the Trans-body. For further details see J. Biol. Chem 274, 24066-24073 (1999). Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see J. Mol. Biol.332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol.369, 1015-1028 (2007) and US20040132028A1. A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single variable domains were derived from the variable domain of the antibody heavy chain from camelids (nanobodies or VHH fragments). Furthermore, the term single- variable domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks. Fibronectin is a scaffold which can be engineered to bind to antigen. Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the β-sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng. Des. Sel.18, 435- 444 (2005), US20080139791, WO2005056764 and US6818418B1. Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site. For further details see Expert Opin. Biol. Ther.5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges - examples of microproteins include KalataBI and conotoxin and knottins. The microproteins have a loop which can be engineered to include up to 25 amino acids without affecting the overall fold of the microprotein. For further details of engineered knottin domains, see WO2008098796. The term "Fv" or "Fv fragment" or "Fv region" as used herein refers to a polypeptide that comprises the VH and VL domains of a single antibody. The term "Fc" or "Fc region", as used herein refers to the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below. By "Fc polypeptide" or “Fc-derived polypeptide” as used herein is meant a polypeptide that comprises all or part of an Fc region. Fc polypeptides herein include but are not limited to antibodies, Fc fusions and Fc fragments. Also, Fc regions according to the invention include variants containing at least one modification that alters (enhances or diminishes) an Fc associated effector function. Also, Fc
P62011579wo 18 regions according to the invention include chimeric Fc regions comprising different portions or domains of different Fc regions, e.g., derived from antibodies of different isotype or species. Fc thus refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cγ2 (CH2) and Cγ 3 (CH3) and the hinge between Cγ 1 and Cγ 2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226, P230 or A231 to its amino-terminus, wherein the numbering is according to the EU index. The "CH2 domain" of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The "CH3 domain" comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid residue at about position 341 to an amino acid residue at about position 447 of an IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced "protuberance" ("knob") in one chain thereof and a corresponding introduced "cavity" ("hole") in the other chain thereof; see US Patent No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as herein described. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The "knob-into-hole" technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc region, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc region. In a further specific embodiment, the subunit of the Fc region comprising the knob modification additionally comprises the amino acid substitution
P62011579wo 19 S354C, and the subunit of the Fc region comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). The numbering is according to EU index of Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. A "region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C- terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)). The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody- dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation. An "activating Fc receptor" is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16A), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt accession no. P08637, version 141), also referred to as CD16 or CD16A. In humans, CD16 consists of two isoforms, CD16A and CD16B, encoded by two highly homologous genes. CD16A is a transmembrane protein expressed by lymphocytes and some monocytes, whereas CD16B is linked to the plasma membrane via a GPI anchor and primarily expressed by neutrophils. Therefore, when reference is made herein to CD16 in the context of expression on NK cells herein, usually CD16A is meant unless otherwise indicated. By "variable region" as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including Vκ and Vλ) and/or VH genes that make up the light chain (including κ and λ) and heavy chain immunoglobulin genetic loci respectively. A light or heavy chain variable region (VL or VH) comprise four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The term "hypervariable region" or "HVR," as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in
P62011579wo 20 the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53- 55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol.196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen-binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol.196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody. Table A. CDR defintions1 CDR Kabat Chotia AbM2 VH CDR1 31-35 26-32 26-35 VH CDR2 50-65 52-58 50-58 VH CDR3 95-102 95-102 95-102 VL CDR1 24-34 26-32 24-34 VL CDR2 50-56 50-52 50-56 VL CDR3 89-97 91-96 89-97 1 Numbering of all CDR definitions in Table A is according to the numbering conventions set forth by Kabat et al. (see below). 2 "AbM" with a lowercase "b" as used in Table A refers to the CDRs as defined by Oxford Molecular’s "AbM" antibody modeling software. Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological
P62011579wo 21 Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDRL2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31- 35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci.13:1619- 1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. As used herein, the term "affinity matured" in the context of antigen-binding molecules (e.g., antibodies) refers to an antigen-binding molecule that is derived from a reference antigen-binding molecule, e.g., by mutation, binds to the same antigen, preferably binds to the same epitope, as the reference antibody; and has a higher affinity for the antigen than that of the reference antigen- binding molecule. Affinity maturation generally involves modification of one or more amino acid residues in one or more CDRs of the antigen-binding molecule. Typically, the affinity matured antigen-binding molecule binds to the same epitope as the initial reference antigen-binding molecule. The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and m respectively. The term "specifically binds" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding region or antigen-binding protein can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen- binding protein (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD. Affinity can be determined in a manner known per se, depending on the specific combination of antigen-binding protein and antigen of interest. Avidity is herein understood to refer to the strength of binding of a target molecule with multiple binding sites by a larger complex of binding agents, i.e. the strength of binding of multivalent binding. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding protein and the valency, i.e. the number of binding sites present on the antigen-binding protein. Affinity, on the other hand refers to simple monovalent receptor ligand systems. Typically, an antigen-binding region or a region that has affinity for an extracellular portion of an antigen of a bispecific antigen binding protein of the invention thereof will bind its target molecule (antigen) with a dissociation constant (KD) of about 10-6 to 10-12 M or less, and preferably 10-8 to 10-
P62011579wo 22 12 M or less, and/or with a binding affinity of at least 10-6 M or 10-7 M, preferably at least 10-8 M, more preferably at least 10-9 M, such as at least 10-10, 10-11, 10-12 M or more. Any KD value greater than 10-4 M (i.e. less than 100 μM) is generally considered to indicate non-specific binding. Thus, an antigen-binding region that “specifically binds” an antigen, is an antigen-binding domain that binds the antigen with a KD value of no more than 10-4 M, as may be determined as herein described below. Preferably, an antigen-binding region or a region that has affinity for a surface antigen expressed on NK cells of a multispecific antigen binding protein of the invention will bind to the target molecule with an affinity less than 800, 400, 200, 100, 50, 10 or 5 nM, more preferably less than 1 nM, such as less than 500, 200, 100, 50, 10 or 5 pM. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention (see e.g. Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds.. Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589- 601 (1983)). Specific illustrative embodiments are described in the following. A "KD" or "KD value" can be measured by using an ELISA as described in the Examples herein or by using surface plasmon resonance assays using a BIAcore™-2000 or a BIAcore™- 3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ∼10 - 50 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10mM sodium acetate, pH 4.8, into 5 µg/ml (∼0.2 µM) before injection at a flow rate of 5µl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of the antibody or Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25µl/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneous fitting the association and dissociation sensorgram. The equilibrium dissociation constant (KD) is calculated as the ratio koff/kon. See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds 106 M-1 S-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette. The term "humanized antibody" or "humanized immunoglobulin" refers to an immunoglobulin comprising a human framework, at least one and preferably all complementarity determining regions (CDRs) from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least about 85%, at least 90%, and at least 95% identical. Hence, all parts of a humanized immunoglobulin, except possibly
P62011579wo 23 the CDRs, are substantially identical to corresponding parts of one or more native human immunoglobulin sequences. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. See, e.g., Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370 (each of which is incorporated by reference in its entirety). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Mol. Immunol., 28:489498 (1991); Studnicka et al., Prot. Eng.7:805814 (1994); Roguska et al., Proc. Natl. Acad. Sci. 91:969973 (1994), and chain shuffling (U.S. Pat. No. 5,565,332), all of which are hereby incorporated by reference in their entireties. One class of antigen-binding regions for use in the invention comprises immunoglobulin single variable domains (ISVDs) with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring single variable domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring single variable domain sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the prior art on humanization including e.g. Jones et al. (Nature 321:522- 525, 1986); Riechmann et al., (Nature 332:323-329, 1988); Presta (Curr. Op. Struct. Biol.2:593- 596, 1992), Vaswani and Hamilton (Ann. Allergy, Asthma and Immunol., 1:105-1151998); Harris (Biochem. Soc. Transactions, 23:1035-1038, 1995); Hurle and Gross (Curr. Op. Biotech., 5:428- 433, 1994), and specific prior art relating to humanization of VHHs such as e.g. Vincke et al. (2009, J. Biol. Chem. 284:3273–3284). Again, it should be noted that such humanized single variable domains of the invention can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring single variable domain as a starting material. "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the
P62011579wo 24 number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence. As an alternative to humanization, human antibodies can be generated. By “human antibody” is meant an antibody containing entirely human light and heavy chains as well as constant regions, produced by any of the known standard methods. For example, transgenic animals (e.g., mice) are available that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region PH gene in chimeric and germ- line mutant mice results in the complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ line mutant mice will result in the production of human antibodies after immunization. See, e.g., Jakobovits et al., Proc. Nat. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Human antibodies may also be generated by in vitro activated B cells or SCID mice with its immune system reconstituted with human cells. Once a human antibody is obtained, its coding DNA sequences can be isolated, cloned and introduced into an appropriate expression system i.e. a cell line, preferably from a mammal, which subsequently express and liberate it into a culture media from which the antibody can be isolated. The term “tumor associated antigen” (TAA) as used herein means any antigen including but not limited to a protein, glycoprotein, ganglioside, carbohydrate, lipid that is associated with cancer. Such antigen can be expressed on malignant cells or in the tumor microenvironment such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates. Expressly included in the term TAA are homologues of a wild-type TAA that differs therefrom as a result of tumor-specific mutations (which can be patient-specific or shared) and that result in altered amino acid sequences, i.e. so-called neoantigens. A “nucleic acid construct” or “nucleic acid vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term “nucleic acid construct” therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms
P62011579wo 25 “expression vector” or expression construct" refer to nucleic acid molecules that are capable of effecting expression of a nucleotide sequence or gene in host cells or host organisms compatible with such expression vectors or constructs. These expression vectors typically include regulatory sequence elements that are operably linked to the nucleotide sequence to be expressed to effect its expression. Such regulatory elements usually at least include suitable transcription regulatory sequences and optionally, 3’ transcription termination signals. Additional elements necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism of the invention whereas an expression construct will usually integrate in the host cell’s genome for it to be maintained. Techniques for the introduction of nucleic acid into cells are well established in the art and any suitable technique may be employed, in accordance with the particular circumstances. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. adenovirus, AAV, lentivirus or vaccinia. For microbial, e.g. bacterial, cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduced nucleic acid may be on an extra-chromosomal vector within the cell or the nucleic acid may be integrated into the genome of the host cell. Integration may be promoted by inclusion of sequences within the nucleic acid or vector which promote recombination with the genome, in accordance with standard techniques. The introduction may be followed by expression of the nucleic acid to produce the encoded fusion protein. In some embodiments, host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid, so that the encoded fusion protein polypeptide is produced, when an inducible promoter is used, expression may require the activation of the inducible promoter. As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. The term “selectable marker” is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. The term “reporter” may be used interchangeably with
P62011579wo 26 marker, although it is mainly used to refer to visible markers, such as green fluorescent protein (GFP). Selectable markers may be dominant or recessive or bidirectional. As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3- dimensional structure or origin. The term “signal peptide” (sometimes referred to as signal sequence) is a short peptide (usually 16-30 amino acids long) present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. At the end of the signal peptide there is usually a stretch of amino acids that is recognized and cleaved by signal peptidase either during or after completion of translocation (from the cytosol into the secretory pathway, i.e. ER) to generate a free signal peptide and a mature protein. Signal peptides are extremely heterogeneous, and many prokaryotic and eukaryotic signal peptides are functionally interchangeable even between different species however the efficiency of protein secretion may depend on the signal peptide. Suitable signal peptides are generally known in the art e.g. from Käll et al. (2004 J. Mol. Biol.338: 1027– 1036) and von Heijne (1985, J Mol Biol.184 (1): 99–105). The term “gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, a coding region and a 3’ non-translated sequence (3’ end) comprising a polyadenylation site. “Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide. The term “homologous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. When used to indicate the relatedness of two nucleic acid sequences the term “homologous” means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend
P62011579wo 27 on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later. The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other. As used herein, the terms "therapies" and "therapy" can refer to any protocol(s), method(s) and/or agent(s) that can be used in the prevention, treatment, management or amelioration of the disease. A preferred disease is a cancer, preferably a cancer comprising tumor cells expressing PD-L1. The terms "treat", "treating" and "treatment" can refer to the reduction or amelioration of the progression, severity, and/or duration of a disease, preferably a cancer, preferably a cancer comprising tumor cells expressing PD-L1, and/or reduces or ameliorates one or more symptoms of the disease. As used herein, the term "effective amount" refers to the amount of a therapy, i.e. the (bispecific) antigen binding protein as defined herein, which is sufficient to reduce the severity, and/or duration of a disease, ameliorate one or more symptoms thereof, prevent the advancement of the disease, or cause regression of the disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of the disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent). Preferably, the disease is a cancer, preferably a cancer comprising tumor cells expressing PD-L1. The medical use herein described is formulated as an antigen binding protein, preferably a bispecific antigen binding protein, for use as a medicament for treatment of the stated disease(s), but could equally be formulated as a method of treatment of the stated disease(s) using a (bispecific) antigen binding protein as defined herein, a (bispecific) antigen binding protein as defined herein for use in the preparation of a medicament to treat the stated disease(s), and use of a (bispecific) antigen binding protein as defined herein for the treatment of the stated disease(s) by administering an effective amount as defined herein. Such medical uses are all envisaged by the present invention.
P62011579wo 28 Detailed description of the invention The immune system has evolved to identify and eliminate aberrant cells, such as tumor cells. Recognition of these cells is enabled via interaction between peptide-major histocompatibility complexes (MHCs) expressed on cancer cells and T cell receptors (TCRs) on T cells. This process is furthermore regulated to a large extent by a set of co-stimulatory and co-inhibitory receptors and their ligands, termed immune checkpoints. Among these modulators, programmed death ligand 1 (PD-L1; also known as B7-H1 and CD274) and its receptor, PD-1, have emerged as therapeutic targets and have exhibited considerable clinical efficacy in various cancers. Despite many successes, immune checkpoint blockage therapies have however also been linked with certain challenges, such as acquired resistance and adverse side effects. E3 ligase-induced targeted protein degradation has emerged as a promising strategy in both oncology and immuno-oncology (I/O), offering unique advantages over traditional blocking antibodies. The current invention concerns the inventive concept of using bispecific antigen binding proteins for targeted internalisation and subsequent degradation of membrane-bound proteins. The bispecific antigen binding proteins of the invention can simultaneously bind an RNF128 transmembrane E3 ubiquitin ligase (RNF128) and an extracellular portion of a tumor associated antigen (TAA) such as PD-L1. Induced proximity (i.e. “forced dimerization”) of the ubiquitin ligase with the desired TAA will result in ubiquitination of the target followed by its removal from the cell surface and subsequent degradation. E.g. as a consequence, cancer cell growth is compromised. A schematic representation of an exemplary embodiment of the invention is provided in Figure 5. This approach allows for sustained and comprehensive suppression of disease-associated proteins, potentially minimizing off-target effects and overcoming limitations associated with traditional inhibitors. In scenarios where the target lacks suitable binding sites for blocking antibodies or exhibits complex structural features, E3 ligase-induced degradation provides an alternative avenue for therapeutic intervention. In the indications of immuno-oncology, this innovative approach enables precise immunomodulation by selectively degrading proteins associated with immune checkpoints, such as PD-1 and PD-L1. By leveraging the cell's natural protein degradation machinery, targeted protein degradation allows for adjustable control over the tumor microenvironment, offering potential benefits in overcoming resistance mechanisms and shaping the intricate balance between anti-tumor immunity and immune tolerance. The advantages of this approach include at least the following: i) The heterobifunctional molecules of the invention allow for strong gains in potency, requiring only sub-stoichiometric amounts of the molecule compared to their target molecules when compared to conventional ‘occupancy-based’ therapeutics. ii) The required specific binding of two proteins, i.e. a transmembrane E3 ubiquitin ligase as well as a TAA also reduces potential off-target toxicity. Preferably, ubiquitin ligases that localize to the plasma membrane and display increased expression in cancer cells will be employed.
P62011579wo 29 iii) Targeting protein degradation leads to a prolonged pharmacodynamic effect as compared to standard antibodies, due to the time required to synthesize sufficient amounts of a new transmembrane protein. iv) The bispecific antigen binding proteins bind to the extracellular protein parts and thus do not need to cross the cell membrane. Accordingly in a first aspect, the invention pertains to a bispecific antigen binding protein comprising: i) a first an immunoglobulin single variable domain (ISVD) that specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128); and, ii) a second ISVD that specifically binds an extracellular portion of a tumor associated antigen (TAA). The inventors have discovered that incorporation of non-blocking antibodies in the bispecific antigen binding proteins as described herein allows for full removal of a target from the cell surface. The use of non-blocking antibodies has a couple of advantages: - It allows distinguishing the effects of degradation versus blocking strategies; and - In comparison to conventional blocking antibodies, the use of non-blocking PD-L1 binders in the bispecific antigen binding proteins as described herein offer the possibility for tissue-specific therapeutic effects (thereby limiting toxicity). When non-blocking binding arms are used, degradation and functional inactivation of the target will ONLY happen in tissues where both target and E3 are expressed. In one embodiment at least one of the first and second ISVD comprises the binding domain of a non-blocking antibody. In one embodiment, the immunoglobulin variable regions can comprise or consist of a single chain antigen binding domain such as a scFv, a VH domain, a VL domain, or an immunoglobulin single variable domain (ISVD) such as a dAb, a V-NAR domain or a VHH domain. Preferably the bispecific antigen binding protein comprises a first and second ISVD. The first ISVD of the bispecific antigen binding protein as described herein specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128). The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. In one embodiment, the first ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID
P62011579wo 30 NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9. The second ISVD of the bispecific antigen binding protein as described herein specifically binds a TAA. In one embodiment, the antigen-binding region that specifically binds a TAA is an antigen-binding region derived from immunoglobulin or non-immunoglobulin scaffolds as defined above. Preferably, the antigen-binding region that specifically binds a TAA comprises or consists of at least one immunoglobulin variable domain. More preferably, the antigen-binding region that specifically binds a TAA comprises or consists of a Fab that specifically binds a TAA or an immunoglobulin single variable domain (ISVD) that specifically binds a TAA. In one embodiment, the antigen-binding region that specifically binds a TAA is an antigen-binding region that binds the TAA with a KD value of no more than 10-4 M, as may be determined as herein described above. In one embodiment, the antigen-binding region that specifically binds a TAA comprises or consists of a human or humanized immunoglobulin variable region or immunoglobulin single variable region as herein defined above. As used herein, the term tumor-associated antigen (TAA) refers to an antigen that is differentially expressed by cancer/tumor cells as compared to normal, i.e. non-tumoral cells. Alternatively, a TAA can be an antigen that is expressed by non-tumoral cells (e.g. immune cells) having a pro-tumoral effect (e.g. an immunosuppressive effect), and can thereby be exploited in order to target cancer cells. A TAA can thus be any antigen that potentially stimulates apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, or expressed at lower levels or less frequently, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other TAAs are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations, including neo-antigens. Still other TAAs antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Still other TAAs can be expressed on immune cells capable of contributing to or mediating a pro-tumoral effect, e.g. cell that contributes to immune evasion, a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell.
P62011579wo 31 The TAAs are usually normal cell surface antigens which are either overexpressed or expressed at abnormal times or are expressed by a targeted population of cells. Ideally the target TAA is expressed only on proliferative cells (e.g., tumor cells) or pro-tumoral cells (e.g. immune cells having an immunosuppressive effect), however this is rarely observed in practice. As a result, target antigens are in many cases selected on the basis of differential expression between proliferative/disease tissue and healthy tissue. Examples of TAAs include Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), a Siglec family member, for example CD22 (Siglec2) or CD33 (Siglec3), CD79, CD123, CD138, CD171, CTLA-4 (CD152), PD1, PSCA, L1-CAM, EpCAM, PSMA (prostate specific membrane antigen), BCMA, TROP2, STEAP1, CD52, CD56, CD80, CD70, E-selectin, EphB2, EPHA4, Melanotransferrin, Mud 6 and TMEFF2. Examples of TAAs also include Immunoglobulin superfamily (IgSF) proteins such as cytokine receptors, Killer-lg Like Receptor, CD28 family proteins, for example, Killer-lg Like Receptor 3DL2 (KIR3DL2), B7.1, B7.2, B7-H3, B7-H4, B7-H6, PD-L1, IL-6 receptor. Examples of TAAs further include MAGE, MART-1/Melan-A, gp100, major histocompatibility complex class l-related chain A and B polypeptides (MICA and MICB), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017- 1A/GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1, nectins (e.g. nectin-4), major histocompatibility complex class l-related chain A and B polypeptides (MICA and MICB), proteins of the UL16-binding protein (ULBP) family, proteins of the retinoic acid early transcript-1 (RAET1) family, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, CEACAM5, etv6, amll, prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens, e.g. MAGE-A3, GAGE-family of tumor antigens, anti-Müllerian hormone Type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, ROR1 (also known as Receptor Tyrosine Kinase-Like Orphan Receptor 1 or NTRKR1 (EC 2.7.10.1), SLAMF7, TRAILR1, TRAILR2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, e.g. MUC1 or MUC1-C, VEGF, VEGF receptors, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR/ERBB1), members of the human EGF-like receptor family, e.g., HER-2/neu, HER-3/ERBB3, HER-4/ERBB4 or a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, cMET, integrin receptors, α5β3 integrins, α5β1 integrins, αllbβ3-integrins, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, IL-1 accessory Protein, CSF1R (tumor-associated monocytes and macrophages), a-fetoprotein, mesothelin, Isoform 2 of Claudin-18 (Claudin 18.2), folate receptor alpha (FRα, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, α-catenin, β-catenin and γ-catenin, Plexin-A1, TNFRSF10B, AXL, EDNRB, OLR1, ADAM12, PLAUR, CCR6, p120ctn, PRAME, NY-ESO-1, cdc27, CDCP1, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2,
P62011579wo 32 FcRL5/FcRH5, Flt3, muc16, muc17, mmp9, FAP, Lewis-Y, EGFRvIII, GPC3, GPRC5D, gpA33, 5T4, SSTR2, CD73, CD25, CD45, and CD133, although this is not intended to be exhaustive. Thus, in one embodiment, the bispecific antigen binding protein as described herein comprises a second ISVD that specifically binds to a TAA selected from the group consisting of: PD-L1, PD1, PSCA, L1-CAM, EpCAM, Her2 (ErbB2/Neu), Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, Glycoprotein NMB, CanAg, CD22 (Siglec2), CD33 (Siglec3), CD79, CD123, CD138, CD171, CTLA-4 (CD152), PSMA (prostate specific membrane antigen), BCMA, TROP2, STEAP1, CD52, CD56, CD80, CD70, E-selectin, EphB2, EPHA4, Melanotransferrin, Mud 6, TMEFF2, Killer lg-Like Receptor, Killer lg- Like Receptor 3DL2 (KIR3DL2), B7.1, B7.2, B7-H3, B7-H4, B7-H6, IL-6 receptor, IL-1 accessory Protein, MAGE, MART-1/Melan-A, gp100, MICA, MICB, adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1, nectin-4, a UL16- binding protein (ULBP), a RAET1 protein, carcinoembryonic antigen (CEA), CEACAM5, etv6, aml1, prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-A3, a GAGE-tumor antigen, anti-Müllerian hormone Type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, NTRKR1 (EC 2.7.10.1), SLAMF7, TRAILR1, TRAILR2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC1, MUC1-C, VEGF, VEGFR2, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR/ERBB1), HER-3/ERBB3, HER-4/ERBB4, a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, cMET, CA125, integrin receptors, a5β3 integrins, a5β1 integrins, αllbβ3-integrins, PDGF alpha receptor, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, IL-1 accessory protein, CSF1R, α- fetoprotein, mesothelin (MSLN), Isoform 2 of Claudin-18 (Claudin 18.2, CLDN18), folate receptor alpha (FRα, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, α-catenin, β-catenin and γ-catenin, Plexin-A1, TNFRSF10B, AXL, EDNRB, OLR1, ADAM12, PLAUR, CCR4, CCR6, p120ctn, PRAME, NY-ESO-1, cdc27, CDCP1, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, a GM2 ganglioside, a GD2 ganglioside, a human papillomavirus protein, imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, FcRL5/FcRH5, Flt3, muc16, muc17, mmp9, FAP, Lewis-Y, EGFRvIII, GPC3, GPRC5D, gpA33, 5T4, SSTR2, CD73, CD25, CD45, and CD133. In one embodiment, the bispecific antigen binding protein as described herein comprises at a second ISVD that is obtained/obtainable from a cytotoxic monoclonal antibody against a TAA as is known in the art. In one embodiment, the at least one antigen-binding region at least comprises the six CDR sequences that are obtained/obtainable from a monoclonal antibody against a TAA as is known in the art. In one embodiment, the at least one antigen-binding region at least comprises the variable light (VL) domain and variable heavy (VH) domain sequences that are obtained/obtainable from a monoclonal antibody against a TAA as is known in the art. Examples of such monoclonal antibodies against TAAs include: trastuzumab (to HER2), pertuzumab (to HER2), margetuximab (to HER2), rituximab (to CD20), tositumomab (to CD20), ibritumomab (to CD20),
P62011579wo 33 obinutuzumab (to CD20), ofatumumab (to CD20), alemtuzumab (to CD52), blinatumomab (to CD19), inebilizumab (to CD19), tafasitamab (to CD19), daratumumab (to CD38), isatuximab (to CD38), polatuzumab (to CD79b), talacotuzumab (to CD123), dinutuximab (to GD2), naxitamab (to GD2), bevacizumab (to VEGF-A), elotuzumab (to SLAMF7), enfortumab (to nectin-4) sacituzumab (to TROP2), mogamulizumab (to CCR4), ipilimumab (to CTLA-4), tremelimumab (to CTLA-4), durvalumab (to PD-L1), pidilizumab (to PD-1), pembrolizumab (to PD-1), nivolumab (to PD-1), cemiplimab (to PD-1), avelumab (to PD-L1), atezolizumab(to PD-L1), cosibelimab (to PD-L1), cetuximab (to EGFR), necitumumab (to EGFR), panitumumab (to EGFR), amivantamab (bispecific to EGFR and cMet), onartuzumab (monovalent to cMet), olaratumab (to PDGFRα), enoblituzumab (to B7-H3), vobramitamab (to B7-H3), zolbetuximab (to isoform 2 of Claudin-18), mirvetuximab (to folate receptor alpha, FRα), farletuzumab (to folate receptor alpha, FRα), tisotumab (tissue factor, CD142), rovalpituzumab (to DLL3), omburtamab (to B7-H3) and ramucirumab (to VEGFR2). In a further aspect, the invention provides for a bispecific antigen binding protein comprising: i) a first an immunoglobulin single variable domain (ISVD) that specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128), wherein the first ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9. and,
P62011579wo 34 ii) a second ISVD that specifically binds an extracellular portion of PD-L1, wherein the second ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12; e) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 15; and, f) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 18. In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12.
P62011579wo 35 In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 15. In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 18. In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ
P62011579wo 36 ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12. In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 15. In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 18. In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID
P62011579wo 37 NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12. In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 15. In one embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 18. In a preferred embodiment, the bispecific antigen binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3 and a second ISVD comprising a CDR1 comprising an amino acid sequence as set forth in SEQ ID
P62011579wo 38 NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12. Preferably, the bispecific antigen binding protein comprises a first ISVD that specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128) comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9. More preferably, the bispecific antigen binding protein comprises a first ISVD that specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128) comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6. Even more preferably, the bispecific antigen binding protein comprises a first ISVD that specifically binds an extracellular portion of a RNF128 transmembrane E3 ubiquitin ligase (RNF128) comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3. Preferably, the bispecific antigen binding protein comprises a second ISVD that specifically binds an extracellular portion of PD-L1, wherein the second ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 18. More preferably, the bispecific antigen binding protein comprises a second ISVD that specifically binds an extracellular portion of PD-L1, wherein the second ISVD comprises a CDR1 comprising an amino acid sequence
P62011579wo 39 as set forth in SEQ ID NO: 13 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 15. Even more preferably, the bispecific antigen binding protein comprises a second ISVD that specifically binds an extracellular portion of PD-L1, wherein the second ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12. In one embodiment, the bispecific binding protein as described herein comprises a first ISVD that comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 19; b) the amino acid sequence as set forth in SEQ ID NO: 20; and c) the amino acid sequence as set forth in SEQ ID NO: 21; and, ii) ) the second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: d) the amino acid sequence as set forth in SEQ ID NO: 22; e) the amino acid sequence as set forth in SEQ ID NO: 23; and f) the amino acid sequence as set forth in SEQ ID NO: 24. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with
P62011579wo 40 the amino acid sequence as set forth in SEQ ID NO: 19 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 22. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 23. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 24. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 20 and ii) a second ISVD comprises an amino
P62011579wo 41 acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 22. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 20 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 23. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 20 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 24. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least
P62011579wo 42 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 22. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 23. In one embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 24. In a preferred embodiment, the bispecific antigen binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19 and ii) a second ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least
P62011579wo 43 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity the amino acid sequence as set forth in SEQ ID NO: 22. Preferably the first ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 21. More preferably the first ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 20. Even more preferably the first ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 19. Preferably the second ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 24. More preferably the second ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 23. Even more preferably, the second ISVD of the bispecific binding protein as described herein comprises heavy an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least
P62011579wo 44 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 22. In one embodiment, the first and the second ISVD of the bispecific binding protein as described herein have the same percentage sequence identity of their respective heavy chains. In one embodiment, at least one of the first and second ISVD is humanized as defined herein above. In one embodiment, at least the first ISVD is humanized. The first humanized ISVD preferably comprises a CDR1, CDR2 and CDR3 selected from the group consisting of a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9. Preferably, the first humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3. Preferably, the first humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3.
P62011579wo 45 Preferably, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 114; b) the amino acid sequence as set forth in SEQ ID NO: 115; c) the amino acid sequence as set forth in SEQ ID NO: 116; and d) the amino acid sequence as set forth in SEQ ID NO: 117. Preferably, the first humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 and has an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 114; b) the amino acid sequence as set forth in SEQ ID NO: 115; c) the amino acid sequence as set forth in SEQ ID NO: 116; and d) the amino acid sequence as set forth in SEQ ID NO: 117. Preferably, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 114; b) the amino acid sequence as set forth in SEQ ID NO: 115; and c) the amino acid sequence as set forth in SEQ ID NO: 117. Preferably, the first humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 and has an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
P62011579wo 46 least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 114; b) the amino acid sequence as set forth in SEQ ID NO: 115; and c) the amino acid sequence as set forth in SEQ ID NO: 117. In one embodiment, at least the second ISVD is humanized. The second humanized ISVD preferably comprises a CDR1, CDR2 and CDR3 selected from the group consisting of d) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12; e) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 15; and, f) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 18. Preferably, the second humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 12. Preferably, the second humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 12.
P62011579wo 47 Preferably, the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 107; b) the amino acid sequence as set forth in SEQ ID NO: 108; c) the amino acid sequence as set forth in SEQ ID NO: 110; and d) the amino acid sequence as set forth in SEQ ID NO: 111. Preferably, the first humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 12, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 13 and has an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 107; b) the amino acid sequence as set forth in SEQ ID NO: 108; c) the amino acid sequence as set forth in SEQ ID NO: 110; and d) the amino acid sequence as set forth in SEQ ID NO: 111. Preferably, the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 108; and b) the amino acid sequence as set forth in SEQ ID NO: 110. Preferably, the first humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 10, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 12, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 13 and has an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
P62011579wo 48 least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 108; and b) the amino acid sequence as set forth in SEQ ID NO: 110. Preferably, the first and the second ISVD are humanized. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 114 and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 108. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 114 and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 110. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 115 and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at
P62011579wo 49 least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 108. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 115 and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 110. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 116 and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 108. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 116 and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at
P62011579wo 50 least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 110. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 117 and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 108. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 117 and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 110. In one embodiment, the bispecific antigen binding protein as described herein is a bivalent bispecific antibody comprising: i) a first polypeptide comprising a first human Fc fragment and the first ISVD; and, ii) a second polypeptide comprising a second human Fc fragment and the second ISVD, wherein preferably, the antibody contains no antibody light chains. In one embodiment, each of the Fc fragments comprises a CH2 domain and a CH3 domain. In one embodiment, the immunoglobulin Fc region at least comprises at least one of a CH2 and CH3 domain and a hinge region. In one embodiment, the immunoglobulin Fc region comprises or consists of a hinge region and a CH2 and CH3 domain. In one embodiment, the first and/or second ISVD may be fused to the Fc fragment via a hinge region or via a linker. In antibodies, the "hinge" or "hinge region" or "hinge domain" refers to the flexible portion of a heavy chain located between the CH1 domain and the CH2 domain. It is
P62011579wo 51 approximately 25 amino acids long, and is divided into an "upper hinge," a "middle hinge" or "core hinge," and a "lower hinge." A "hinge subdomain" refers to the upper hinge, middle (or core) hinge or the lower hinge. Hinge regions of an IgG1, IgG2, IgG3 and IgG4 are well known and described in the art. Suitable linker-amino acid sequences for linking the various functional domains and regions in a multispecific antigen binding protein as described herein, are known in the art (e.g. from Chen et al., 2013, Adv Drug Deliv Rev.65(10): 1357–1369). Linker amino acid sequence can be rigid but are usually flexible. Flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties. Preferred flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). An example of preferred (and widely used) flexible linker has the sequence of (GGGGS)n (SEQ ID NO: 46). By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions. Specific examples of GS linkers include (GGGGS)4 (SEQ ID NO: 47), GGGSGGG (SEQ ID NO: 48), GGSGGGGSGG (SEQ ID NO: 49) and G. Besides the GS linkers, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but can contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility, such as e.g. the flexible linkers KESGSVSSEQLAQFRSLD (SEQ ID NO: 50) and EGKSSGSGSESKST (SEQ ID NO: 51), that have been applied for the construction of a bioactive scFvs. Additional examples of flexible linkers are represented by SEQ ID Nos: 52-95. Examples of rigid linkers can be found in SEQ ID Nos: 96-99. In one embodiment, a “knob-into-holes” approach is used in which the CH3 domain interface of the antibody Fc region is mutated so that the antibodies preferentially form heterodimers (further including the attached light chains). These mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. For example, one heavy chain comprises a T366W substitution and the second heavy chain comprises a T366S, L368A and Y407V substitution, see, e.g. Ridgway et al (1996) Protein Eng., 9, pp.617-621; Atwell (1997) J. Mol. Biol., 270, pp.26-35; and W02009/089004, the disclosures of which are incorporated herein by reference. In another approach, one heavy chain comprises a F405L substitution, and the second heavy chain comprises a K409R substitution, see, e.g., Labrijn et al. (2013) Proc. Natl. Acad. Sci. U.S.A., 110, pp.5145-5150. In another approach, one heavy chain comprises T350V, L351Y, F405A, and Y407V substitutions and the second heavy chain comprises T350V, T366S, K392L, and T394W substitutions, see, e.g. Von Kreudenstein et al., (2013) mAbs 5:646-654. In
P62011579wo 52 another approach, one heavy chain comprises both K409D and K392D substitutions and the second heavy chain comprises both D399K and E356K substitutions, see, e.g. Gunasekaran et al., (2010) J. Biol. Chem.285:19637-19646. In another approach, one heavy chain comprises D221E, P228E and L368E substitutions and the second heavy chain comprises D221R, P228R, and K409R substitutions, see, e.g. Strop et al., (2012) J. Mol. Biol.420: 204-219. In another approach, one heavy chain comprises S364H and F405A substitutions and the second heavy chain comprises Y349Tand, T394F substitutions, see, e.g. Moore et al., (2011) mAbs 3: 546-557. In another approach, one heavy chain comprises a H435R substitution, and the second heavy chain optionally may or may not comprise a substitution, see, e.g. US Patent no.8,586,713. When such hetero- multimeric antibodies have Fc regions derived from a human lgG2 or lgG4, the Fc regions of these antibodies can be engineered to contain amino acid modifications that permit CD16 binding. In some embodiments, the antibody may comprise mammalian antibody-type N-linked glycosylation at residue N297 (Kabat EU numbering). Accordingly, in one embodiment the bispecific antigen binding protein as described herein, wherein the first Fc fragment and the second Fc fragment comprise different amino acid sequence comprising complementary knob-into-hole modifications promoting association of the first and the second polypeptide chains of the Fc region to form a heterodimeric Fc region. It is understood that when the bispecific antigen binding protein comprises heterodimeric heavy chains, the "knob-into-hole" technology as described above can be applied, wherein the CH3 domain of the first chain is modified to have a "protuberance" ("knob") and the second chain is modified to have a corresponding "cavity" ("hole"). In one embodiment, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions. In one embodiment, the bivalent bispecific antibodies as described herein have effector functions, preferably enhanced effector functions. In one embodiment, the bivalent bispecific antibodies as described herein comprising an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues), or amino acids 233- 239 (see e.g. US6737056), or at position 235, 243, 300 and 396 (see e.g. US10711069). In one embodiment, an bivalent bispecific antibodies as described herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the antibody comprises an Fc region, the oligosaccharide attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26- 32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl
P62011579wo 53 glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In one embodiment, modifications of the oligosaccharide in an bivalent bispecific antibody as described herein may be made in order to create antibody variants with certain improved properties. In one aspect, antibody variants are provided having a non-fucosylated oligosaccharide, i.e. an oligosaccharide structure that lacks fucose attached (directly or indirectly) to an Fc region. Such non-fucosylated oligosaccharide (also referred to as "afucosylated" oligosaccharide) particularly is an N-linked oligosaccharide which lacks a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one embodiment antibody variants are provided having an increased proportion of non- fucosylated oligosaccharides in the Fc region as compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e. no fucosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues, relative to the sum of all oligosaccharides attached to Asn 297 (e. g. complex, hybridand high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO2006082515, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues). Such antibodies having an increased proportion of non- fucosylated oligosaccharides in the Fc region may have improved FcyRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See, e.g., WO2003035835; WO2003055993. Examples of cell lines capable of producing antibodies with reduced fucosylation include: ^ Lecl3 CHO cells deficient in protein fucosylation (see Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);WO2003035835; and WO2004056312, especially at Example 11) ^ knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003085107), ^ cells with reduced or abolished activity of a GDP-fucose synthesis or transporter protein (see, e.g., WO2003085119, WO2003084570, WO2003085118, WO2003085102). In one embodiment, antibody variants are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function as described above. Examples of such antibody variants are described, e.g., in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO9954342; WO2004065540, WO2003011878. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO199730087; WO199858964; and WO199922764. In one embodiment, the bivalent bispecific antibodies lacks or has reduced effector functions.
P62011579wo 54 "Silenced Fc” (Fc-silent) refers to a genetically engineered Fc domain comprising mutations that abrogate binding of the Fc domain to Fc receptors (FcγR, FcR) while maintain the ability of the Fc domain to binds to neonatal Fc receptor (FcRn). Such silenced Fc exhibits extended half-life as described in Borrok M.J, et al. J Pharm Sci.2017. In one embodiment, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half- life of the antibody in vivo is important yet certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell mediated cytotoxicity (ADCC)) are unnecessary or deleterious. In one embodiment, an antibody variant comprises an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In one embodiment, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant further comprises D265A and/or P329G in an Fc region derived from a human IgG1 Fc region (see e.g. Xu, et al. Cellular immunology 2000, 200 (1), 16–26) In one embodiment, the substitutions are L234A, L235A and P329G (LALAPG) in an Fc region derived from a human IgG1 Fc region (See, e.g., WO2012130831). In one embodiment, the substitutions are L234A, L235A and P329S (LALAPS) in an Fc region derived from a human IgG1 Fc region (See, e.g.,WO1999051642). In one embodiment, the substitutions are L234A, L235A and G236R (STR) in an Fc region derived from a human IgG1 Fc region (See, e.g.,WO2021234402) In one embodiment, the substitutions are L234A, L235A and D265A (LALADA) in an Fc region derived from a human IgG1 Fc region or F234, L235E and D265A in an Fc region derived from a human IgG4 Fc region (see, e.g. WO2021234402). In one embodiment, the antibodies may have a modification at position N297 to reduce or eliminate ADCC activity, such as N297G, N297A or N297Q (see e.g. WO2014153063). Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238,265,269,270,297, 327 and 329 (US6737056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US7332581). Certain antibody variants with improved or diminished binding to certain selected FcRs are described (See, e.g., US6737056; WO2004056312, and Shields et al., J Biol. Chem.9(2): 6591- 6604(2001). In some such cases, the antibody lacks effector function. In one embodiment, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US6194551, WO9951642, and Idusogie et al. J Immunol.164: 4178-4184 (2000).See also Duncan & Winter, Nature 322:738-40 (1988), Damelang et al., Frontiers in Immunology 2024, vol 14 and WO1988007089 and WO9429351 concerning other examples of Fc region variants. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to
P62011579wo 55 ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in WO1988004936. Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat. Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO2006029879 and WO2005100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US20050014934. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (See, e.g., US7371826; WO2002060919). Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q/M428L (see e.g. US7365168) and M252Y/S254T/T256E + H433K/N434F (see e.g. WO2019147973), have been shown to increase the binding affinity to neonatal Fc receptor (FcRn) and the half-life of IgG1 in vivo. By this the serum half-life of an Fc-containing molecule could be further extended. In one embodiment, an antibody variant comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 253, and/or 310, and/or 435 of the Fc-region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region with the amino acid substitutions at positions 253, 310 and 435. In one embodiment, the substitutions are I253A, H310A and H435A in an Fc region derived from a human IgG1 Fc-region (see, e.g., Grevys, A, et al., J. Immunol.194 (2015) 5497-5508). In one embodiment, an antibody variant comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 310, and/or 433, and/or 436 of the Fc region (EU numbering of residues). In one embodiment, the antibody variant comprises an Fc region with the amino acid substitutions at positions 310, 433 and 436.
P62011579wo 56 In one embodiment, the substitutions are H310A, H433A and Y436A in an Fc region derived from a human IgG1 Fc-region (see, e.g., WO2014177460). In one embodiment, an antibody variant comprises an Fc region with one or more amino acid substitutions which increase FcRn binding, e.g., substitutions at positions 252, and/or 254, and/or 35256 of the Fc region (EU numbering of residues). In one embodiment, the antibody variant comprises an Fc region with amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T and T256E in an Fc region derived from a human IgG1 Fc-region. See e.g. teachings of WO2002060919 or WO2013165690. In one embodiment, the substitutions are M428L and N434S, n an Fc region derived from a human IgG1 Fc-region. See e.g. teachings of EP3138853B1. Fc region residues critical to the mouse Fc-mouse FcRn interaction have been identified by site- directed mutagenesis (see e.g. Dall'Acqua, W.F., et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU index numbering) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol.26 (1996) 2533; Firan, M., et al., Int. Immunol.13 (2001) 993; Kim, J.K., et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J.K., et al., Eur. J. Immunol.29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are crucial for the interaction (Firan, M., et al., Int. Immunol.13 (2001) 993; Shields, R.L., et al., J. Biol. Chem.276 (2001) 6591-6604). In Yeung, Y.A., et al. (J. Immunol.182 (2009) 7667-7671) various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined. FcRn binding and in vivo clearance/half-life determinations can be performed using methods known in the art (see, e.g., Petkova, S.B. et al.,Int'l. Immunol. 18(12):1759-1769 (2006); WO2013120929). A bispecific antigen binding protein as described herein can have a one or more biological activities, including e.g. antigen binding, endocytosis, lysosomal degradation, tumor cell killing and induction the secretion of cytokines. In one embodiment, thebispecific antigen binding protein as described herein has at least one biological activity selected from: a) the bispecific antigen binding protein induces endocytosis and lysosomal degradation of PD-L1 when brought into contact with a cell expressing PD-L1 and RNF128; and, b) the bispecific antigen binding protein induces at least one of secretion of a cytokine and killing of tumor cells expressing PD-L1 and RNF128, when added to a coculture of the tumor cells and human PBMCs, wherein the secreted cytokine includes at least one of IL-2 and interferon γ. In a further aspect, the invention relates to a pharmaceutical composition comprising a bispecific antigen binding protein as described herein, and a pharmaceutically acceptable carrier (excipient). The pharmaceutically acceptable carrier such as an adjuvant, or vehicle, is for administration of the polypeptide to a subject. Said pharmaceutical composition can be used in the methods of treatment described herein below by administration of an effective amount of the composition to a subject in need thereof. The term "subject", as used herein, refers to all animals
P62011579wo 57 classified as mammals and includes, but is not restricted to, primates and humans. The subject is preferably a male or female human of any age or race. The term "pharmaceutically acceptable carrier", as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see e.g. “Handbook of Pharmaceutical Excipients”, Rowe et al eds. 7th edition, 2012, www.pharmpress.com). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; 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, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions such as sodium; metal complexes (e.g. Zn2+-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Supplementary active compounds can also be incorporated into the pharmaceutical composition of the invention. Thus, in a particular embodiment, the pharmaceutical composition of the invention may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a chemotherapeutic agent, a cytokine, an analgesic agent, a thrombolytic or an immunomodulating agent, e.g. an immunosuppressive agent or an immunostimulating agent. The effective amount of such other active agents depends, among other things, on the amount of the polypeptide of the invention present in the pharmaceutical composition, the type of disease or disorder or treatment, etc. In one embodiment, the polypeptide of the invention is prepared with carriers that will protect said compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems, e.g. liposomes. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions, including targeted liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811 or WO2010/095940.
P62011579wo 58 The administration route of the polypeptide of the invention can be parenteral. The term "parenteral" as used herein includes intravenous, intra-arterial, intralymphatic, intraperitoneal, intramuscular or subcutaneous. The intravenous or intramuscular forms of parenteral administration are preferred. By "systemic administration" is meant intravenous, intraperitoneal and intramuscular administration. The amount of the polypeptide required for therapeutic or prophylactic effect will, of course, vary with the polypeptide chosen, the nature and severity of the condition being treated and the patient. In addition, the polypeptide may suitably be administered by pulse infusion, e.g., with declining doses of the polypeptide. Preferably the dosing is given by injections, most preferably intravenous, intramuscular or subcutaneous injections, depending in part on whether the administration is brief or chronic. Thus, in a particular embodiment, the pharmaceutical composition of the invention may be in a form suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the appropriate unit dosage form. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CremophorEM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g a polypeptide of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
P62011579wo 59 In a particular embodiment, said pharmaceutical composition is administered via intravenous (IV), intramuscular (IM) or subcutaneous (SC) route. Adequate excipients can be used, such as bulking agents, buffering agents or surfactants. The mentioned formulations will be prepared using standard methods for preparing parenterally administrable compositions as are well known in the art and described in more detail in various sources, including, for example, “Remington: The Science and Practice of Pharmacy” (Ed. Allen, L. V.22nd edition, 2012, www.pharmpress.com). It is especially advantageous to formulate the pharmaceutical compositions, namely parenteral compositions, in dosage unit form for ease administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound (polypeptide of the invention) calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or condition to be treated and its severity, the potency of the specific polypeptide of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the polypeptide of the invention will generally be administered in the range of from 0.001 to 1,000 mg/kg body weight/day, preferably about 0.01 to about 100 mg/kg body weight/day, most preferably from about 0.05 to 10 mg/kg body weight/day, such as about 1, 10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. In another aspect there is provided a bispecific antigen binding protein as described herein for use as a medicament. In one embodiment, the multispecific antigen binding is used as protein as described herein is used as an active ingredient, component or substance in a medicament. In one aspect, the invention pertains to a use of a multispecific antigen binding protein as described herein for the manufacture of a medicament, e.g. a pharmaceutical preparation comprising the bispecific antigen binding protein as an active ingredient, for the treatment, prevention or diagnosis of a disease in a subject in need thereof. In one aspect, the invention pertains to a bispecific antigen binding protein as described herein, or a pharmaceutical preparation comprising the multispecific antigen binding protein as an active ingredient, for use in the treatment, prevention or diagnosis of a disease in a subject in need thereof.
P62011579wo 60 In one aspect, the invention pertains to a method for the treatment of a disease in a subject in need thereof, wherein the method comprises the step of administering to the subject (an effective amount of) a bispecific antigen binding protein as described herein, or a pharmaceutical preparation comprising the bispecific antigen binding protein as an active ingredient. The disease to be treated, prevented or diagnosed using the bispecific antigen binding protein can be a cancer, an infectious disease, an inflammatory disease or an autoimmune disease. In one embodiment, the disease to be treated, prevented or diagnosed using the bispecific antigen binding protein is a cancer, e.g. a cancer as described below. In one embodiment, the cancer to be treated, prevented or diagnosed using the bispecific antigen binding protein is a cancer comprising tumor cells expressing PD-L1. In one embodiment, the cancer is solid cancer or a hematologic cancer. In one embodiment, the solid cancer is selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers. In one embodiment, the hematologic cancer is chosen from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia. In one embodiment, a bispecific antigen binding protein as described herein can be used as a monotherapy (i.e. without other therapeutic agents). In another embodiment, a bispecific antigen binding protein as described herein can be used in combined treatments. In one embodiment, a bispecific antigen binding protein as described herein is used in combination with another immunotherapy, e.g. a cellular immunotherapy. The bispecific antigen
P62011579wo 61 binding protein can thus be used in combination with the adoptive transfer of immune cells, including the adoptive transfer of T cells, e.g. CAR T cells, or NK cells. The NK cells can e.g. be enriched or expanded by methods known in the art or can be ex vivo NK cells as herein described. In one embodiment, a bispecific antigen binding protein as described herein can be used in combined treatments with one or more other therapeutic agents. The additional therapeutic agent or agents will normally be administered in amounts and treatment regimens typically used for that agent in a monotherapy for the particular disease or condition being treated. Such therapeutic agents when used in the treatment of cancer, include, but are not limited to anti-cancer agents and chemotherapeutic agents. Exemplary therapeutic agents that may be used as part of a combination therapy in treating cancer, include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine flutamide, drogenil, butocin, carmofur, razoxane, sizofilann, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony stimulating factor-1, colony stimulating factor-2, denileukin diftitox, interleukin-2, and luteinizing hormone releasing factor. An additional class of agents that may be used as part of a combination therapy in treating cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. Yet other agents that may be used as part of a combination therapy in treating cancer are cytotoxic monoclonal antibodies against TAA, including cytotoxic monoclonal antibody against a TAA known in the art such as: trastuzumab (to HER2), pertuzumab (to HER2), rituximab (to CD20), tositumomab (to CD20), ibritumomab (to CD20), obinutuzumab (to CD20), ofatumumab (to CD20), alemtuzumab (to CD52), blinatumomab (to CD19), inebilizumab (to CD19), tafasitamab (to CD19), daratumumab (to CD38), isatuximab (to CD38), polatuzumab (to CD79b), dinutuximab (to GD2), naxitamab (to GD2), bevacizumab (to VEGF-A), elotuzumab (to SLAMF7), enfortumab (to nectin- 4) sacituzumab (to TROP2), mogamulizumab (to CCR4), ipilimumab (to CTLA-4), tremelimumab (to CTLA-4), durvalumab (to PD1), pidilizumab (to PD-1), pembrolizumab (to PD-1), nivolumab (to PD-1), cemiplimab (to PD-1), avelumab (to PD-L1), cetuximab (to EGFR), necitumumab (to EGFR), panitumumab (to EGFR), olaratumab (to PDGFRα) and ramucirumab (to VEGFR2). In some embodiments the administration of the bispecific antigen binding protein and the other therapeutic agent can elicit an additive or synergistic effect on immunity and/or on therapeutic efficacy. In one embodiment, a bispecific antigen binding protein as described herein is used as at least one of an neoadjuvant therapy and an adjuvant therapy, in addition to a primary therapy
P62011579wo 62 comprising e.g. surgery and/or radiation therapy. As an neoadjuvant therapy, the bispecific antigen binding protein is administered before the primary treatment, e.g. to help reduce the size of a tumor (such that less extensive surgery and/or radiation therapy is required), kill cancer cells that have spread (e.g. micrometastatic disease) and/or reduce the risk of tumor cells spreading post-surgery. As an adjuvant therapy, the bispecific antigen binding protein is administered after the primary treatment, e.g. to treat minimal residual disease (destroy remaining cancer cells). The use of the m bispecific antigen binding protein as an neoadjuvant therapy and/or an adjuvant therapy lowers relapse rates. In the neoadjuvant therapy and/or adjuvant therapy, the bispecific antigen binding protein can be used as monotherapy or in in combined treatments as described above. In one aspect, the invention relates to a nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of a bispecific antigen binding protein as described herein. The nucleotide sequence encoding such a polypeptide chain preferably encodes a signal peptide operably linked to the polypeptide chain. A nucleic acid molecule comprising one or more of the nucleotide sequences encoding a polypeptide chain, further preferably comprises regulatory elements for (or conducive to) the expression of the polypeptide chain in an appropriate host cell, which regulatory elements are operably linked to the nucleotide sequence. In one aspect, the invention relates to a host cell comprising the nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of a bispecific antigen binding protein as described herein. In one embodiment, the host cell is an isolated cell or a cultured cell. Among the host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, for example Escherichia coli or bacilli. Suitable yeast cells include Saccharomyces cerevisiae and Pichia pastoris. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-1, COS-7 line of monkey kidney cells (Gluzman et al., 1981, Cell 23:175), L cells, HEK 293 cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, BHK cell lines, e.g. BHK21, BSC-1, Hep G2, 653, SP2/0, and the CVI/EBNA cell line derived from the African green monkey kidney cell line CVI as described by McMahan et al. (1991, EMBO J.10: 2821). The host cell may be any suitable species or organism capable of producing N-linked glycosylated polypeptides, e.g. a mammalian host cell capable of producing human or rodent IgG type N-linked glycosylation. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985). Host cells comprising the nucleic acid molecule of the invention can be cultured under conditions that promote expression of the polypeptide. Thus, another aspect the invention relates to a method for producing a bispecific antigen binding protein as described herein. The method preferably comprises culturing a host cell as described above such that one or more nucleotide sequences are expressed and the bispecific antigen binding protein is produced. The method preferably comprises the step of cultivating a host cell comprising one or more of the nucleotide sequences encoding a polypeptide chain of the
P62011579wo 63 bispecific antigen binding protein. The host cell is preferably cultured under conditions conducive to expression of the one or more polypeptide chains. The method can further comprise the step of recovering the bispecific antigen binding protein. The bispecific antigen binding protein can be recovered by conventional protein purification procedures, including e.g. protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, size exclusion chromatograpy or affinity chromatography, using e.g. strepavidin/biotin (see e.g. Low et al., 2007, J. Chromatography B, 848:48-63; Shukla et al., 2007, J. Chromatography B, 848:28-39), ion-exchange chromatography on strong or weak cation or anion media, or hydrophobic interaction chromatography, to name a few by example. In a further aspect, the invention relates to a method for producing a pharmaceutical composition comprising a bispecific antigen binding protein as described herein, the method comprising the steps of a) producing the bispecific antigen binding protein in a method as defined above; and b) formulating the bispecific antigen binding protein with a pharmaceutically acceptable carrier as defined above, to obtain a pharmaceutical composition. In another aspect, the invention pertains to a novel specific RNF128 binding protein. The RNF128 binding protein according to the invention is described in more detail herein, when described as being part of a bi-specific antigen binding protein of the invention. The RNF128 binding protein as described herein is however not limited to being a part of said bispecific antigen binding protein. The RNF128 binding protein according to the invention comprises a first ISVD as defined herein. Preferably the RNF128 binding protein comprises an ISVD that specifically binds an extracellular portion of RNF128, wherein the first ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth
P62011579wo 64 in SEQ ID NO: 8 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 9. Preferably, the immunoglobulin single variable domain of the RNF128 binding protein comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, wherein the CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, the CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and the CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3. Preferably, the immunoglobulin single variable domain of the RNF128 binding protein comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of: a) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3; b) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 4, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 6; and, c) a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 7, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 9. Preferably, the immunoglobulin single variable domain of the RNF128 binding protein comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, wherein the CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1, the CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2, and the CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 3. The ISVD of the RNF128 binding protein preferably comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of a) the amino acid sequence as set forth in SEQ ID NO: 19, b) the amino acid sequence as set forth in SEQ ID NO: 20; and c) the amino acid sequence as set forth in SEQ ID NO: 21.
P62011579wo 65 Preferably, the ISVD of the RNF128 binding protein preferably comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 19. The ISVD of the specific RNF128 binding protein is preferably a humanized ISVD. Preferably, the humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 3. Preferably, the humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3. The humanized ISVD preferably comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 114; b) the amino acid sequence as set forth in SEQ ID NO: 115; c) the amino acid sequence as set forth in SEQ ID NO: 116; and d) the amino acid sequence as set forth in SEQ ID NO: 117. Preferably, the first humanized ISVD comprises a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 and has an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: a) the amino acid sequence as set forth in SEQ ID NO: 114; b) the amino acid sequence as set forth in SEQ ID NO: 115;
P62011579wo 66 c) the amino acid sequence as set forth in SEQ ID NO: 116; and d) the amino acid sequence as set forth in SEQ ID NO: 117. The RNF128 binding protein can be an antibody, wherein the antibody comprises a first polypeptide comprising a first human Fc fragment and a (first) ISVD as defined herein. The antibody further preferably comprises a second polypeptide comprising a second human Fc fragment and a further ISVD. The further ISVD can be identical to the (first) ISVD comprised in the first polypeptide, or can be a different ISVD. Preferably, the antibody contains no antibody light chains. Preferably the Fc fragment comprises a CH2 domain and a CH3 domain as defined herein. In an aspect, the invention concerns a pharmaceutical composition as described herein, wherein the pharmaceutical composition comprises a specific RNF128 binding protein as described herein. The pharmaceutical composition and/or specific RNF128 binding protein can be for use as a medicament as described herein. The pharmaceutical composition and/or specific RNF128 binding protein can be for use in the treatment of a cancer, preferably a cancer comprising tumor cells expressing RNF128 as described herein. In an aspect, the invention pertains to a nucleic acid molecule as described herein, wherein the nucleic acid molecule comprises one or more nucleotide sequences encoding the specific RNF128 binding protein, Preferably the one or more nucleotide sequences are operably linked to regulatory sequences as described herein for expression of the RNF128 binding protein in a host cell. The invention further pertains to a host cell as described herein, wherein the host cell comprises said nucleic acid molecule. In an aspect, the invention further relates to a method as described herein for producing the specific RNF128 binding protein, wherein the method comprises culturing the host cell under conditions suitable for expression of the one or more nucleotide sequences and production of the specific RNF128 binding protein. Optionally the method further comprises one or more steps of recovering the specific RNF128 binding protein and formulating the protein with a pharmaceutically acceptable carrier. The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings and examples. Modifications and alternative implementations of some parts or elements are possible and are included in the scope of protection as defined in the appended claims. Description of the figures Figure 1. Characterization of PD-L1-binding VHHs (a) Detection of binding of newly generated myc-tagged anti-PD-L1 VHHs (1B8, 1G7, 2F3) to overexpressed FLAG-tagged PD-L1 on the cell surface of HEK293T cells, using immunofluorescence confocal microscopy. Cells were treated with 50 nM VHH for 16 hours (h). One representative image was shown for NT condition treated with VHHs for non-specific binding. Images were taken with a 20x lens. NT = non-transfected control, (b) Detection of binding of newly generated myc-tagged anti-PD-L1 VHHs (1B8, 1G7, 2F3) to HEK293T cells transfected with FLAG- PD-L1 and treated with 50 nM VHH for 16 h. VHH-myc was detected using Alexa 488-conjugated
P62011579wo 67 anti-myc antibody and mean fluorescence intensity (MFI) at the cell surface was determined with flow cytometry. IgG control antibody was used as a negative control. MFI levels normalized to non- treated control cells are shown. (c) Detection of binding of newly generated myc-tagged anti-PD-L1 VHHs (1B8, 1G7, 2F3) to endogenous PD-L1 at the surface of MDA-MB-231 cells, upon 16h treatment with 50 nM anti-PD-L1 VHHs. VHHs were detected with anti-myc antibody. MFI levels normalized to non-treated control cells are shown, and (d). The capacity of newly generated anti- PD-L1 VHHs to block PD1/PD-L1 pathway activity was analyzed using a Raji-PD-L1/Jurkat luciferase reporter assay (Invivogen). A PD-L1 blocking antibody (Clone #2340D, R&D Systems) was used as a positive control. Co-cultures of Raji and Jurkat cells were supplemented with 10uM anti-PD-L1 VHHs for 6h. Luciferase activity reflects the level of pathway activation. Luciferase activity was normalized to control Raji Null cells. Figure 2. Characterization of PD-L1-binding VHHs (a) Detection of binding of a newly generated set of myc-tagged anti-PD-L1 VHHs to overexpressed FLAG-tagged PD-L1, on the cell surface of HEK293T cells, using immunofluorescence confocal microscopy. Cells were treated with 50 nM VHH for 16 h. Images were taken with a 20x lens. Representative image for background binding of VHHs on NT cells is presented in Figure 1A. NT = non-transfected control (b) Heatmap of the relative binding of anti-PD-L1 VHHs to cells that overexpress PD-L1 (HEK293T) or display endogenous PD-L1 (MDA-MB-231), as assessed by flow cytometry, and (c) PD-L1 pathway blocking capacity,analyzed using a Raji-PD-L1/Jurkat reporter assay (Invivogen). A PD-L1 blocking antibody (Clone #2340D, R&D Systems) was used as a positive control. The co-cultures of Raji and Jurkat cells were incubated with VHHs for 6 h. Pathway inhibition was assessed by measuring luciferase activity. Luciferase activity was normalized to control Raji Null cells Figure 3. Characterization of RNF43-binding VHHs Detection of binding of newly generated (a) FLAG- or (b) V5-tagged - anti-RNF43 VHHs to overexpressed myc-tagged RNF128, on the cell surface of HEK293T cells, using immunofluorescence confocal microscopy. Cells were treated with 50 nM VHHs for 16 h, in the presence of 10 nM Bafilomycin A1 to block lysosomal degradation and increase signals for RNF128. Images were taken with a 20x lens. NT = non-transfected control. Figure 4. Characterization of RNF128-binding VHHs Detection of binding of newly generated (a) FLAG- or (b) V5-tagged - anti-RNF128 VHHs to overexpressed myc-tagged RNF128, on the cell surface of HEK293T cells, using immunofluorescence confocal microscopy. Cells were treated with 50 nM VHHs for 16 h, in the presence of 10 nM Bafilomycin A1 to block lysosomal degradation and increase signals for RNF128. Images were taken with a 20x lens. NT = non-transfected control. Figure 5. Schematic depiction of SureTACs’ mode of action
P62011579wo 68 The SureTAC molecule simultaneously binds PD-L1 and a membrane-bound E3 ligase. Following the binding, the E3 ligase ubiquitinates PD-L1, inducing its internalization and subsequent lysosomal degradation. Figure 6. Schematic figure of heterobispecific SureTACs molecules using a knob-in-hole strategy. Bi-specific SureTAC assembled using ‘’knob-in-hole’’ heterodimerization strategy. VHHs for PD-L1 and the E3 ligase are fused to the knob and hole variants. Figure 7. SureTACs-induced surface removal and degradation of endogenous PD-L1 (a) Levels of endogenous PD-L1 expression at the surface of colorectal cancer cells (HT29) pre- treated with 1 ng / mL of IFN^ for 1.5 days, followed by treatment with 10 nM of indicated SureTACs for 16 h, (b) Representative histograms for PD-L1 staining of flow cytometry data, (c) Assessment of PD-L1 protein levels using Western blotting. HT29 cells were treated with indicated SureTACs (10 nM) for 16 h. Actin loading control is shown, (d) HT29 cells were pre-treated with 1 ng / ml IFN^ for 1.5 days, followed by treatment with 10 nM or 100 nM of indicated SureTACs for 16 h. PD-L1 levels are shown as mean fluorescence intensity (MFI) and were normalized to PD-L1 levels of non- treated cells, (e) mean fluorescent intensity (MFI) of PD-L1 levels at the cell surface ofHT29 cells treated with 1 ng/mL IFN^ for 1.5 days followed by treatment with a dose range of indicated SureTACs (in nM) for 16 h, determined by flow cytometry, (f) levels of SureTAC binding (V5 staining) of HT29 cells treated with 1 ng / mL IFN^ for 1.5 days followed by treatment with a dose range of indicated SureTACs (in nM) for 16 h determined by flow cytometry, and (g) Timelapse montage of PD-L1 levels in mouse colorectal cancer cell line MC38, treated with 1 ng / mL of INFy 24 h before the start of the experiment. PD-L1 was visualized with a PE-labelled anti-PD-L1 antibody (1:1000; BD Biosciences) using a 1 h incubation pre-imaging. Excess antibody was washed away before the start of the experiment. Indicated SureTACs (10 nM) were added at the start of the imaging montage. Timelapse is shown as a 25 minutes (min) jump per frame display. Figure 8. Durable effects and efficiency of SureTACs as compared to existing blocking antibodies (a) Six cell lines of various cancer origins were treated with 1 ng / mL of INFy for 1.5 days, followed by treatment with 10 nM of indicated SureTACs for 16 h. PD-L1 surface levels are indicated as mean fluorescence intensity (MFI). PD-L1 levels are normalized to non-treated control samples per cell line. Significance was assessed by 2-way ANOVA analysis and data were corrected using Dunnett multiple comparison analysis. mRNA expression levels of RNF43 and RNF128 were assessed by qPRC (b) PD-L1 surface levels, indicated as mean fluorescence intensity (MFI). Mouse colorectal cancer cells (MC38) were treated for 16 h with 10 nM of control SureTACs and PD-L1-targeting SureTACs employing RNF128. The levels were assessed using flow cytometry, at indicated times after wash-out of SureTACs reagents, and normalized to non-treated samples, (c) PD-L1 surface levels , determined by flow cytometry and normalized to non-treated control samples. MC38 cells were treated with 10 nM, 1 nM and 0.1 nM control or PD-L1-targeting SureTACs for indicated times and medium was not replaced for 96 h, (d) Assessment of PD-L1 surface levels in
P62011579wo 69 HT29 cells pretreated with 1 ng / mL IF^N. upon treatment with SureTACs that employ different RNF128 VHHs (4E2; newly identified RNF128 VHH in this work and B8; previously described RNF128 VHH), compared to SureTACs containing an Atz-based PD-L1 blocking binding arm and SureTACs containing newly identified 1B8.2 PD-L1 blocking VHH. PD-L1 surface levels were determined by flow cytometry and normalized to non-treated control samples. and (e) V5 levels as MFI. Binding of SureTACs to the HT29 cell surface was assessed by V5 staining and flow cytometry. Figure 9. Functional effects of STs in a mixed lymphocyte reaction (a) IL2 and (b) IFN^ cytokine secretion in hPBMCs that were activated by SEB (50 ng / mL) and incubated with indicated STs (5 nM, 10 nM or 50 nM) for 66 h. Plots display IL2 or IFN^ concentration released in pg / mL, (c) Bar graph displaying the percentage of LDH release normalized to max release (at 100%). HT29 colorectal cancer cells were preincubated for 4 h with indicated STs (10 nM and 50 nM), then hPBMCs were added to the co-culture (ratio 3:1 Effector:Tumor cells) and tumor cell lysis was assessed by LDH secretion according to manufacturer’s protocol (Thermofisher Scientific , and (d) Graph representing the average of two separate experiments using different hPBMC donors. HT29 colorectal cancer cells were preincubated for 4 h with indicated STs (10 nM and 50 nM), then hPBMCs were added to the co-culture (ratio 3:1 Effector:Tumor cells) and tumor cell lysis was assessed by LDH secretion according to manufacturer’s protocol (Thermofisher Scientific). Figure 10: PK study and in vivo efficacy studies for PD-L1/RNF128 SureTAC. (a) Half-life of SureTac 04 (ST04) PD-L1 (2F3) x RNF128 (4E2) is comparable to monoclonal antagonistic PD-L1 antibodies. Serum concentrations of ST04 PD-L1 (2F3) x RNF128 (4E2), ST45 PD-L1 (2F3) x GFP, ST78 RNF128 (4E2) x GFP and ST74 (GFPxGFP) were analysed over time (h) following single intravenous injection with 10mg/kg of SureTACs indicated in C57BL/6JRj female mice. Mice were injected at the same time point (n=20 per group) with the indicated SureTAC, blood was sampled 3 times per mouse (5 mice per time point). Time points pre-dose, 15 min, 30 min, 1h, 2h, 1 day, 2 day, 3 day, 5 day, 10 day and 14 days. Antibody concentration in the serum was determined to calculate SureTAC half-lives; summarized in the table. (b) - (c) PD-L1/RNF128 SureTAC inhibits tumor outgrowth in dose dependent manner. (b) MC38 mouse colorectal cancer cells were injected sub-cutaneous in C57BL/6 mice. Random staggered inclusion upon tumour volume 80-100mm3. Mice (n=10 per group) were treated with control ST74 GFP x GFP or ST04 PD-L1 (2F3) x RNF128 (4E2) at indicated concentrations. Set tumor size endpoint was 1200mm3. Graph calculations non-linear regression curve fit (y=100) (c) Kaplan Meier plot depicts the treatment groups described for (b), loss of survival is indicated when mouse reached set endpoint. Figure 11: (a) Alignment of 2F3 (PD-L1 binder) and 4E2 (RNF128 binder) to the most similar human germline frameworks and suggested humanization versions (top: SEQ ID NO: 22, 100, 106- 108, 101, 109-111, 103, 112, 102 and 113, bottom: SEQ ID NO: 104, 114-117, 103, 118, 102 and 119), (b) Modelling of a PD-L1 x RNF128 bispecific SureTAC. (c) - (d) Wireframe overlay of 6
P62011579wo 70 different humanized versions of the PD-L1 (c) and RNF128 (d) binder. The humanization versions in (a) were produced as a fusion to an Fc part of human IgG1, and tested for equilibrium binding by ELISA on the antigens PD-L1 (e) and RNF128 (f). Examples ^ Example 1 bi-specific antigen binding proteins generation, panel of unique VHHs To examine proximity-induced PD-L1 endocytosis and lysosomal degradation at the endogenous level, we used a knob-in-hole (KiH) strategy to generate bispecific antibodies (SureTACs) with one arm that engages PD-L1 and another arm that binds an E3 ligase (RNF128, RNF43, RNF130 or RNF167). To generate SureTACs, various single chain antibodies (VHH; using llama immunization) were generated against PD-L1, RNF43, RNF128. These VHHs were incorporated into the KiH format to generate heterobispecific SureTACs antibodies (Figure 6). As an example, we here below describe the development and characterization of llama anti-PD-L1 and anti-RNF128 human/mouse cross-reactive antibodies Human and mouse RNF128 protein was produced in HEK293 cells fused to a hexahistidine tag, and subsequently isolated by immobilized metal affinity chromatography (IMAC). 2 llamas were primed with 50µg human and mouse RNF128 and subsequently boosted with 50 µg mouse RNF128. Peripheral blood lymphocytes were isolated from which mRNA was extracted. RNA was transcribed into cDNA using reverse transcriptase and random hexamers. IG-H cDNA fragments (both conventional and heavy chain) were amplified using primers annealing at the IGH leader sequence region and the CH2 region. The VHH specific fragment was excised and purified from an agarose gel and subsequently used as a template for a nested PCR with cloning primers. The resulting fragments were cloned into a phagemid vector as fusion with M13gpD after an amber stop codon, ligated and transformed into TG1 E. coli amber suppressor cells (K-12 supE thi-1 Δ(lac- proAB) Δ(mcrB-hsdSM)5, (rK-mK-) F' [traD36 proAB+ lacIq lacZΔM15]) by electroporation. Bacteriophages were produced by infecting with helper phage VCSM13 and precipitated with PEG- NaCL according to standard methods. Antigens were coated to MaxiSorp plates at 5, 0.5 and 0.05 µg/ml concentrations. After 2 rounds of panning individual clones were picked and the reactivity of the periplasmic extract was evaluated in a standard ELISA with either human or mouse antigen as a target. A significant proportion of these clones exhibit cross-reactivity towards mouse and human RNF128, while a smaller subset of clones display specificity solely towards human RNF128. These clones were sequences by Sanger sequencing. Different clusters of VHH molecules were sequence-annotated and selected for a diversity of antibody paratopes. A selection of clones were produced in BL21 E.coli (E. coli B dcm ompT hsdS(rB-mB-) gal) and the secreted VHH with a C- terminal HIS-tag were purified by immobilized metal affinity chromatography (IMAC). The concentration of the VHH was measured and the equilibrium constant KD was determined by
P62011579wo 71 ELISA. 20 unique anti-RNF128 VHHs were identified, for 12 binding to RNF128-overexpressing cells was shown via immunofluorescence (IF) (Figure 4) 7 distinct binders exhibited a KD of 0.5-30 nM against human and 10-500 nM against mouse RNF128. Epitope binning was performed using SPR technology and two epitope bins were confirmed.5 binders were selected for further evaluation (O4A5, O4E2, O4H4, O4D7 and L5H4). Table 1 SEQ ID RNF128 binders sequence NO: O4A5 20 EVQLVESGGGLVQAGGSLRLSCVASSTNIFSSYIMGWFRQAPGKEREFVAAINWNGRSTDYVDSVK GRFTISRDNAKKSVYLQMNSLKSEDTAVYYCVAKLGTQSDYWGQGTQVTVSS O4E2 19 EVQLVESGGGLVQAGGSLRVSCAASGRTFKYYAMGWFRQAPGKEREFVAVIGWSGGSTDYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGLSGLEPDYWGQGTQVTVSS O4H4 21 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYADSVK GRFTISRDNAKNTMYLQMNDLKPEDTAVYYCVADADLVAARPTYWGQGTQVTVSS O4D7 25 EVQLVESGGGLVQAGGSLRLSCAASGSISSFEGARWYRQAPGKTREWIASIFSYGRTTYADFVKGR FTITRDNANNAVYLQMNSLKPEDTAVYYCAVVQSGRPYYGTQGTQVTVSS L5H4 26 EVQLVESGGGLVQAGGSLRLSCTASGRGVSSYAMGWFREAVGKERELVAVINWNGRSTDYADSVK GRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAADASYGSNQIPDYWGQGTQVTVSS VHH binders against human and mouse PD-L1 were isolated in a similar way using commercial recombinant human PD-L1 (R&D Systems Catalog #: 9049-B7) and mouse PD-L1 (R&D Systems Catalog #: 9048-B7).18 unique anti-PD-L1 VHHs were identified, affinities ranging from 0,02-10 nM for human PD-L1 of <10 nM with had cross-reactivity for mouse PD-L1 (KD 8-50 nM). Functional analysis indicated we generated 2 blockers (O1G8 and O1B8.2) and 16 non-blockers of PD-L1 downstream signaling. Epitope binning was performed using SPR technology, and 3 binders were selected for further evaluation (O2F3, O1G7, O1B8.2). These VHHs bind unique epitopes on PD-L1.1B8.2 is a blocker, 2F3 and 1G7 are non-blockers Table 2 SEQ ID PD-L1 binders sequence NO: O2F3 22 EVQLVESGGGLVQTGGSLTLSCAASGGAFNSVAIAWFRQAPGKEREFVARIRLNTETDFYADS VIGRFTISTDNAKNTVSLQMNSLKPEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTQVTVS S O1G7 23 EVQLVESGGGLVEAGGSLRLSCAASGRNSYYMAWFRQAPGKDREFVAASRWSDGSTYYSDF VKGRFAISGEGGRNTLNLQMNNLKPEDTAVYYCAASKGYIYPSSGPEIAATYDYWGQGTQVTV SS O1B8.2 24 EVQLVESGGGSVQAGASLRLSCVVSGPADSTYITAWFRQRPGKDREFVAHISRGGAPYHADS VKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCAANAGFLTTLASQYPYWGQGTQVTVSS
P62011579wo 72 Development and characterization of PD-L1 and RNF128 cross-linking bispecific SureTAC antibodies DNA encoding the VHH variable domain antibody generated by gene synthesis and fused to the hinge-Fc region of a human immunoglobin. Optionally a linker sequence was used. Typically, a human IgG1 or IgG4 sequence were used. The synthesized genes fragments were inserted into mammalian expression vectors containing the corresponding heavy or light constant domains. Species and isotypes included human IgG1 and IgG4. Optional, C-terminal tags were fused to the antibody sequence for analytical purposes (e.g. HIS-tag, V5-tag, myc-tag, FLAG-tag). Recombinant antibodies were produced by transient transfection of Expi293 cells with mammalian expression vectors encoding the antibody heavy chains. Bispecific antibodies were generated using knob-into-hole technology (WO1996027011). Knob-into-Hole (KiH) refers to a set of heterodimerization mutations in the CH3 domain of an IgG Fc part. Thus creating a KNOB chain that specifically heterodimerizes with a HOLE chain. Heavy chains were encoded on separate vectors, and were transfected using a 1:1 ratio. Antibodies were purified from the cell culture supernatant by protein-A affinity chromatography, followed by a single polishing step based on hydroxyapatite, ion exchange, hydrophobic interaction or size exclusion to remove the homodimers. Homodimeric side products were typically less than 15% in spent medium and efficiently removed in downstream processing. Backbones typically included mutations to reduce effector function (e.g. L234A, L235A, P329G or P329S and/or N297G). Optionally, the Fc part contained half-life extending mutations such as M252Y, S254T and T256E (WO2002060919, WO2013165690) or M428L and N434S (EP3138853B1). Typical Fc-fusion partners used are given in table 3 Table 3: hinge and Fc sequences Description SEQ Fc derived fusion parts sequences ID NO: hIgG4 27 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1 28 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1-KO1 29 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1-KO1- 30 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE KNOB VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1-KO1- 31 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE HOLE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
P62011579wo 73 hIgG1-KO1- 32 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEV HE1-KNOB HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1-KO1- 33 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEV HE1-HOLE HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1-KO1- 34 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE HE2-KNOB VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK hIgG1-KO1- 35 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE HE2-HOLE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLT VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK hIgG1-KO2- 36 DKTHTCPPCPAPSTRGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE KNOB VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1-KO2- 37 DKTHTCPPCPAPSTRGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE HOLE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Either PD-L1 binders or RNF128 binders could be fused to either the KNOB or the HOLE Fc part without any difference in functionality. Table 4 lists some VHH-Fc fusions that were evaluated using human IgG1 based Fc with Knock-out mutations set KO1 (LALAPS) combined with KNOB or HOLE mutations. Table 4 SEQ ID RNF128 binders sequence with hIgG1-KO1-HOLE fusion NO: O4A5- 38 EVQLVESGGGLVQAGGSLRLSCVASSTNIFSSYIMGWFRQAPGKEREFVAAINWNGRSTDYVDS Fc-KO1- VKGRFTISRDNAKKSVYLQMNSLKSEDTAVYYCVAKLGTQSDYWGQGTQVTVSSDKTHTCPPCP KNOB APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK O4E2- 39 EVQLVESGGGLVQAGGSLRVSCAASGRTFKYYAMGWFRQAPGKEREFVAVIGWSGGSTDYADS Fc-KO1- VKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGLSGLEPDYWGQGTQVTVSSDKTHTCPPC KNOB PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK O4H4- 40 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYADS Fc-KO1- VKGRFTISRDNAKNTMYLQMNDLKPEDTAVYYCVADADLVAARPTYWGQGTQVTVSSDKTHTCP KNOB PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK O4D7- 41 EVQLVESGGGLVQAGGSLRLSCAASGSISSFEGARWYRQAPGKTREWIASIFSYGRTTYADFVKG Fc-KO1- RFTITRDNANNAVYLQMNSLKPEDTAVYYCAVVQSGRPYYGTQGTQVTVSSDKTHTCPPCPAPE KNOB AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
P62011579wo 74 L5H4- 42 EVQLVESGGGLVQAGGSLRLSCTASGRGVSSYAMGWFREAVGKERELVAVINWNGRSTDYADS Fc-KO1- VKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAADASYGSNQIPDYWGQGTQVTVSSDKTHTC KNOB PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID PD-L1 binders sequence NO: O2F3- 43 EVQLVESGGGLVQTGGSLTLSCAASGGAFNSVAIAWFRQAPGKEREFVARIRLNTETDFYADSVI Fc-KO1- GRFTISTDNAKNTVSLQMNSLKPEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTQVTVSSDK HOLE THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK O1G7- 44 EVQLVESGGGLVEAGGSLRLSCAASGRNSYYMAWFRQAPGKDREFVAASRWSDGSTYYSDFVK Fc-KO1- GRFAISGEGGRNTLNLQMNNLKPEDTAVYYCAASKGYIYPSSGPEIAATYDYWGQGTQVTVSSDK HOLE THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK O1B8.2- 45 EVQLVESGGGSVQAGASLRLSCVVSGPADSTYITAWFRQRPGKDREFVAHISRGGAPYHADSVK Fc-KO1- GRFTISRDNAKNTVYLQMNNLKPEDTAVYYCAANAGFLTTLASQYPYWGQGTQVTVSSDKTHTC HOLE PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALSAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK Transient expression levels were typically found to be around 200 mg/L, resulting in >100 mg/L purified heterodimeric product. All selected VHH parts had a Tm melting temperature of >65°C, typically >80°C. The bispecific SureTAC molecules maintained the affinities of the individual VHH molecules selected (1-2 mM for PD-L1 binders selected, 2-7 mM for the RNF128 binders). The bispecific SureTAC molecules were stable in standard buffer for more than 3 freeze/thaw cycles, maintaining monodispersity as judged by analytical size exclusion chromatography, and binding characteristics as judged by ELISA experiments. PD-L1/RNF128 SureTACs robustly induce PD-L1 endocytosis and lysosomal degradation To evaluate degrader activity of SureTACs directed against endogenous PD-L1, we employed colorectal cancer cell line HT29 that expresses both PD-L1 and each of the four selected E3 ligases (Figure 8a), after mining of online databases of E3 expression to study expression in healthy and tumor tissue (TCGA), (Figure 10a) HT29 cells were treated with 1 ng / ml IFN^ for 36 h to induce high levels of PD-L1 expression at the cell surface, after which cells were treated with 10 nM of SureTACs for 16 h. SureTACs that engage PD-L1 (VHH: 2F3) and RNF128 (VHH: 4E2, 4A5 and 4H4) induced robust elimination of PD-L1 from the cell surface as shown by flow cytometry (Figure 7a and b), on total protein levels (Figure 7c) and live imaging (Figure 7g). PD-L1 cell surface removal is already visible after 2 h of SureTAC treatment (Figure 8c). A PD-L1/RN128 SureTAC that employs a previously reported RNF128 binder (B8, described in WO2020139950) was unable to remove PD-L1 from the cell surface, indicating that our RNF128 binders are surprisingly successful for degrader activity (Figure 8d and e). Furthermore, three different PD-L1 binders that we characterized (blocking: 1B8.2 and non-blocking: 2F3, 1G7) performed equally well in down- regulating PD-L1 when combined with an optimal RNF128 binder (4E2) (Figure 7a and c). With
P62011579wo 75 these results, we are the first to show that a non-blocking binder can be employed in a membrane E3 degrader strategy to fully eradicate cell surface expression. Using a non-blocking PD-L1 binder holds potential for tissue specific PD-L1 degradation and lower overall toxicity when compared to blocking strategies. Relevant characteristics of PD-L1/RNF128 SureTACs - Treatment of cells with 10 nM PD-L1/RNF128 SureTAC removed PD-L1 from the cell surface of colorectal cancer cells within 2 h and surface levels remained undetectable for up to 1 week (end of experiment), indicating a durable response (Figure 8c). - Doses of 1 nM PD-L1/RNF128 SureTAC were sufficient to remove PD-L1 from the cell surface of colorectal cells within 24 h and surface levels remained undetectable for up to 1 week (end of experiment), indicating a durable response (Figure 8c). - Doses of 0.1 nM PD-L1/RNF128 SureTAC induced a maximum of 50% PD-L1 down-regulation, which was maintained for up to 1 week (end of experiment), indicating a durable response (Figure 8c). - We examined the degrader capacity of our most optimal PD-L1/RNF128 SureTAC against a panel of cell lines that express various mRNA levels of RNF128 (Figure 8a). Only cell lines in which RNF128 expression was detected, displayed PD-L1 cell surface removal and degradation (Figure 8a). Two cell lines that displayed no detectable RNF128 expression, remained non-responsive. These results thus demonstrate that the degrader activity of our SureTACs reagents depends on endogenous E3 ligase expression. - We treated HT29 cells with PD-L1/RNF128 SureTACs for 16 h to completely remove PD-L1 levels from the cell surface. After washing away all SureTACs reagents from the medium, cells were monitored for restoration for PD-L1 levels at the cell surface. We noticed that (partial) restoration of PD-L1 levels took 24 h and longer (after 24 h PD-L1 levels back to 50%, after 72 h back to ~60%, to date we have not seen full recovery of PD-L1 surface levels in our assays), indicating that SureTACs-induced PD-L1 degradation may have a prolonged pharmacological effect (Figure 8b). Comparison of E3s SureTACs employing our optimal RNF128 binding arms (4E2, 4A5 and 4H4) outperformed all other SureTACs that employed binders to RNF43, RNF130 or RNF167 at 10nM dosing (Figure 7a, b and c). See also Figure 10b were PD-L1 down-regulation by RNF128 was much more prominent than the effect of RNF43. For PD-L1/RNF130 SureTACs, increasing the concentration to 100 nM increased the performance of this PD-L1 degrader (Figure 7d). RNF43 was employed previously in a heterobispecific antibody format for degradation of PD-L1 (papers James Wells, and Garcia). In our hands, however, PD- L1/RNF43 SureTACs only achieve a maximum of 50% PD-L1 degradation (Figure 7a, b, c, e, f and Figure 8a).
P62011579wo 76 PD-L1/RNF128 SureTACs-mediated PD-L1 degradation induces cytokine secretion and tumor cell killing To assess functional effects of SureTACs-mediated PD-L1 cell surface removal and degradation, PD-L1(1G7/2F3)/RNF128(4E2) SureTACs were added to mixed lymphocyte reactions in concentrations of 5 nM, 10 nM and 50 nM. Treatment with similar concentrations of Atezolizumab (Atz; benchmark PD-L1 blocker) was used as a positive control. PD-L1/RNF128 SureTACs treatment induced secretion of the cytokine IL-2 to similar levels as Atz, while IFN^ secretion was even enhanced by SureTACs treatment compared to Atz, at all tested concentrations (Figure 9a and b). These results indicate that PD-L1/RNF128 SureTACs potently induce T cell activation, to similar or increased levels as the benchmark antibody Atz. Next, the functional effects of PD-L1/RNF128 SureTACs treatment was assessed in a tumor lysis assay. In this setup, human colorectal cancer cells (HT29) were co-cultured with human PBMCs and treated with or without 10 nM or 50 nM control antibodies, Atz or PD- L1(1G7/2F3)/RNF128(4E2) SureTACs. In this assay, PD-L1/RNF128 SureTACs induced up to 70% tumor killing (LDH release), at 10 nM and 50 nM concentrations, while Atz-induced killing maximally reached 50% at 50 nM (Figure 9c and d). These results indicate that PD-L1-targeting by PD- L1/RNF128 SureTACs outperforms Atz in this experimental setup for tumor cell killing. Conclusion Combination of membrane E3 ligases can be successfully employed to drive removal of various transmembrane targets. PD-L1 targeting bi-specific binders, down-regulate, significantly, the surface levels of PD-L1 and enhance tumor cell killing. By using non-blocking PD-L1 binders and tissue or cancer specific E3 ligase, reducing toxicity is achieved, while reducing resistance can be achieved by not employing an active site of PD-L1. Finally, the pharmacological window is enhanced by fully removing PD-L1 proteins instead of blocking them. ^ Example 2 In vivo efficacy study MC38 mouse colorectal cancer cells were injected sub-cutaneous (s.c.) in C57BL/6JRj (female) mice. Mice were randomly included in groups upon reaching tumour volume 80-100mm3. MC38 tumor-bearing mice were treated with control ST74 GFP x GFP (n=10) or ST04 PD-L1 (2F3) x RNF128 (4E2) (n=10 per concentration) at indicated concentrations every three days, compounds were injected intra peritoneally (i.p.). Tumor volume measurements were performed every three days (same as treatment days). Mice were sacrificed at set tumor size endpoint of approximately 1200mm3. Tumor volume per time point was calculated as non-linear regression curve (Formula: Y=Y0*exp(k*x) with Y0=100) fit with y set at 100 (inclusion of mice in to experiment was at an average of tumor size 100mm3 ).
P62011579wo 77 Conclusion The in vivo efficacy study shows that PD-L1-targeting SureTAC ST04 PD-L1 (2F3) x RNF128 (4E2) inhibits in vivo tumor outgrowth of a subcutaneously injected MC38 (mouse colorectal cancer) tumor at a dose of 10mg/kg as compared to control SureTAC GFP x GFP which does not impair tumor outgrowth. ^ Example 3 Humanization of a PD-L1 x RNF128 bispecific SureTAC. SureTAC ST04 consists of PD-L1 binder – Fc(KO) fusion O2F3-Fc-KO1-HOLE (SEQ ID NO: 43) and RNF128 binder – Fc(KO) fusion O4E2-Fc-KO1-KNOB (SEQ ID NO: 39). A 3D model of the fusion protein was constructed based on predictions from AlphaFold 2-Multimer (Jumper at al.2021 “Highly accurate protein structure prediction with AlphaFold”, Nature 596(7873); 583-589), run on a local server. The model was refined using Protean3D and PyMol3 software and is depicted in Figure 11(b) (space-filling representation). Using BLAST software we probed a local database containing human germline V-regions extracted from V-BASE and IMGT For O2F3 both the IGHV3-48 (SEQ ID NO: 100) and VH3-13374 (SEQ ID NO: 101) were withheld, while for O4E2, IGHV3-23*01 (SEQ ID NO: 104) showed a high homology. For both sequences, also 2 different pre-humanized framework sequences were tested: one containing an engineered extra disulfide bond stabilizing CDR2 (preHum_ds, SEQ ID NO: 103), and a general acceptor framework for camelid antibodies as described in Vincke et al., 2009: “General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold”, Journal of Biological Chemistry 284(5); 3273-3284 (preHum-Nb, SEQ ID NO: 102). Figure 11(a) shows the alignment of O2F3 and O4E2 to the chosen human germline frameworks and the pre-humanized general acceptor frameworks FR1-FR2 and FR3, as well as different versions of humanized sequences. The consensus sequence from SEQ ID NO:105 was used for FR4. The humanized sequences and their respective SEQ ID NO’s are listed in Table 7 and 8 below. The proposed humanized sequences were structure-predicted as described above, and aligned with the original sequences using Protean3D. These models are shown as a wireframe overlay in Figure 11(c) for O2F3 and Figure 11(d) for O4E2. The Root Mean Square Deviation (RMSD) from the camelid sequence was taken as indicative for a good model, and RMSD >1.5 Angstrom sequences were discarded. The sequences were also compared for a “humanness” calculation, as described in Abhinandan et al.2007: “Analysing the degree of humanness of antibody sequences”, J Mol Biol 369(3); 852-62, essentially calculating a Z-score from the standard deviation of a mean sequence identity (see Table 5 and 6, below).
P62011579wo 78 Table 5. Humanization of PD-L1 binder O2F3 RMSD HU (Z) KD(eq) SD SEQ ID Name Frame (Å) (nM) (nM) NO: O2F3-v0 Lama glama 0.018 -1.0 0.22 0.01 22 O2F3-v1 IGHV3-48 1.574 +0.1 * * 106 O2F3-v1.1 IGHV3-48 1.317 -0.1 1.16 0.05 107 O2F3-v2.1 IGHV3-48 0.901 -0.5 0.36 0.03 108 O2F3-v3.1 VH3-13374 1.687 +0.2 * * 109 O2F3-v4.1 VH3-13374 0.766 -0.4 0.36 0.01 110 O2F3-v7 IGHV3-48 1.297 -0.3 0.67 0.14 111 O2F3-v5 preHum 1.395 -0.4 >100 * 112 O2F3-v6.1 preHum 1.188 +0.0 >100 * 113 Table 6. Humanization of RNF128 binder O4E2 RMSD HU (Z) KD(eq) SD SEQ ID Name Frame (Å) (nM) (nM) NO: O4E2-v0 Lama glama 0.010 +0.1 1.05 0.10 19 O4E2-v1 IGHV3-23*01 0.729 +1.2 1.22 0.13 114 O4E2-v2 IGHV3-23*01 0.999 +0.8 0.79 0.06 115 O4E2-v3 IGHV3-23*01 0.547 +0.8 0.99 0.04 116 O4E2-v3.2 IGHV3-23*01 0.424 +1.0 1.05 0.04 117 O4E2-v4 preHum 0.542 +1.0 >100 * 118 O4E2-v5 preHum 1.101 +1.0 7.74 0.80 119 The humanized binders were produced in HEK293 cells using transient transfection as a single arm VHH-Fc(KO) fusion, purified and tested for equilibrium binding by ELISA on the respective antigens. Figure 11(e) and (f) show the respective dilution curves, while the calculated KD values are indicated in Table 5 and Table 6. Conclusion For O2F3 (PD-L1 binder) it can be seen that conservation of the camelid sequence at the site where the human VH interacts with the VL domain is important in both human frameworks selected, but even more, conservation of the Threonine at position 62 in SEQ ID NO: 22 is relevant to correct folding and activity. None of the general use pre-humanized frameworks gave an active antibody function. V2.1 (SEQ ID NO: 108) and V4.1 (SEQ ID NO: 110) gave a KD value close to the camelid antibody (both 0.36nM versus 0.22nM) with an acceptable level of humanization. O4E2 (RNF128 binder) already showed a high degree of humanness. Some variants dramatically increased the humanness as well as increasing the equilibrium constant. V2 (SEQ ID NO: 115) in
P62011579wo 79 particular increased humanness to +0.8 (versus +0.1), and increased binding (KD 0.79 versus 1.05). V3.2 (SEQ ID NO: 117) is of particular interest as it has a high humanization score, while preserving the binding. As can be appreciated from Figure 11(f), most variations using the IGHV3- 23*01 framework gave comparable results. V1 (SEQ ID NO: 114) reaching a high score on humanness by mere CDR grafting is also performing well. But also with O4E2, the pre-humanized frameworks failed to give a comparable antibody activity, indicating that not all frameworks will give the results described. Table 7. Amino acid sequence humanized PD-L1 binders SEQ ID Humanized PD-L1 binders sequence NO: 106 EVQLVESGGGLVQPGGSLRLSCAASGGAFNSVAIAWFRQAPGKGLELVARIRLNTETDFYADS VIGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTLVTVS O2F3-v1 S 107 EVQLVESGGGLVQPGGSLRLSCAASGGAFNSVAIAWFRQAPGKGLELVARIRLNTETDFYADS VIGRFTISTDNAKNSLYLQMNSLRAEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTLVTVS O2F3-v1.1 S 108 EVQLVESGGGLVQPGGSLRLSCAASGGAFNSVAIAWFRQAPGKEREFVARIRLNTETDFYADS VIGRFTISTDNAKNSLYLQMNSLRAEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTLVTVS O2F3-v2.1 S 109 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSVAIAWVRQAPGKGLVWVSRIRLNTETDFYADS VIGRFTISTDNAKNTLYLQMNSLRAEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTLVTVS O2F3-v3.1 S 110 EVQLVESGGGLVQPGGSLRLSCAASGGAFNSVAIAWFRQAPGKEREFVARIRLNTETDFYADS VIGRFTISTDNAKNTLYLQMNSLRAEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTLVTVS O2F3-v4.1 S 111 EVQLVESGGGLVQPGGSLRLSCAASGGAFNSVAIAWFRQAPGKEREFVARIRLNTETDFYADS VIGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTLVTVS O2F3-v7 S 112 EVQLVESGGGLVQPGGSLRLSCAASGGAFNSVAIAWFRQAPGKEREGVCARIRLNTETDFYAD SVIGRFTCSRDNAKNTLYLQMNSLRAEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTLVTV O2F3-v5 SS 113 EVQLVESGGGLVQPGGSLRLSCAASGGAFNSVAIAWFRQAPGKGLEAVARIRLNTETDFYADS VIGRFTISTDNSKNTLYLQMNSLRAEDTAVYYCAAETVASGSIYSLQTPSRYASWGQGTLVTVS O2F3-v6.1 S Table 8. Amino acid sequence humanized RNF128 binders SEQ ID Humanized PD-L1 binders sequence NO: 114 EVQLLESGGGLVQPGGSLRLSCAASGFTFKYYAMGWFRQAPGKGLEFVAVIGWSGGSTDYAD O4E2-v1 SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGLSGLEPDYWGQGTLVTVSS 115 EVQLLESGGGLVQPGGSLRLSCAASGFTFKYYAMGWFRQAPGKEREFVAVIGWSGGSTDYAD O4E2-v2 SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGLSGLEPDYWGQGTLVTVSS 116 EVQLLESGGGLVQPGGSLRLSCAASGRTFKYYAMGWFRQAPGKEREFVAVIGWSGGSTDYA O4E2-v3 DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGLSGLEPDYWGQGTLVTVSS 117 EVQLLESGGGLVQPGGSLRLSCAASGRTFKYYAMGWFRQAPGKGLEFVAVIGWSGGSTDYAD O4E2-v3.2 SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGLSGLEPDYWGQGTLVTVSS 118 EVQLVESGGGLVQPGGSLRLSCAASGGAFNYYAMGWFRQAPGKEREGVCAVIGWSGGSTDY O4E2-v4 ADSVKGRFTCSRDNAKNTLYLQMNSLRAEDTAVYYCAAGLSGLEPDYWGQGTLVTVSS 119 EVQLVESGGGLVQPGGSLRLSCAASGGAFNYYAMGWFRQAPGQGLEAVAVIGWSGGSTDYA O4E2-v5 DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGLSGLEPDYWGQGTLVTVSS