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WO2025240659A2 - Activatable constructs, compositions and methods - Google Patents

Activatable constructs, compositions and methods

Info

Publication number
WO2025240659A2
WO2025240659A2 PCT/US2025/029426 US2025029426W WO2025240659A2 WO 2025240659 A2 WO2025240659 A2 WO 2025240659A2 US 2025029426 W US2025029426 W US 2025029426W WO 2025240659 A2 WO2025240659 A2 WO 2025240659A2
Authority
WO
WIPO (PCT)
Prior art keywords
activatable
activatable construct
amino acids
construct
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/029426
Other languages
French (fr)
Other versions
WO2025240659A3 (en
Inventor
Ellaine Anne Mariano FOX
Maryann LABOE
Madan M. Paidhungat
Trang T. T. VU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytomx Therapeutics Inc
Original Assignee
Cytomx Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cytomx Therapeutics Inc filed Critical Cytomx Therapeutics Inc
Publication of WO2025240659A2 publication Critical patent/WO2025240659A2/en
Publication of WO2025240659A3 publication Critical patent/WO2025240659A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present disclosure relates to the field of biotechnology, and more specifically, to activatable constructs, and to methods of making and using the polypeptides and activatable constructs in a variety of therapeutic, diagnostic, and prophylactic applications.
  • Antibody-based therapies have emerged as treatments for a variety of disorders, but in some cases, toxicities due to broad target expression have limited their therapeutic effectiveness. In addition, antibody -based therapeutics have exhibited other limitations such as rapid clearance from the circulation following administration.
  • the present disclosure provides an activatable construct including a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB1 includes a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), wherein the HVD1 is covalently coupled directly or indirectly to a first masking moiety (MM1) via a first cleavable moiety (CM1) , wherein the LVD1 is covalently coupled directly or indirectly to a second masking moiety (MM2) via a second cleavable moiety (CM2), a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 includes a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2), and a first dimerization domain (DD1) and a second dimerization domain (DD2).
  • the AB1 is coupled directly or indirectly to the DD1.
  • the AB2 is coupled directly or
  • the AB2 is not coupled to a MM. In some aspects, the AB2 is coupled directly or indirectly to a third masking moiety (MM3) via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
  • MM3 third masking moiety
  • CM3 third cleavable moiety
  • the AB2 is further coupled directly or indirectly to a fourth masking moiety (MM4) via a fourth cleavable moiety (CM4), wherein the MM3 and MM4 inhibit binding of the AB2 to the second antigen.
  • MM4 masking moiety
  • CM4 fourth cleavable moiety
  • the present disclosure provides a composition containing the activatable construct according to any of the aspects disclosed herein.
  • the present disclosure provides a container, vial, syringe, injector pen, or kit comprising at least one dose of a composition comprising the activatable construct according to any of the aspects disclosed herein.
  • the present disclosure provides a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising the activatable construct according to any of the aspects disclosed herein.
  • the subject has been identified or diagnosed as having a cancer, an autoimmune disease, or an inflammatory disorder.
  • the present disclosure provides a nucleic acid encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein.
  • the nucleic acid encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein comprises a vector.
  • the nucleic acid encodes a polypeptide according to any one of the sequences in Table 16.
  • the present disclosure provides a cell transfected with the nucleic acid or vector encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein.
  • the cell is a mammalian cell.
  • the present disclosure provides a method of producing the activatable construct according to any of the aspects disclosed herein.
  • the method comprises culturing a cell comprising a nucleic acid or vector disclosed herein in a liquid culture medium to produce the activatable construct according to any of the aspects disclosed herein and recovering the activatable construct from the liquid culture medium.
  • the method comprises purifying the activatable construct recovered from the liquid culture medium to obtain a purified activatable construct.
  • the method comprises combining the purified activatable construct with a carrier to form a pharmaceutical composition.
  • FIGs. 1A-1D provide schematics of antibody constructs.
  • FIG. 1A is a schematic of an activatable construct with the masking moiety attached to the light chain with a cleavable moiety.
  • FIG. IB is a schematic of an activatable construct with the masking moiety attached to the heavy chain with a cleavable moiety.
  • FIG. 1C is a schematic of an activatable construct with masking moi eties attached to each light chain with a cleavable moiety and masking moi eties attached to each heavy chain with a cleavable moiety (wherein the masking moieties and cleavable moieties may be the same or different).
  • FIG. 1A is a schematic of an activatable construct with the masking moiety attached to the light chain with a cleavable moiety.
  • FIG. 1C is a schematic of an activatable construct with masking moi eties attached to each light chain with a cleavable moiety and mask
  • ID is a schematic of an activatable construct with a masking moiety attached to the light chain with a cleavable moiety and another masking moiety (the same or different from the first masking moiety) attached to the heavy chain with a cleavable moiety (the same or different from the first cleavable moiety), and another masking moiety attached to a scFv with a cleavable moiety.
  • FIGs. 2A-2D show results of an ELISA binding assay to determine the shift in ability of the molecules to bind human Integrin alpha 3 beta 1 (ITGA3B1) antigen coated on a plate.
  • FIG. 2A provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 2325 to bind human ITGA3B1 antigen coated on a plate.
  • FIG. 2B provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 2331 to bind human ITGA3B1 antigen coated on a plate.
  • FIG. 2C provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 2333 to bind human ITGA3B1 antigen coated on a plate.
  • FIG. 2D provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 2337 to bind human ITGA3B1 antigen coated on a plate.
  • FIGs. 3A-3D provide the results of an ELISA binding assay to determine the shift in ability of the molecules to bind epidermal growth factor receptor (EGFR) antigen coated on a plate.
  • FIG. 3A provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 3954 to bind EGFR antigen coated on a plate.
  • FIG. 3B provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask CF41 to bind EGFR antigen coated on a plate.
  • FIG. 3C provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 3954 on the heavy chain and Mask CF41 on the light chain to bind EGFR antigen coated on a plate.
  • FIG. 3D provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 3954 on the light chain and Mask CF41 on the heavy chain to bind EGFR antigen coated on a plate.
  • FIGs. 4A-4B are schematics of activatable constructs comprising a single masked Fab domain and a dual masked scFv domain, including an additional masking moiety attached to an Fc domain (FIG. 4A) or an additional masking moiety attached to a light chain constant region (FIG. 4B)
  • FIGs. 5A-5C are schematics of activatable constructs comprising a dual masked Fab domain and a dual masked scFv domain, including an additional masking moiety attached to an Fc domain (FIG. 5A) or an additional masking moiety attached to a light chain constant region (FIG. 5B).
  • FIG. 5C A single masked scFv is depicted in FIG. 5C.
  • FIGs. 6-9 show configurations of exemplary activatable molecules.
  • the molecules are designed such that the activated molecules resulting from the activation of the activatable molecules do not comprise half-life extending moieties and thus have a shorter half-life than counterpart molecules that are the same as the activated molecules but comprising the half-life extending moieties.
  • a and “an” refers to one or more (i.e., at least one) of the grammatical object of the article.
  • a cell encompasses one or more cells.
  • the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • the foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives.
  • the term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • an “activatable construct” may refer to a polypeptide, multiple polypeptides or a nucleic acid, e.g., polynucleotide, or mRNA encoding the polypeptide or multiple polypeptides.
  • a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, composition, or method is intended to and does find direct support for embodiments related to constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.
  • nucleic acid sequence encoding includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.
  • polynucleotide as used herein shall mean a polynucleotide of genomic, cDNA, RNA, mRNA, or synthetic origin or some combination thereof, which by virtue of its origin the “polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, and/or (3) does not occur in nature as part of a larger sequence.
  • polynucleotides include the nucleic acid molecules encoding heavy chain immunoglobulin molecules, and nucleic acid molecules encoding light chain immunoglobulin molecules.
  • polypeptide and “recombinant polypeptide” are used interchangeably herein to refer to a polymer of amino acids that exists in a form that is not found in nature.
  • a “polypeptide” includes an expressed polymer of amino acids with or without post-translational modifications.
  • the polypeptide comprises one or more glycosyl groups attached thereto.
  • Polypeptides employed herein may be encoded by cDNA, recombinant RNA, recombinant DNA, messenger RNA, or a polynucleotide of synthetic origin or some combination thereof.
  • polypeptide (1) is not in a naturally occurring organism (e.g., is not an endogenous polypeptide of a naturally occurring organism) and (2) is present in a form not found in nature.
  • cleavable moiety and “CM” are used interchangeably herein to refer to a peptide, the amino acid sequence of which encodes a substrate for a sequence-specific protease.
  • Cleavable moieties that are suitable for use as a CM include any of the protease substrates that are known the art. Exemplary cleavable moieties are described in more detail below.
  • MM mass moiety
  • N-terminally positioned when referring to a position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence means that the first domain or sequence is located closer to the N-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some aspects, there may be additional sequences and/or domains between the first domain or sequence and the second domain or sequence.
  • C-terminally positioned when referring to a position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence means that the first domain or sequence is located closer to the C-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some aspects, there may be additional sequences and/or domains between the first domain or sequence and the second domain or sequence.
  • N-terminal to C-terminal direction refers to the N-terminus to the C-terminus of the polypeptide elements disclosed herein in the left to right direction.
  • exogenous refers to any material introduced from or originating from outside a cell, a tissue, or an organism that is not produced by or does not originate from the same cell, tissue, or organism in which it is being introduced.
  • transduced refers to a process by which an exogenous nucleic acid is introduced or transferred into a cell.
  • a “transduced,” “transfected,” or “transformed” cell e g., mammalian cell
  • exogenous nucleic acid e.g., a vector
  • nucleic acid refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some aspects of any of the nucleic acids described herein, the nucleic acid is DNA. In some aspects of any of the nucleic acids described herein, the nucleic acid is RNA.
  • the phrase “specifically binds,” or “immunoreacts with” means that the activatable construct reacts with one or more antigenic determinants of the desired target antigen and does not react with other polypeptides, or binds at much lower affinity or potency, e.g., a KD or ECso about or greater than 10’ 6 M.
  • treatment refers to ameliorating at least one symptom of a disorder.
  • the disorder being treated is a cancer.
  • the disorder being treated is an autoimmune disease.
  • the disorder being treated is an inflammatory disorder.
  • linker refers to a peptide, the amino acid sequence of which is not a substrate for a protease. Exemplary linkers are described in more detail below.
  • linking region refers to the stretch of amino acid residues between the C-terminus of the antigen binding domain and the amino acid residue that is N-terminally adjacent to the proximal point of interaction between the dimerization domains (i.e., the linking region does not include the C-terminal amino acid of the antigen binding domain or the N-terminal amino acid of the DD that forms the proximal point of interaction to the DD of the corresponding second monomer).
  • the linking region is the stretch of amino acid residues between the C-terminus of the antigen binding domain and the first N-terminal cysteine residue of the Fc that participates in the disulfide linkage with the second Fc domain (e.g., Cysteine 226 of an IgGl or IgG4 Fc domain, according to EU numbering).
  • the linking region is the stretch of amino acid residues between the C-terminus of the heavy chain constant domain (e.g., a CHI domain) and the first N-terminal cysteine residue of the Fc that participates in the disulfide linkage with the second Fc domain
  • the dimerization domain is not a polypeptide
  • the linking region is the stretch of amino acid residues following the C- terminus of the antigen binding domain until the last amino acid.
  • the linking region of the biotin-containing monomer is the stretch of amino acid residues between the C-terminus of the antigen binding domain and the biotin molecule
  • the linking region of the streptavidin-containing monomer is the stretch of amino acid residues between the C-terminus of the antigen binding domain and the streptavidin molecule.
  • the term “mask linking region” or “MLR” refers to the stretch of amino acid residues between a MM and an antigen binding domain (AB).
  • the MLR spans from the N- terminus of a AB to the C-terminus of a AB .
  • the MLR may include a MM, a MM and a linker, or a MM and two linkers.
  • the MLR spans 15 to 22 amino acids.
  • the MLR spans 16 to 21 amino acids.
  • the MLR spans 17 to 20 amino acids.
  • the MLR spans 18 to 20 amino acids.
  • the MLR spans 15, 16, 17, 18, 18, 20, 21, or 22 amino acids.
  • the term “masking efficiency” refers to the activity (e.g., EC50) of the activatable construct (i.e., intact and uncleaved) divided by the activity of a control construct, wherein the control antibody or antigen binding domain may be either cleavage product of the activatable construct or the antibody or antigen binding domain used as the activatable construct, for example corresponding activatable antibody that lacks MMs.
  • variable domain may be used interchangeably with variable light (VL) domain.
  • heavy chain variable domain may be used interchangeably with variable heavy (VH) domain.
  • light chain constant region LC
  • heavy chain constant region HC
  • CH CH and may be used interchangeably.
  • the present disclosure includes single chain polypeptides capable of forming dimer complexes as well as dimerized complexes of a first monomer construct and a second monomer construct. Dimerization of the monomeric components is facilitated by a dimerization domain capable of forming a dimer or a first dimerization domain and a second dimerization domain capable of forming a dimer.
  • the present disclosure includes homodimers, e g., wherein each of the first and second monomer constructs are identical, as well as heterodimers, where the first and second monomer constructs different from each other.
  • a polypeptide such as an antibody (including, for example, a light chain, a heavy chain, an scFv, and the like), antibody fragment, or an Fc domain
  • a polypeptide may be a wild-type polypeptide (e.g., a naturally-existing polypeptide) or a variant of the wild-type polypeptide.
  • a variant may be a polypeptide modified by substitution, insertion, deletion and/or addition of one or more amino acids of the wild-type polypeptide, provided that the variant retains the basic function or activity of the wild-type polypeptide.
  • a variant may have altered (e.g., increased or decreased) function or activity comparing with the wild-type polypeptide.
  • activatable antigen binding domain constructs having a masking format that significantly inhibits binding of the antigen binding domain to its target, yet allows for substantial recovery of binding activity following activation.
  • activatable constructs according to any one of the aspects described herein have significantly greater masking efficiency than a corresponding activatable construct having only a single masking moiety associated with an antigen binding domain, i.e., only a single masking moiety per antigen binding domain.
  • activatable constructs according to the present disclosure have greater masking efficiency than a corresponding activatable construct having a masking moiety coupled directly or indirectly to either a heavy chain variable domain or a light chain variable domain, but not a masking moiety coupled directly or indirectly to both a heavy chain variable domain and a light chain variable domain of the same antigen binding domain.
  • the present disclosure provides an activatable construct, comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), wherein the HVD1 is covalently coupled directly or indirectly to a first masking moiety (MM1) via a first cleavable moiety (CM1), wherein the LVD1 is covalently coupled directly or indirectly to a second masking moiety (MM2) via a second cleavable moiety (CM2); a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2), and a first dimerization domain (DD1) and a second dimerization domain (DD2), wherein the MM1 and the MM2 inhibit binding of the AB 1 to the first antigen.
  • the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide.
  • the first polypeptide comprises the MM2, the CM2, and the LVD1.
  • the LVD1 is coupled directly or indirectly to the C- terminus of the MM2 via the CM2.
  • the second polypeptide comprises the MM1, the CM1, the HVD1, and the DD1
  • the HVD1 is coupled directly or indirectly to the MM1 via the CM1 and directly or indirectly to the DD1.
  • the third polypeptide comprises the AB2 and the DD2. In some aspects, the AB2 is not coupled to a MM. In some aspects, the AB2 is coupled directly or indirectly to a third masking moiety (MM3) via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the third polypeptide comprises from N-terminus to the C-terminus, the LVD2, the HVD2, and the DD2. In some aspects, the third polypeptide comprises from N-terminus to the C-terminus, the HVD2, the LVD2, and the DD2
  • the third polypeptide comprises the MM3, the CM3, the LVD2, the HVD2, and the DD2.
  • the HVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the LVD2.
  • the LVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the HVD2.
  • the first polypeptide comprises the MM2, the CM2, and the LVD1.
  • the second polypeptide comprises the MM1, the CM1, the HVD1, and the DD1.
  • the second polypeptide further comprises a third masking moiety (MM3), a third cleavable moiety (CM3), and the AB2, and the MM3 inhibits binding of the AB2 to the second antigen.
  • the second polypeptide comprises in an N-terminal to C-terminal direction the MM1, the CM1, the HVD1, the HVD2, the LVD2, the CM3, the MM3, and the DD1.
  • the second polypeptide comprises in an N-terminal to C-terminal direction the MM1, the CM1, the HVD1, the LVD2, the HVD2, the CM3, the MM3, and the DD1.
  • the third polypeptide comprises the DD2. In some aspects, the third polypeptide does not comprise a variable domain.
  • the activatable construct further comprises a third polypeptide. In some aspects, the third polypeptide comprises the HVD2 and the DD2. In some aspects, the activatable construct further comprises a fourth polypeptide. In some aspects, the fourth polypeptide comprises the LVD2. In some aspects, the third polypeptide comprises the MM3 coupled directly or indirectly to the HVD2 via the CM3 and the fourth polypeptide does not comprise a MM, wherein the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the fourth polypeptide comprises the MM3 coupled directly or indirectly to the LVD2 via the CM3 and the third polypeptide does not comprise a MM, wherein the MM3 inhibits binding of the AB2 to the second antigen.
  • the fourth polypeptide comprises a fourth masking moiety (MM4) and a fourth cleavable moiety (CM4).
  • the first polypeptide comprises the MM2, the CM2, and the LVD1
  • the second polypeptide comprises the MM1, the CM1, the HVD1, and the DD1
  • the third polypeptide comprises the MM3, the CM3, the HVD2, and the DD2
  • the fourth polypeptide comprises the MM4, the CM4, and the LVD2.
  • the first polypeptide comprises the MM2, the CM2, the LVD1, and the LC1
  • the second polypeptide comprises the MM1, the CM1, the HVD1, the HC1, and the DD1
  • the third polypeptide comprises the MM3, the CM3, the HVD2, the HC2, and the DD2
  • the fourth polypeptide comprises the MM4, the CM4, the LVD2, and the LC2.
  • the present disclosure provides an activatable construct, comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB 1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), a first light chain constant domain (LC1), a first heavy chain constant domain (HC1); a first masking moiety (MM1) coupled directly or indirectly to a first cleavable moiety (CM1), wherein the MM1 is coupled directly or indirectly to the HVD1, the LVD1, the HC1, or the LC1 via the CM1; a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2); a second masking moiety (MM2) coupled directly or indirectly to a second cleavable moiety (CM2), wherein the MM2 is coupled directly or indirectly to the HV
  • the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide. In some aspects, the activatable construct further comprises a third cleavable moiety (CM3) and a third masking moiety (MM3). In some aspects, the activatable construct further comprises a fourth cleavable moiety (CM4) and a fourth masking moiety (MM3).
  • the first polypeptide comprises the MM1, CM1, LVD1, and the LC1.
  • the second polypeptide comprises the HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1.
  • the third polypeptide comprises the MM3, CM3, and the DD2.
  • the MM3 inhibits binding of the AB2 to the second antigen.
  • the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
  • the MM1 inhibits binding of the AB1 to the first antigen.
  • the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1.
  • the second polypeptide comprises the LVD1, LC1, CM3, and the MM3.
  • the third polypeptide comprises the DD2.
  • the MM3 inhibits binding of the AB2 to the second antigen.
  • the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
  • the MM1 inhibits binding of the AB1 to the first antigen.
  • the first polypeptide comprises the MM1, CM1, LVD1, and the LC1.
  • the second polypeptide comprises the MM4, CM4, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1.
  • the third polypeptide comprises the MM3, CM3, and the DD2.
  • the MM4 inhibits binding of the AB1 to a first antigen.
  • the MM1 and the MM4 inhibit binding of the AB 1 to the first antigen.
  • the MM3 inhibits binding of the AB2 to the second antigen.
  • the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
  • the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1.
  • the second polypeptide comprises the MM4, CM4, LVD1, LC1, CM3, and the MM3.
  • the third polypeptide comprises the DD2.
  • the MM4 inhibits binding of the AB1 to the first antigen.
  • the MM1 and the MM4 inhibit binding of the AB 1 to the first antigen.
  • the MM3 inhibits binding of the AB2 to the second antigen.
  • the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
  • the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1.
  • the second polypeptide comprises the MM3, CM3, LVD1, and the LC1.
  • the third polypeptide comprises the DD2.
  • the MM3 inhibits binding of the AB1 to the first antigen.
  • the MM1 and the MM3 inhibit binding of the AB 1 to the first antigen.
  • the MM2 inhibits binding of the AB2 to the second antigen.
  • FIG. 6 shows an exemplary activatable protein 100 comprising three polypeptides.
  • the first polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM1 101, an optional linker 102, the CM1 103, an optional linker 104, and the ABl's light chain fragment 105.
  • the second polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM3 151, an optional linker 152, the CM3 153, an optional linker 154, and the ABl's heavy chain fragment 121, a linker 122, the AB2's heavy chain variable region 123, a linker 124, the AB2's light chain variable region 125, a linker 126, the CM2 127, an optional linker 128, the MM2 129, a linker 130, and the first Fc domain 131.
  • the third polypeptide comprises the second Fc domain 141.
  • 105 is the ABl's heavy chain fragment and 121 is the ABl's light chain fragment.
  • 123 is the AB2's light chain variable region and 125 is the AB2's heavy chain variable region.
  • the first Fc domain 131 is an Fc domain hole mutant and the second Fc domain 141 is an Fc domain knob mutant.
  • the first Fc domain 131 is an Fc domain knob mutant and the second Fc domain 141 is an Fc domain hole mutant.
  • FIG. 7 shows another exemplary activatable protein 200 comprising three polypeptides.
  • the first polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM1 201, an optional linker 202, the CM1 203, an optional linker 204, the ABl's light chain fragment 205, a linker 206, the AB2's heavy chain variable region 207, a linker 208, and the AB2's light chain variable region 209.
  • the second polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM3 251, an optional linker 252, the CM1 253, an optional linker 254, the ABl's heavy chain fragment 221, an optional linker 222, the CM2 223, an optional linker 224, the MM2 225, a linker 226, and the first Fc domain 227.
  • the third polypeptide comprises the second Fc domain 241.
  • 205 is the ABl's heavy chain fragment and 221 is the ABl's light chain fragment.
  • FIG. 7 is the ABl's heavy chain fragment and 221 is the ABl's light chain fragment.
  • 207 is the AB2's light chain variable region and 209 is the AB2's heavy chain variable region.
  • the first Fc domain 227 is an Fc domain hole mutant and the second Fc domain 241 is an Fc domain knob mutant.
  • the first Fc domain 227 is an Fc domain knob mutant and the second Fc domain 241 is an Fc domain hole mutant.
  • FIG. 8 shows another exemplary activatable protein 300 comprising three polypeptides.
  • the first polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM1 301, an optional linker 302, the CM1 303, an optional linker 304, the ABl's light chain fragment 305, a linker 306, the AB2's heavy chain variable region 307, a linker 308, and the AB2's light chain variable region 309.
  • the second polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM3 351, an optional linker 352, the CM3 353, an optional linker 354, the ABl's heavy chain fragment 321, an optional linker 322, the CM2 323, an optional linker 324, and the first Fc domain 325.
  • the third polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the second Fc domain 341, a linker 342, and the MM2 343.
  • 305 is the ABl's heavy chain fragment and 321 is the ABl's light chain fragment.
  • FIG. 8 is the ABl's heavy chain fragment and 321 is the ABl's light chain fragment.
  • the first Fc domain 325 is an Fc domain hole mutant and the second Fc domain 341 is an Fc domain knob mutant.
  • the first Fc domain 325 is an Fc domain knob mutant and the second Fc domain 341 is an Fc domain hole mutant.
  • FIG. 9 shows another exemplary activatable protein 400 comprising three polypeptides.
  • the first polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM1 401, an optional linker 402, the CM1 403, an optional linker 404, the ABl's light chain fragment 405, a linker 406, the AB2's heavy chain variable region 407, a linker 408, and the AB2's light chain variable region 409.
  • the second polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM3 451 , an optional linker 452, the CM3 453, an optional linker 454, the ABTs heavy chain fragment 421, an optional linker 422, the CM2 423, an optional linker 424, and the first Fc domain 425.
  • the third polypeptide in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM2441, a linker 442, and the second Fc domain 443.
  • 405 is the ABFs heavy chain fragment
  • 421 is the ABFs light chain fragment.
  • 407 is the AB2's light chain variable region and 409 is the AB2's heavy chain variable region.
  • the first Fc domain 425 is an Fc domain hole mutant and the second Fc domain 443 is an Fc domain knob mutant.
  • the first Fc domain 425 is an Fc domain knob mutant and the second Fc domain 443 is an Fc domain hole mutant.
  • the present disclosure also includes a nucleic acid, e.g., polynucleotide, or mRNA encoding the polypeptide or multiple polypeptides, for providing activatable constructs.
  • a nucleic acid e.g., polynucleotide, or mRNA encoding the polypeptide or multiple polypeptides
  • the antigen binding domains employed herein are formed by combination of a heavy chain variable domain and a light chain variable domains.
  • HVD1 and LVD1 together form AB 1.
  • HVD2 and LVD2 together form AB2.
  • AB1 and AB2 bind to the same antigen (i.e., the first antigen and the second antigen are the same),
  • HVD1 and HVD2 are identical, and LVD1 and LVD2 are identical.
  • HVD1 is different from HVD2 and/or LVD1 is different from LVD2.
  • AB1 and AB2 bind different antigens (i.e., the first antigen and the second antigen are different).
  • the set of HVD1 and LVD1 is different from the set of HVD2 and LVD2.
  • the activatable construct further comprises a first heavy chain constant domain (HC1) coupled to the
  • the HVD1 is coupled directly or indirectly to the C- terminus of the MM1 via the CM1 and directly or indirectly to the N-terminus of the DD1 via the HC1 .
  • the activatable construct further comprises a first light chain constant domain (LC1) coupled to the C-terminus of the LVD1.
  • the LVD1 is coupled directly or indirectly to the C-terminus of the MM2 via the CM2, wherein the LVD1 is coupled directly or indirectly to the N-terminus of the LC1, and wherein the HC1 is coupled, directly or indirectly, to the LC1.
  • the AB1, the AB2, or both comprise an antibody fragment or an antigen binding fragment.
  • the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody.
  • antigen-binding fragments include a VH domain, a VHH domain, a VNAR domain, and a single chain fragment variable (scFv), BiTE or a component thereof, a (scFv)2, a NANOBODY®, a nanobody -HS A, VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab’)2, a diabody, a CROSSMAB®, a DAF (two-in-one), a DAE (four-in-one), a DUTAMAB®, a DT- IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a FcAb, a kl-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scF
  • the activatable construct further comprises a second heavy chain constant domain (HC2) coupled to the C-terminus of the HVD2. In some aspects, the activatable construct further comprises a second light chain constant domain (LC2) coupled to the C- terminus of the LVD2.
  • the HVD2 is coupled directly or indirectly to the C- terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the HC2.
  • the LVD2 is coupled directly or indirectly to the C-terminus of the MM4 via the CM4. In some aspects, the LVD2 is coupled directly or indirectly to the N-terminus of the LC2 and the HC2 is coupled directly or indirectly to the LC2.
  • the HVD1 together with the HC1 and the LVD1 together with the LC1 comprise an Fab domain, wherein the HVD1-HC1 is coupled directly or indirectly to the DD1 .
  • the HVD2 and LVD2 form a scFv domain coupled directly or indirectly to the DD2.
  • the HVD2 and LVD2 are not coupled to a MM.
  • at least one of the HVD2 or the LVD2 in the scFv domain is coupled directly or indirectly to the MM3 via the CM3 and the other HVD2 or LVD2 is coupled directly or indirectly to the DD2.
  • the activatable construct comprises an antibody.
  • an activatable construct of the present disclosure is a homodimer comprising two identical polypeptide chains.
  • an activatable construct is a heterodimer comprising two different polypeptide chains.
  • an activatable construct is a bispecific activatable antibody.
  • an activatable construct is a multi-specific activatable antibody.
  • Antibodies to any of a variety of targets may be employed, such as those described herein and also in, for example, WO2019/075405, WO2018/165619, WO2018/222949, WO2018/085555, WO20 17/011580, WO2016/179257, WO2016/179285, WO2016/179335, WO2016/ 149201, WO20 16/ 118629, WO2021/061867, WO2016/014974, W02020/092881, W02020/086665, WO2013/163631, WO2014197612, WO2014/052462, WO2013/192550, WO2020/252358, WO20 19/046652, WO2015/066279, WO2019/173771, WO2020/252349, WO2019/13374, W02024/015830, WO2023/183888, WO2023/192606, WO2023/064955, which are incorporated herein by reference in their entire
  • activatable constructs include those having the following structures in an N- terminal to C-terminal direction: a) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 ; and iii) HVD2-LVD2-DD2; b) i) MM2-CM2-LVD 1 -LC 1 ; ii)MMl-CMl-HVDl-HCl-DDl; and iii) LVD2-HVD2-DD2; c) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-DD1; and iii) MM3- CM3-HVD2-LVD2-DD2; d) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; and ii
  • activatable constructs include those having the following structures in an N-terminal to C-terminal direction: k) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; l) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-LVD2-HVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; m) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; ii) LVD1-LC1-CM3-MM3; and iii) DD2; n) i) MM 1 -CM 1 -HVD 1 -HC 1 -LVD2-HVD2 -CM2 -MM2 -DD2;
  • each between elements of an activatable construct is a direct or indirect linkage (e.g., via a linker).
  • the first antigen and the second antigen are the same molecule or the same epitope.
  • the activatable construct specifically binds to a single epitope.
  • the MM1, the MM2, the MM3, and the MM4 comprise the same amino acid sequence.
  • the MM1 and the MM4 comprise the same amino acid sequence.
  • the MM2 and the MM3 comprise the same amino acid sequence.
  • the second polypeptide chain and the third polypeptide chains comprise the same amino acid sequence.
  • the first polypeptide chain and the fourth polypeptide chains comprise the same amino acid sequence.
  • first antigen and the second antigen are different epitopes on the same molecule. In some aspects, the first antigen and the second antigen are different molecules or different epitopes.
  • the MM1 and the MM2 comprise the same sequence.
  • the MM3 comprises a different amino acid sequence than the MM1, the MM2, or both the MM1 and the MM2.
  • the MM3 comprises a different amino acid sequence than the MM1 and the MM2.
  • the MM1 and the MM3 are different amino acid sequences.
  • the activatable construct is bispecific. In some aspects, the activatable construct comprises a bispecific antibody. In some aspects, the second polypeptide chain and the third polypeptide chain are different amino acid sequences. In some aspects, the first polypeptide chain and the fourth polypeptide chain are different amino acid sequences. In some aspects, the first polypeptide chain and the fourth polypeptide chain comprise the same amino acid sequence and the second polypeptide chain and third polypeptide chain comprise the same amino acid sequence. In some aspects, the first polypeptide chain and the fourth polypeptide chain comprise the same amino acid sequence and the second polypeptide chain and third polypeptide chain comprise a different amino acid sequence. In some aspects, the amino acid sequences of the HVD1-HC1 domains are different amino acid sequences than the HVD2-HC2 domains.
  • the CM1 and the CM3 comprise the same amino acid sequence. In some aspects, the CM1 and the CM3 are different amino acid sequences. In some aspects, the second polypeptide chain and the third polypeptide chain are different amino acid sequences.
  • the DD1 and the DD2 comprise the same amino acid sequence. In some aspects, the DD1 and the DD2 are different amino acid sequences.
  • the activatable construct further includes a third antigen binding domain (AB3) having specific binding affinity for a third antigen.
  • the AB3 comprises a heavy chain variable domain 3 (HVD3) and a light chain variable domain 3 (LVD3).
  • the AB3 is coupled directly or indirectly to a fifth masking moiety (MM5) via a fifth cleavable moiety (CM5) and the MM5 inhibits binding of the AB3 to the third antigen.
  • the AB1 is coupled directly or indirectly to the N-terminus of the DD1.
  • the AB2 is coupled directly or indirectly to the N-terminus of the DD2.
  • the AB3 is coupled directly or indirectly to the C-terminus of the DD1 or the DD2.
  • the AB3 comprises a scFv, wherein the AB3 comprises: i) MM5-CM5-HVD3-LVD3-X; or ii) MM5-CM5-LVD3-HVD3-X; or wherein X represents attachment to the N-terminus or the C-terminus of the DD1 or the DD2, and each is a direct or an indirect covalent linkage.
  • X represents attachment to the N-terminus of the DD2.
  • X represents attachment to the C- terminus of the DD2.
  • X represents attachment to the N-terminus of the DD 1.
  • X represents attachment to the C-terminus of the DD1.
  • the C-terminus of the DD1 or the DD2 is attached to the N-terminus of: iii) LVD3-HVD3-CM5-MM5; or iv) HVD3-LVD3-CM5-MM5;
  • constructs may also be depicted as X-LVD3-HVD3-CM5-MM5 or X-HVD3- LVD3-CM5-MM5, wherein X represents attachment to the C-terminus of the DD1 or the DD2.
  • a masked AB 3 is attached to the N-terminus or the C-terminus of one of the DD1 or DD2 of any of the activatable constructs disclosed herein.
  • activatable constructs with an AB3 include those having the following structures in an N-terminal to C- terminal direction: a) i) MM2-CM2-LVD1-LC1; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 -LVD3 -HVD3 -CM5 -MM5 ; iii) MM3-CM3-HVD2-HC2-DD2; and iv) MM4-CM4-LVD2-LC2; b) i)MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2-LVD3-HVD3-CM5-MM5; and iv) MM4-CM4-LVD2-LC2; c) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 MM
  • MM5 MM3-CM3-DD2
  • j i) MM1-CM1-LVD1-LC1
  • MM3-CM3-DD2-LVD3-HVD3-CM5-MM5 k) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5- MM5; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2; l) i) MM 1 -CM 1 -HVD 1 -HC 1 -HVD2-L VD2-CM2-MM2-DD 1 ; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and i
  • the activatable construct further comprises a fourth antigen binding domain (AB4) having specific binding affinity for a fourth antigen.
  • the AB4 comprises a heavy chain variable domain 4 (HVD4) and a light chain variable domain 4 (LVD4).
  • the AB4 is coupled directly or indirectly to a sixth masking moiety (MM6) via a sixth cleavable moiety (CM6) and the MM6 inhibits binding of the AB4 to the fourth antigen.
  • the AB3 is coupled directly or indirectly to the C-terminus of the DD1 and the AB4 is coupled directly or indirectly to the C-terminus of the DD2.
  • the AB4 comprises a scFv, wherein the AB4 comprises: i) MM6-CM6-HVD4-LVD4-Y; or ii) MM6-CM6-LVD4-HVD4-Y; wherein Y represents attachment to the N-terminus or the C-terminus of the DD2, and each is a direct or an indirect covalent linkage.
  • Y represents attachment to the N-terminus of the DD2.
  • Y represents attachment to the C-terminus of the DD2.
  • Y represents attachment to the N-terminus of the DD1.
  • Y represents attachment to the C-terminus of the DD1.
  • the C-terminus of the DD1 or the DD2 is attached to the N-terminus of: iii) LVD4-HVD4-CM6-MM6; or iv) HVD4-LVD4-CM6-MM6;
  • constructs may also be depicted as Y- LVD4-HVD4-CM6-MM6 or Y-HVD4- LVD4-CM6-MM6, wherein Y represents attachment to the C-terminus of the DD1 or the DD2.
  • a masked AB4 is attached to the N-terminus or the C-terminus of one of the DD1 or DD2 of any of the activatable constructs disclosed herein.
  • Examples of activatable constructs with an AB4 include those having the following structures in an N-terminal to C- terminal direction: a) i) MM2-CM2-LVD1-LC1; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 -LVD3 -HVD3 -CM5 -MM5 ; iii) MM3-CM3-HVD2-HC2-DD2-LVD4-HVD4-CM6-MM6; and iv) MM4-CM4-LVD2-LC2; b) i)MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 -LVD4-HVD4-CM6-MM
  • the activatable constructs of the present disclosure may bind to one or more of a variety of targets.
  • Antigen binding domains that bind to specific binding partners are disclosure herein as well as in the relevant literature.
  • At least one of the AB1, the AB2, the AB3, or the AB4 in the activatable construct is a specific binding partner for any target (antigen) listed in Table 1.
  • At least one of the AB1, the AB2, the AB3, or the AB4 in the activatable construct is a specific binding partner for one of CD3, ITGA3B1, or EGFR, and at least one other ABs is a specific binding partner for any target listed in Table 1.
  • the activatable constructs engage immune effector cells and a tumor associated antigen.
  • a first antigen or first antigen binding domain target or epitope is a tumor associated antigen.
  • a second antigen or second antigen binding domain target or epitope is a surface antigen on an immune effector cells, such as, for example, a leukocyte, such as a T-cell, a natural killer (NK) cell, a mononuclear effector cell (such as, for example, a myeloid mononuclear cell), a macrophage, and the like.
  • the activatable construct may comprise an immune effector cell engaging bispecific activatable antibody, which crosslinks an immune effector cell with another cell (e.g., a cell associated with a disease such as cancer or infection).
  • the activatable construct may comprise a leukocyte cell-engaging bispecific activatable antibody, a T cell engaging bispecific activatable antibody, a NK cell-engaging bispecific activatable antibody, a macrophage cellengaging bispecific activatable antibody, a mononuclear cell-engaging bispecific activatable antibody, or a myeloid mononuclear cell-engaging bispecific activatable antibody.
  • the activatable antibody may comprise a T cell engaging bispecific antibody.
  • the AB 1 and the AB2 in the activatable construct are a specific binding partner for any target listed in Table 1, wherein the AB1 and AB2 are specific binding partners for the same target. In some aspects, the AB1 and the AB2 in the activatable construct are specific binding partners for any target listed in Table 1, wherein the AB1 and AB2 are specific binding partners for different targets. In an exemplary embodiment, AB1 of the activatable construct is specific binding partner for one of CD3, ITGA3B1, or EGFR and AB2 is a specific binding partner for any target listed in Table 1. In another exemplary embodiment, AB2 of the activatable construct is specific binding partner for one of CD3, ITGA3B1, or EGFR and AB l is a specific binding partner for any target listed in Table 1.
  • the antigen binding domain may be an antibody or a fragment thereof, e.g., a monoclonal antibody, single chain antibody, Fab fragment, F(ab')2 fragment, single-chain variable fragment (scFv), diabody (a noncovalent dimer of scFv), single chain antibody (scab), a VHH, a domain antibody (dAb) or single domain antibody (nanobody, e.g., single domain heavy chain antibody, single domain light chain antibody).
  • a single domain antibody may be an antibody fragment that is a single monomeric variable antibody domain.
  • a single domain antibody may have similar affinity to antigens as a corresponding full-length antibody.
  • Some exemplary antibodies are disclosed in Table 3.
  • Other antibodies may be identified or generated using known methods, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art.
  • the present disclosure includes activatable constructs having an engineered dimerization domain for modified effector functions, e.g., so as to enhance or reduce such function to improve the effectiveness of the antibody, antibody domain, or fragment thereof.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibodydependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti -Cancer Drug Design, 3: 219-230 (1989).
  • Fc mutations are made to remove glycosylation sites, thereby reducing Fc function.
  • the antigen binding domains may be a scFv. In some embodiments, the antigen binding domains may be a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.
  • the present disclosure includes structures having combinations of one or more polypeptides comprising any of the domains listed above, e.g., one or more of SDA, Fv, ScFv, Fab, scFab, VHH, and dAb, with one or more selected from SDA, Fv, scFv, Fab, VHH, scFab, and dAb.
  • antibody is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigenbinding domains that specifically bind to an antigen or epitope.
  • antibody specifically includes, e.g., intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific, and multi-specific antibodies.
  • One example of an antibody is an antigen-binding domain formed by a VH -VL dimer. Additional examples of an antibody are described herein. Additional examples of an antibody are known in the art. There are five main heavy-chain classes or isotypes, some of which have several subtypes, and these determine the functional activity of an antibody molecule.
  • immunoglobulin M immunoglobulin M
  • immunoglobulin D immunoglobulin D
  • immunoglobulin G immunoglobulin G
  • immunoglobulin A immunoglobulin A
  • immunoglobulin E immunoglobulin E
  • Illustrative antibodies include any one or combination of the HVD1, the HVD2, the HVD3, or HVD4 comprising a sequence selected from:
  • QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPE SGSVKYNEGFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDY WGQGTTVTVSS (SEQ ID NO: 680); or QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVT VSS (SEQ ID NO: 702).
  • any one or combination of the LVD1, the LVD2, the LVD3, or the LVD4 comprises a sequence selected from:
  • the HVD1-HC1, the HVD2-HC2, or both comprises a sequence selected from:
  • the LVD1-LC1, the LVD2-LC2, or both comprises a sequence selected from:
  • DIQMTQ SPS SLS AS VGDRVTITC S AS S SIS SNYLHW YQQKPGKVPKLLIYRT SNL A SGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 692); or
  • Activatable constructs of the present disclosure may employ a variety of masking moieties (MMs).
  • MMs inhibit interaction of the antigen binding domain with its antigen binding partner.
  • the MM may bind to the AB 1 and/or AB2 and limit the interaction of the antigen binding domain with its antigen binding partner.
  • the MMs contemplated by this disclosure may be from 1-50 amino acids in length (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, or 40 amino acids, or no greater than 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids).
  • the MMs may be from 8 to 15 amino acids in length.
  • any of the MMs are no more than 40 amino acids in length.
  • the MM is no more than 20 amino acids in length.
  • the MM is no more than 19, 18, 17, 16, or 15 amino acids in length.
  • the MM is not a latency associated peptide.
  • the MM is not a cytokine.
  • the MM is not a receptor for a cytokine.
  • the MM is not an albumin.
  • the MM excludes proteins or polypeptides having more than 50 amino acids, In some aspects, the MM excludes proteins or polypeptides having more than 25 amino acids, In some aspects, the MM excludes proteins or polypeptides having more than 20 amino acids, In some aspects, the MM excludes proteins or polypeptides having more than 15 amino acids.
  • a MM may be a receptor for AB1 or AB2 or a fragment of a receptor therefor. In some aspects, a MM may be a scFv that binds selectively to AB 1 or to AB2.
  • the MMs may contain genetically encoded or genetically non-encoded amino acids.
  • genetically non-encoded amino acids are but not limited to D-amino acids, P-amino acids, and y-amino acids.
  • the MMs contain no more than 50%, 40%, 30%, 20%, 15%, 10%, 5% or 1% of genetically non-encoded amino acids.
  • any one or combination of MMs described herein each independently is from 2 to 50 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40 or 50 amino acids).
  • any one or combination of MMs described herein e.g., MM1 and MM2; MM1, MM2, and MM3; MM1, MM2, MM3, and MM4; MM1, MM2, MM3, MM4, and MM5; or the MM1, MM2,
  • MM3, MM4, MM5, and MM6 may each independently comprise a sequence selected from the group consisting of: XCXXRXXFEXXDCVX, where X equals any amino acid (SEQ ID NO: 716); TCPTRWHFETTDCVM (SEQ ID NO: 717); ICGSRLDFELNDCVM (SEQ ID NO: 718); WCRDRSHFETGDCVM (SEQ ID NO: 719); ICTSRWEFENRDCVM (SEQ ID NO: 720); VCRDRNEFEVGDCVM (SEQ ID NO: 721); TCKNRLEFERGDCVM (SEQ ID NO: 722); VCSSRLEFEQKDCVM (SEQ ID NO: 723); WCRDREHFEKGDCVM (SEQ ID NO: 724); YCANRYEFEYGDCVM (SEQ ID NO: 725); VCRTRWHFETTDCVM (SEQ ID NO: 716), TCPTRWHFETTDCVM (SEQ ID NO: 717); IC
  • VCSNRAEFEWGDCVM SEQ ID NO: 727
  • VCASRWHFENTDCVM SEQ ID NO: 728
  • NCASRWHFENEDCVM SEQ ID NO: 729
  • VCSGRLEFELGDCVM SEQ ID NO: 730
  • VCSSRWEFETNDCVM SEQ ID NO: 731
  • FCRDRLDFDTMDCVM SEQ ID NO: 732
  • CCMDRLEFERGDCVM SEQ ID NO: 733
  • VCGSRNEFETGDCVM SEQ ID NO: 734
  • MCSGRLEFETGDCVM SEQ ID NO: 735
  • ECKTRQDFEMHDCVY SEQ ID NO: 736
  • QCMSRFAFEIGDCVM SEQ ID NO: 737
  • TCLSRYEFETTDCVM SEQ ID NO: 738
  • VCRTRWHFETTDCVM SEQ ID NO: 739
  • ICHDPYMNIDYTCKL SEQ ID NO: 740
  • VMCYWEGWGFGRC VCSNRAE
  • CISPRGCPDGPYVMY (SEQ ID NO: 749); CISPRGCPDGPYVM (SEQ ID NO: 750); CISPRGCEPGTYVPT (SEQ ID NO: 751); CISPRGCPGQIWHPP (SEQ ID NO: 752); GSHCLIPINMGAPSC (SEQ ID NO: 753); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 754); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 755);
  • ADHVFWGSYGCISPRGCPG (SEQ ID NO: 756); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 757); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 758); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 759); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 760);
  • CPYYTLSYCGCISPRGCPG (SEQ ID NO: 761); CNHVYFGTCGCISPRGCPG (SEQ ID NO: 762); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 763); CHHFTLTTCGCISPRGCPG (SEQ ID NO: 764); YNPCATPMCCISPRGCPG (SEQ ID NO: 765);
  • CNHHYFYTCGCISPRGCG (SEQ ID NO: 766); CNHHYHYYCGCISPRGCG (SEQ ID NO: 767); CNHVYFGTCGCISPRGCG (SEQ ID NO: 768); CHHVYWGHCGCISPRGCG (SEQ ID NO: 769); CPHFTTTSCGCISPRGCG (SEQ ID NO: 770);
  • CNHFTLTTCGCISPRGCG (SEQ ID NO: 771); CHHFTLTTCGCISPRGCG (SEQ ID NO: 772); CPYYTLSYCGCISPRGCG (SEQ ID NO: 773); CPHVSFGSCGCISPRGCG (SEQ ID NO: 774); ADHVFWGSYGCISPRGCG (SEQ ID NO: 775); YNPCATPMCCISPRGCG (SEQ ID NO: 776); CHHVYWGHCGCISPRGCG (SEQ ID NO: 777);
  • MMYCGGNEVLCGPRV (SEQ ID NO: 792); GYRWGCEWNCGGITT (SEQ ID NO: 793); GYLWGCEWNCGGITT (SEQ ID NO: 794); MMYCGGNEIFCEPRG (SEQ ID NO: 795); GYGWGCEWNCGGSSP (SEQ ID NO: 796); and MMYCGGNEIFCGPRG (SEQ ID NO: 797).
  • any one or combination of MMs described herein each independently comprise a sequence selected from the group consisting of: VCRDRNEFEVGDCVM (SEQ ID NO: 721); TCLSRYEFETTDCVM (SEQ ID NO: 738); VCSNRAEFEWGDCVM (SEQ ID NO: 727); VCSGRLEFELGDCVM (SEQ ID NO: 730); CISPRGCPDGPYVMY (SEQ ID NO: 749); or LSCEGWAMNREQCRA (SEQ ID NO: 783).
  • VCRDRNEFEVGDCVM SEQ ID NO: 721
  • TCLSRYEFETTDCVM SEQ ID NO: 738
  • VCSNRAEFEWGDCVM SEQ ID NO: 727
  • VCSGRLEFELGDCVM SEQ ID NO: 730
  • CISPRGCPDGPYVMY SEQ ID NO: 749
  • LSCEGWAMNREQCRA SEQ ID NO: 783
  • Illustrative MMs include those provided as SEQ ID NOs: 84-108 (for use in an anti-PDL- 1 activatable antibody) disclosed in WO 2019/165143A1, as well as those disclosed in WO 2019/075405A1, WO 2009/025846, WO 2010/096838, WO 2010/081173, WO 2013/163631, WO 2013/192546, WO 2013/192550, WO 2014/026136, WO 2014/052462, WO 2014/107599, WO 2014/197612, WO 2015/013671, WO 2015/048329, WO 2015/066279, WO 2015/116933, WO 2016/014974, WO 2016/118629, WO 2016/149201, WO 2016/179285, WO 2016/179257, WO 2016/179335, WO 2017/011580, PCT/US2017/059740, US Provisional Application Serial Numbers 62/469,429, 62/572,467, and
  • the MMs may be identified through a screening procedure from a library of candidate activatable constructs having variable MMs.
  • a CM can be selected to provide for a desired enzyme/target combination, and the amino acid sequence of the MM can be identified by the screening procedure described below to identify a MM that provides for a switchable phenotype.
  • a random peptide library e.g., of peptides comprising about 2 to about 40 amino acids or more
  • MMs with specific binding affinity for an antigen binding domain can be identified through a screening procedure that includes providing a library of peptide scaffolds consisting of candidate MMs wherein each scaffold is made up of a transmembrane protein and the candidate MM.
  • the library may then be contacted with an entire or portion of a protein such as a full length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing a protein (also capable of binding the binding partner of interest), and identifying one or more candidate MMs having detectably bound protein.
  • the screening may be performed by one more rounds of magnetic-activated sorting (MACS) or fluorescence-activated sorting (FACS), as well as determination of the binding affinity of MM towards the antigen binding domain and subsequent determination of the masking efficiency, e.g., as described in US20200308243A1, which is incorporated herein by reference in its entirety.
  • Methods for screening MMs to obtain MMs unique for the antigen binding domain and those that specifically and/or selectively bind a binding domain of a binding partner/target are provided herein and can include protein display methods.
  • protease cleavable substrates may be employed in the activatable constructs of the present disclosure.
  • Suitable substrates may be readily identified by a number of known methods, including those described in Sandersjbd, et al. "Protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting.” Biotechnology Journal 12.1 (2017): 1600365; Li, Qing, et al. "Profiling protease specificity: Combining yeast ER sequestration screening (YESS) with next generation sequencing.” ACS chemical biology 12.2 (2017): 510-518; and Zhou, Jie, et al. "Deep profiling of protease substrate specificity enabled by dual random and scanned human proteome substrate phage libraries.” Proceedings of the National Academy of Sciences 117.41 (2020): 25464- 25475).
  • Illustrative cleavable moieties that are suitable for use in the activatable constructs described herein include, for example any of the sequences disclosed in WO 2010/081173; WO 2015/048329; WO 2015/116933 ; WO 2016/118629; WO 2020/118109; WO 2024/030843 ; WO 2024/030845; WO 2024/030847; WO 2024/030858; and WO 2024/030850, the disclosures of which are incorporated herein by reference in their entireties.
  • any one or combination of CMs described herein each independently comprises a substrate for the same or different protease.
  • CMs described herein e g., CM1 and CM2; CM1, CM2, and CM3; CM1, CM2, CM3, and CM4; CM1, CM2, CM3, CM4, and CM5; or CM1, CM2, CM3, CM4, CM5, and CM6 each independently comprises a substrate for the same or different protease.
  • any one or combination of CMs described herein (e.g., CM1 and CM2; CM1, CM2, and CM3; CM1, CM2, CM3, and CM4; CM1, CM2, CM3, CM4, and CM5; or CM1, CM2, CM3, CM4, CM5, and CM6) independently comprise a total of about 3 amino acids to about 25 amino acids.
  • the CM may comprise a total of about 3 amino acids to about 25 amino acids, about 3 amino acids to about 20 amino acids, about 3 amino acids to about 15 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 5 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, or about 20 amino acids to about 25 amino acids.
  • each of the CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a total of about 3 amino acids to about 15 amino acids.
  • any one or combination of CMs described herein independently comprises a substrate for a protease selected from the group consisting of ADAMS, AD AMTS, ADAM8, ADAM9, ADAMI 0, ADAM12, ADAM15, ADAM17/TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, Aspartate proteases, BACE, Renin, Aspartic cathepsins, Cathepsin D, Cathepsin E, Caspases, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Ca
  • CMs each independently comprise a substrate for a protease that is more prevalently found in diseased tissue associated with a cancer.
  • the cancer is selected from the group of: gastric cancer, breast cancer, osteosarcoma, and esophageal cancer.
  • the cancer is breast cancer.
  • the cancer is a HER2-positive cancer.
  • the cancer is Kaposi sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell cancer (RCC), melanoma, neuroblastoma, basal cell carcinoma, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, breast cancer, ovarian cancer, bladder cancer, BCG-resistant non-muscle invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck carcinoma, uterine cancer, cervical cancer, or testicular cancer, and the like.
  • the CM components comprise substrates for protease(s) that is/are more prevalent in tumor tissue
  • any one or combination of CMs described herein each independently comprises a sequence selected from the group consisting of sequences listed in Table 13.
  • any one or combination of CMs described herein each independently comprises a sequence selected from the group consisting of: LSGRSDNH (SEQ ID NO: 5), TGRGPSWV (SEQ ID NO: 6), PLTGRSGG (SEQ ID NO: 7), TARGPSFK (SEQ ID NO: 8), NTLSGRSENHSG (SEQ ID NO: 9), NTLSGRSGNHGS (SEQ ID NO: 10), TSTSGRSANPRG (SEQ ID NO: 11), TSGRSANP (SEQ ID NO: 12), VHMPLGFLGP (SEQ ID NO: 13), AVGLLAPP (SEQ ID NO: 14), AQNLLGMV (SEQ ID NO: 15), QNQA
  • any one or combination of CMs described herein each independently comprises a sequence selected from the group consisting of: ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), ISSGLLSGRSDQH (SEQ ID NO: 54), SGRSDNI (SEQ ID NO: 100), ISSGLLSGRSDNI (SEQ ID NO: 68), ISSGLLSGRSDNP (SEQ ID NO: 57), and PWGLSGRS (SEQ ID NO: 157).
  • ISSGLLSGRSDNH SEQ ID NO: 28
  • LSGRSDDH SEQ ID NO: 33
  • ISSGLLSGRSDQH SEQ ID NO: 54
  • SGRSDNI SEQ ID NO: 100
  • ISSGLLSGRSDNI SEQ ID NO: 68
  • ISSGLLSGRSDNP SEQ ID NO: 57
  • PWGLSGRS SEQ ID NO: 157
  • Cleavable moieties that are suitable for use in the constructs described herein include truncation variants of any one of the aforementioned amino acid sequences, which retain the recognition site for the corresponding protease.
  • These include C-terminal and/or N-terminal truncation variants comprising at least 3 contiguous amino acids of the above-described amino acid sequences, or at least 4, or at least 5, or at least 6, or at least 7 amino acids of the foregoing amino acid sequences that retain a recognition site for a protease.
  • the truncation variant of the above-described amino acid sequences is an amino acid sequence corresponding to any of the above, but that is C- and/or N-terminally truncated by 1 to about 10 amino acids, 1 to about 9 amino acids, 1 to about 8 amino acids, 1 to about 7 amino acids, 1 to about 6 amino acids, 1 to about 5 amino acids, 1 to about 4 amino acids, or 1 to about 3 amino acids, and which: (1) has at least three amino acid residues; and (2) retains a recognition site for a protease.
  • the truncated CM is an N-terminally truncated CM.
  • the truncated CM is a C-terminally truncated CM.
  • the truncated C is a C- and an N-terminally truncated CM.
  • the protease(s) is/are those corresponding to upregulated protease activity, e.g., in a tumor microenvironment.
  • Activatable constructs of the present disclosure may employ any of a variety of dimerization domains.
  • Suitable DDs include both polymeric (e.g., a synthetic polymer, a polypeptide, a polynucleotide, and the like) and small molecule (non-polymeric moieties having a molecular weight of less than about 1 kilodalton, and sometimes less than about 800 daltons) types of moieties.
  • the pair of DDs may be any pair of moieties that are known in the art to bind to each other.
  • Illustrative DDs that are suitable for use in the activatable constructs described herein include, for example any of the DDs disclosed in WO2021207669A1, WO2022197764A2, or WO2024216194A1, which are incorporated by reference herein in their entireties.
  • the DD1 and the DD2 are members of a pair, or the DD is a pair, selected from the group of: a sushi domain from an alpha chain of human IL- 15 receptor (IL15Ra) and a soluble IL-15; barnase and barnstar; a protein kinase A (PKA) and an A-kinase anchoring protein (AKAP); adapter/docking tag molecules based on mutated RNase I fragments; a pair of antigen-binding domains (e.g., a pair of single domain antibodies); soluble N-ethyl- mal eimide sensitive factor attachment protein receptors (SNARE); modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25; a single domain antibody (sdAb) and corresponding epitope; an antigen-binding domain (e.g., a single chain antibody such as a single chain variable fragment (scFv), a single domain antibody, and
  • the DD, or the DD1 and DD2 are non-polypeptide polymers.
  • the nonpolypeptide polymers may covalently bound to each other.
  • the non- polypeptide polymer is a sulfur-containing polymer, e.g., sulfur-containing polyethylene glycol.
  • the DD, or the DD1 and DD2 may be covalently bound to each other via one or more disulfide bonds.
  • the epitope may be a naturally or non-naturally occurring epitope.
  • exemplary non-naturally occurring epitopes include, for example, a non-naturally occurring peptide, such as, for example, a poly-His peptide (e.g., a His tag, and the like).
  • the DD, or the DD1 and the DD2 are a pair of Fc domains.
  • an “Fc domain” refers to a contiguous amino acid sequence of a single heavy chain of an immunoglobulin, e.g., the CH2-CH3 domains of IgG, IgA, or IgD, or the CH2-CH3-CH4 domains of IgE or IgM. A pair of Fc domains associate together to form an Fc region of an immunoglobulin.
  • the pair of Fc domains is a pair of human Fc domains (e.g., a pair of wildtype human Fc domains).
  • the human Fc domains are human IgGl Fc domains (e.g., wildtype human IgGl Fc domains), human IgG2 Fc domains (e.g., wildtype human IgG2 Fc domains), human IgG3 Fc domains (e.g., wildtype human IgG3 Fc domains), or human IgG4 Fc domains (e.g., wildtype human IgG4 Fc domains).
  • the human Fc domains comprise a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 661 .
  • the human Fc domains comprise SEQ ID NO: 661.
  • the pair of Fc domains comprise a knob mutant and a hole mutant of an Fc domain.
  • the knob and hole mutants may interact with each other to facilitate the dimerization.
  • the knob and hole mutants may comprise one or more amino acid modifications within the interface between two Fc domains (e.g., in the CH3 domain).
  • the modifications comprise amino acid substitution T366W and optionally the amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other one of the antibody heavy chains (numbering according to EU index of Kabat numbering system).
  • knob and hole mutants include Fc mutants of SEQ ID NOs: 663 and 664, as well as those described in U.S. Pat. Nos. 5,731,168; 7,695,936; and 10,683,368, which are incorporated herein by reference in their entireties.
  • the dimerization domains comprise a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NOs: 663 and 664, respectively.
  • the DD is a homodimer. In some aspects, the DD is a heterodimer. In some aspects, the DD may comprise SEQ ID NOs: 663 and 664. In some aspects, the DD1 and the DD2 may comprise SEQ ID NOs: 663 and 664, respectively. In some aspects, the DD1 and the DD2 are the same. In some aspects, the human Fc domains include mutations to eliminate glycosylation and/or to reduce Fc-gamma receptor binding.
  • the human Fc domains comprise the mutation N297Q, N297A, or N297G; In some aspects the human Fc domains comprise a mutation at position 234 and/or 235, for example L235E, or L234A and L235A (in IgGl), or F234A and L235A (in IgG4); In some aspects the human Fc domains are IgG2 Fc domains that comprise the mutations V234A, G237A, P238S, H268Q/A, V309L, A330S, or P331 S, or a combination thereof (all according to EU numbering).
  • Ig heavy chain constant region amino acids in which mutations in at least one amino acid leads to reduced Fc function include, but are not limited to, mutations in amino acid 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330, and 331 of the heavy constant region (according to EU numbering).
  • combinations of mutated amino acids are also known in the art, such as, but not limited to a combination of mutations in amino acids 234, 235, and 331, such as L234F, L235E, and P331S or a combination of amino acids 318, 320, and 322, such as E318A, K320A, and K322A.
  • engineered Fc domains include F243L/R292P/Y300L/V305I/P396 IgGl; S239D/I332E IgGl; S239D/I332E/A330L IgGl; S298A/E333A/K334A; in one heavy chain, L234Y/L235Q/G236W/S239M/H268D/D270E/S298A IgGl, and in the opposing heavy chain, D270E/K326D, A330M/K334E IgG; G236A/S239D/I332E IgGl; K326W/E333S IgGl; S267E/H268F/S324T IgGl; E345R/E430G/S440Y IgGl; N297A or N297Q or N297G IgGl; L235E IgGl; L234A/L235A
  • the C-terminal residue of an IgG Fc domain may be a lysine. In some aspects, the C-terminal lysine residue of an IgG Fc domain may be absent.
  • the DD1 comprises an antigen-binding domain and the DD2 comprises a corresponding epitope.
  • the antigen-binding domain is an anti-His tag antigenbinding domain and wherein the DD2 comprises a His tag.
  • the antigen-binding domain is a single chain variable fragment (scFv).
  • the antigen-binding domain is a single domain antibody (sdAb).
  • at least one of the DD1 and the DD2 comprises a dimerization domain substituent selected from the group consisting of a nonpolypeptide polymer and a small molecule.
  • the DD1 and the DD2 comprise non-polypeptide polymers covalently bound to each other.
  • the non-polypeptide polymer is a sulfur-containing polyethylene glycol, and wherein the DD1 and the DD2 are covalently bound to each other via one or more disulfide bonds.
  • at least one of the DD1 and the DD2 comprises a small molecule.
  • the small molecule is biotin.
  • the DD1 comprises biotin and the DD2 comprises an avidin.
  • the DD, or the DD1 and/or DD2 can further include a serum half-life extending moiety (e.g., polypeptides that bind serum proteins, such as immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA)).
  • a serum half-life extending moiety e.g., polypeptides that bind serum proteins, such as immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA)).
  • half-life extending moieties include hexa-hat GST (glutathione S-transferase) glutathione affinity, Calmodulin- binding peptide (CBP), Strep-tag, Cellulose Binding Domain, Maltose Binding Protein, S- Peptide Tag, Chitin Binding Tag, Immuno-reactive Epitopes, Epitope Tags, E2Tag, HA Epitope Tag, Myc Epitope, FLAG Epitope, AU1 and AU5 Epitopes, Glu-Glu Epitope, KT3 Epitope, IRS Epitope, Btag Epitope, Protein Kinase-C Epitope, and VSV Epitope.
  • the DD, or the DD1 and/or DD2 each include a total of about 5 amino acids to about 250 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids,
  • DD1 and DD2 are each an Fc domain that comprises a portion of the hinge region that includes two cysteine residues, a CH2 domain, and a CH3 domain.
  • DD1 and DD2 are each an Fc domain whose N-terminus is the first cysteine residue (reading in the N- to C- direction) in the hinge region that participates in a disulfide linkage with a second Fc domain (e.g., Cysteine 226 of human IgGl or IgG4, using EU numbering).
  • the dimerization domain is an IgG Fc region. In some aspects, the dimerization domain is an IgG Fc region wherein the upper hinge residues have been deleted.
  • the Fc is a variant wherein N-terminal sequences EPKSCDKTHT (SEQ ID NO: 1), ERK, ELKTPLGDTTHT (SEQ ID NO: 2), or ESKYGPP (SEQ ID NO: 3) have been deleted.
  • DD1 is an Fc domain hole mutant and DD2 is an Fc domain knob mutant. In some aspects, DD1 is an Fc domain knob mutant and DD2 is an Fc domain hole mutant.
  • the DD1 and the DD2 are selected from an IgGl Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a mutant or truncation variant thereof. In some aspects, the DD1 and the DD2 are human IgGl Fc domains or a mutant or truncation variant thereof.
  • the N-terminus of the Fc region is the first cysteine residue (reading in the N- to C- direction) in the hinge region that participates in a disulfide linkage with a second Fc domain (e.g., Cysteine 226 of human IgGl or IgG4, using EU numbering), and wherein any linker(s) interposed between the AB1 and the N-terminal cysteine of DD1 (the “linking region”) and any linker(s) interposed between the AB2 and the N-terminal cysteine of DD2 have a combined total length of no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids, preferably no more than 10 amino acids, especially preferably no more than 7 amino acids.
  • a second Fc domain e.g., Cysteine 226 of human IgGl or IgG4, using EU numbering
  • a spacer (or header) comprises one or more glutamine (Q) residues.
  • residues in the spacer minimize aminopeptidase and/or exopeptidase action to prevent cleavage of N-terminal amino acids.
  • Illustrative and non-limiting spacer amino acid sequences may comprise or consist of any of the following exemplary amino acid sequences: QGQSGS (SEQ ID NO: 668); GQSGS (SEQ ID NO: 669); QSGS (SEQ ID NO: 670); SGS; GS; S; QGQSGQG (SEQ ID NO: 671); GQSGQG (SEQ ID NO: 672); QSGQG (SEQ ID NO: 673); SGQG (SEQ ID NO: 674); GQG; QG; G; QGQSGQ (SEQ ID NO: 675); GQSGQ (SEQ ID NO: 676); QSGQ (SEQ ID NO: 677); QGQSG (SEQ ID NO: 678); QGQS (SEQ ID NO: 679); SGQ; GQ; and Q.
  • spacer sequences are omitted.
  • each of DD1 and DD2 independently comprises a sequence that is at least 80% identical, at least 90% identical, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from:
  • one or more linkers are introduced into the activatable construct to provide flexibility at one or more of the junctions between domains, between moi eties, between moi eties and domains, or at any other junctions where a linker would be beneficial.
  • a linker is inserted to facilitate formation and maintenance of a structure in the uncleaved construct. Any of the linkers described herein can provide the desired flexibility to facilitate the inhibition of the binding of a target, or to facilitate cleavage of a CM by a protease.
  • linkers are included in the activatable constructs that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired activatable constructs.
  • Some linkers may include cysteine residues, which may form disulfide bonds and reduce flexibility of the construct. It has been found that reducing the length of the linkers or linking region reduces the activity of the antigen binding domain protein in activatable constructs.
  • linker length is determined by counting, in a N- to C- direction, the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the preceding component, to the C-terminus of the linker adjacent to the N-terminal amino acid of the following component (i.e., where the linker length does not include either the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component).
  • linker length is determined by counting the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the preceding component to C-terminus of the linker adjacent to the first cysteine of an Fc hinge region that participates in the disulfide linkage with a second Fc domain (i.e., where the linker length does not include the C-terminal amino acid of the preceding component or the first cysteine of the Fc hinge region).
  • the linker can include a total of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 24 amino acids, about 1 amino acid to about 22 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 18 amino acids, about 1 amino acid to about 16 amino acids, about 1 amino acid to about 15 amino acids, about 1 amino acid to about 14 amino acids, about 1 amino acid to about 12 amino acids, about 1 amino acid to about 10 amino acids, about 1 amino acid to about 8 amino acids, about 1 amino acid to about 6 amino acids, about 1 amino acid to about 5 amino acids, about 1 amino acid to about 4 amino acids, about 1 amino acid to about 3 amino acids, about 1 amino acid to about 2 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 14 amino acids,
  • the linker includes a total of about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, or about 25 amino acids.
  • activatable constructs that do not comprise any linkers.
  • a linker is rich in glycine (Gly or G) residues.
  • the linker is rich in serine (Ser or S) residues.
  • the linker is rich in glycine and serine residues.
  • the linker has one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs).
  • the linker has one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 622) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences).
  • the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 629) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences). In some aspects, the linker has one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 642) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences).
  • the activatable construct further comprises one or more linkers.
  • the linker is a peptide having a length of 1 to 30, 5 to 25, 10-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some aspects, the linker comprises at least 5 amino acids.
  • a linker is independently selected from the group consisting of: GSSGGSGGSGG (SEQ ID NO: 623); GGGS (SEQ ID NO: 622); GGGSGGGS (SEQ ID NO: 624); GGGSGGGSGGGS (SEQ ID NO: 625); GGGGSGGGGSGGGGS (SEQ ID NO: 626); GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 627); GGGGSGGGGS (SEQ ID NO: 628); GGGGS (SEQ ID NO: 629); GS; GGGGSGS (SEQ ID NO: 630); GGGGSGGGGSGGGGSGS (SEQ ID NO: 631); GGSLDPKGGGGS (SEQ ID NO: 632); PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 633); SKYGPPCPPCPAPEFLG (SEQ ID NO: 634); GKSSGSGSESKS (SEQ ID NO: 635); GST
  • Non-limiting examples of linkers can include a sequence that is at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to GGGS (SEQ ID NO: 622), GSSGGSGGSGG (SEQ ID NO: 623), GGGGSGGGGSGGGGS (SEQ ID NO: 626), GGGGSGS (SEQ ID NO: 630), GGGGSGGGGSGGGGSGS (SEQ ID NO:
  • the linker comprises a sequence of GGGS (SEQ ID NO: 622).
  • an activatable construct includes one, two, three, four, five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences of any of the exemplary linker sequences described herein or known in the art).
  • a linker comprises sulfo-SIAB, SMPB, and sulfo-SMPB, wherein the linkers react with primary amines sulfhydryls.
  • activatable constructs of the present disclosure can be readily constructed from the component parts as described herein.
  • Activatable constructs may be readily designed in accordance with the structures described herein. While the present disclosure contains exemplary figures, sequences, and working examples, those examples are provided for illustrative purposes and are non-limiting. Those skilled in the art will recognize that the component parts described herein are suitable for assembly into various activatable construct structures.
  • the activatable construct has a structure as shown in Figures 1A-1C.
  • the activatable construct comprises an anti-integrin antigen binding domain.
  • the first polypeptide comprises an amino acid sequence selected from:
  • QGQSGQVCRDRNEF EVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
  • the second polypeptide comprises an amino acid sequence selected from:
  • the third polypeptide comprises an amino acid sequence selected from:
  • the fourth polypeptide comprises an amino acid sequence selected from:
  • the light chain and the heavy chain of the first and second polypeptide, respectively, comprise the same MM.
  • the first polypeptide comprises an amino acid sequence of:
  • the first polypeptide comprises an amino acid sequence of:
  • the first polypeptide comprises an amino acid sequence of:
  • the second polypeptide comprises an amino acid sequence of:
  • the first polypeptide comprising an amino acid sequence of: QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLSAS VGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDYTL TISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700); and the second polypeptide comprises an amino acid sequence of:
  • the light chain and the heavy chain of the fourth and third polypeptide comprise the same MM.
  • the fourth polypeptide comprises an amino acid sequence of: QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694); and the third polypeptide comprising an amino acid sequence of:
  • the fourth polypeptide comprises an amino acid sequence of:
  • the fourth polypeptide comprising an amino acid sequence of:
  • first and second polypeptide together with the third and fourth polypeptide may form an activatable construct according to Figure 1C.
  • the activatable construct comprises an anti-EGFR antigen binding domain.
  • the first polypeptide comprises an amino acid selected from:
  • the second polypeptide comprises an amino acid selected from:
  • the third polypeptide comprises an amino acid selected from:
  • the fourth polypeptide comprises an amino acid selected from:
  • the light chain and the heavy chain of the first and second polypeptide, respectively, comprise the same MM.
  • the first polypeptide comprises an amino acid sequence of:
  • the first polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVS PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and the second polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKS
  • the first polypeptide comprises an amino acid sequence of:
  • the first polypeptide comprises an amino acid sequence of: QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVSP GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSV ESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); and the second polypeptide comprises an amino acid sequence of:
  • the light chain and the heavy chain of the fourth and third polypeptide, respectively, comprise the same MM.
  • the fourth polypeptide comprises an amino acid sequence of:
  • the fourth polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVS PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNF YPRE AK VQWKVDNALQ SGNSQE S VTEQD SKD STYSL S STLTL SKAD YEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and the third polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNT
  • the fourth polypeptide comprises an amino acid sequence of:
  • the fourth polypeptide comprising an amino acid sequence of: QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVSP GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSV ESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAI ⁇ VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEI ⁇ HI ⁇ VYACE VTHQGLSSPVTKSFNRGEC (SEQ TD NO: 710); and the third polypeptide comprises an amino acid sequence of:
  • the first and second polypeptide together with the third and fourth polypeptide may form an activatable construct according to Fig. 1C.
  • the activatable construct has a structure as shown in Figs. 4-5.
  • the activatable construct comprises an anti-HER2 antigen binding domain (trastuzamab). The following are illustrative sequences:
  • the first polypeptide comprises an amino acid sequence of:
  • the second polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTT
  • the first polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNT AYLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTL VT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWW A Y
  • the first polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNT A YLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTL VT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQ
  • the first polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNTAYLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTLVT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTV
  • the first polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNTA YLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTL VT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWW A
  • the activatable construct has reduced level of activity for at least one of AB1, AB2, AB3, AB4, or any combination thereof, for example, activity is characterized by a masking efficiency that is greater than 1.5, 2, 5, 6, 7, 8, 9, or 10.
  • the activatable constructs described herein have a masking efficiency that is greater than 10, greater than 100, greater than 1000, or greater than 5000.
  • the activatable construct has greater than 10-fold, 15-fold, 20-fold, 25- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 5000-fold reduced binding activity than a corresponding antibody that lacks MMs.
  • the activatable construct has a masking efficiency that is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than a corresponding activatable antibody that lacks MM3, MM4, or both MM3 and MM4.
  • the activatable construct has a masking efficiency that is greater than 3- fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, or 100-fold greater than a corresponding activatable antibody having only a single MM coupled directly or indirectly to the AB1, AB2, AB3, AB4 or any combination thereof.
  • the activatable construct has a masking efficiency that is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than a corresponding activatable construct with only a single masking moiety per antigen binding domain.
  • the at least one activity is the binding affinity of the AB1 and/or the AB2 for its antigen (i.e., binding partner) as determined using surface plasmon resonance (e.g., performed in phosphate buffered saline at 25 degrees Celsius).
  • the amino acid sequence further comprises an amino acid sequence that encodes a signal peptide, e g., MRAWIFFLLCLAGRALA (SEQ ID: NO 665); MALTFALLVALLVLSCKSSCSVG (SEQ ID NO: 666), METDTLLLWVLLLWVPGSTG (SEQ ID NO: 667), or that encodes a signal peptide known in the art.
  • MRAWIFFLLCLAGRALA SEQ ID: NO 665
  • MALTFALLVALLVLSCKSSCSVG SEQ ID NO: 666
  • METDTLLLWVLLLWVPGSTG SEQ ID NO: 667
  • the present disclosure further includes modifications to the amino acid sequence of the polypeptides described herein or the nucleotide sequences encoding the polypeptides described herein by techniques including but not limited to site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis.
  • Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with acidic side chains e.g., aspartate and glutamate
  • amino acids with basic side chains e.g., lysine, arginine, and histidine
  • non-polar amino acids e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan
  • uncharged polar amino acids e.g., glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine
  • hydrophilic amino acids e.g., arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine
  • hydrophobic amino acids e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine
  • amino acids include: aliphatic-hydroxy amino acids (e.g., serine and threonine), amide family (e.g., asparagine and glutamine), aliphatic family (e.g., alanine, valine, leucine and isoleucine), and aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).
  • aliphatic-hydroxy amino acids e.g., serine and threonine
  • amide family e.g., asparagine and glutamine
  • aliphatic family e.g., alanine, valine, leucine and isoleucine
  • aromatic family e.g., phenylalanine, tryptophan, and tyrosine
  • This disclosure also provides methods and materials for including additional elements in any of the activatable constructs described herein including, for example, a targeting moiety to facilitate delivery to a cell or tissue of interest, an agent (e.g., a therapeutic agent, an antineoplastic agent), a toxin, or a fragment thereof.
  • a targeting moiety to facilitate delivery to a cell or tissue of interest
  • an agent e.g., a therapeutic agent, an antineoplastic agent
  • a toxin e.g., a toxin, or a fragment thereof.
  • the activatable constructs are conjugated to a cytotoxic agent, including, without limitation, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) or a radioactive isotope.
  • a cytotoxic agent including, without limitation, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) or a radioactive isotope.
  • Non-limiting exemplary cytotoxic agents that can be conjugated to any of the activatable constructs described herein include: dolastatins and derivatives thereof (e.g., auri statin E, AFP, monomethyl auri statin D (MMAD), monomethyl auri statin F (MMAF), monomethyl auristatin E (MMAE), desmethyl auristatin E (DMAE), auristatin F, desmethyl auristatin F (DMAE), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycin, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistat
  • Non-limiting exemplary enzymatically active toxins that can be conjugated to any of the activatable constructs described herein include: diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii proteins, dianfhin proteins, Phytoiaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geionin, mitogeliin, restrictocin, phenomycin, neomycin, and tricothecenes.
  • diphtheria toxin exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain abrin A chain
  • modeccin A chain alpha-sarcin
  • Non-limiting exemplary anti -neoplasties that can be conjugated to any of the activatable constructs described herein include: adriamycin, cerubidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabizine, and cytarabine.
  • Non-limiting exemplary antivirals that can be conjugated to any of the activatable constructs described herein include: acyclovir, vira A, and Symmetrel.
  • Non-limiting exemplary antifungals that can be conjugated to any of the activatable constructs described herein include: nystatin.
  • Non-limiting exemplary conjugatable detection reagents that can be conjugated to any of the activatable constructs described herein include: fluorescein and derivatives thereof, fluorescein isothiocyanate (FITC).
  • fluorescein and derivatives thereof fluorescein isothiocyanate (FITC).
  • Non-limiting exemplary antibacterials that can be conjugated to any of the activatable constructs described herein include: aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin.
  • PBDs pyrroleauzodiazepine dimers
  • Non-limiting exemplary radiopharmaceuticals that can be conjugated to any of the activatable constructs described herein include: 123 I , 89 Zr, 125 1, 131 I, "mTc, 2O1 T1, 62 Cu, 18 F, 68 Ga, 13 N, 15 0, 38 K, 82 Rb, in In, 133 Xe, n C, and "mTc (Technetium).
  • Non-limiting exemplary heavy metals that can be conjugated to any of the activatable constructs described herein include: barium, gold, and platinum.
  • Non-limiting exemplary anti-mycoplasmals that can be conjugated to any of the activatable constructs described herein include: tylosine, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.
  • Conjugation can include any chemical reaction that will bind the two molecules so long as the activatable constructs and the other moiety retain their respective activities. Conjugation can include many chemical mechanisms, e g., covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In some aspects, the preferred binding is covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in conjugating any of the activatable constructs described herein.
  • conjugation can include organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzenes, and hexamethylene diamines.
  • the activatable construct can include, or otherwise introduce, one or more non-natural amino acid residues to provide suitable sites for conjugation.
  • an agent and/or conjugate is attached by disulfide bonds (e.g., disulfide bonds on a cysteine molecule) to the antigen-binding domain.
  • disulfide bonds e.g., disulfide bonds on a cysteine molecule
  • glutathione present in the cancerous tissue microenvironment can reduce the disulfide bonds, and subsequently release the agent and/or the conjugate at the site of delivery.
  • the conjugate when the conjugate binds to its target in the presence of complement within the target site (e.g., diseased tissue (e.g., cancerous tissue)), the amide or ester bond attaching the conjugate and/or agent to the linker is cleaved, resulting in the release of the conjugate and/or agent in its active form.
  • the conjugates and/or agents when administered to a subject, will accomplish delivery and release of the conjugate and/or the agent at the target site (e.g., diseased tissue (e.g., cancerous tissue)).
  • These conjugates and/or agents are particularly effective for the in vivo delivery of any of the conjugates and/or agents described herein.
  • the linker is not cleavable by enzymes of the complement system.
  • the conjugate and/or agent is released without complement activation since complement activation ultimately lyses the target cell.
  • the conjugate and/or agent is to be delivered to the target cell (e.g., hormones, enzymes, corticosteroids, neurotransmitters, or genes).
  • the linker is mildly susceptible to cleavage by serum proteases, and the conjugate and/or agent is released slowly at the target site.
  • the conjugate and/or agent is designed such that the conjugate and/or agent is delivered to the target site (e.g., disease tissue (e.g., cancerous tissue)) but the conjugate and/or agent is not released.
  • the target site e.g., disease tissue (e.g., cancerous tissue)
  • the conjugate and/or agent is not released.
  • the conjugate and/or agent is attached to an antigen-binding domain either directly or via a non-cleavable linker.
  • exemplary non-cleavable linkers include amino acids (e.g., D-amino acids), peptides, or other organic compounds that may be modified to include functional groups that can subsequently be utilized in attachment to antigen-binding domains by methods described herein.
  • an activatable construct includes at least one point of conjugation for an agent. In some aspects, all possible points of conjugation are available for conjugation to an agent. In some aspects, the one or more points of conjugation include, without limitation, sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not sulfur atoms involved in intrachain disulfide bonds,, and/or sulfur atoms of cysteine or other amino acid residues containing a sulfur atom. In such cases, residues may occur naturally in the protein construct structure or may be incorporated into the protein construct using methods including, without limitation, site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.
  • any of the activatable constructs described herein are modified to include one or more interchain disulfide bonds.
  • disulfide bonds in the activatable constructs can undergo reduction following exposure to a reducing agent such as, without limitation, TCEP, DTT, or P-mercaptoethanol.
  • a reducing agent such as, without limitation, TCEP, DTT, or P-mercaptoethanol.
  • the reduction of the disulfide bonds is only partial.
  • partial reduction refers to situations where an activatable construct is contacted with a reducing agent and a fraction of all possible sites of conjugation undergo reduction (e.g., not all disulfide bonds are reduced).
  • an activatable construct is partially reduced following contact with a reducing agent if less than 99%, (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less than 5%) of all possible sites of conjugation are reduced.
  • the activatable constructs having a reduction in one or more interchain disulfide bonds is conjugated to a drug reactive with free thiols.
  • This disclosure also provides methods and materials for conjugating a therapeutic agent to a particular location on an activatable construct.
  • any of the activatable constructs described herein are modified so that the therapeutic agents can be conjugated to the activatable constructs at particular locations.
  • an activatable construct can be partially reduced in a manner that facilitates conjugation.
  • partial reduction of the activatable constructs occurs in a manner that conjugation sites in the activatable constructs are not reduced.
  • the conjugation site(s) are selected to facilitate conjugation of an agent at a particular location on the protein construct.
  • Various factors can influence the “level of reduction” of the activatable constructs upon treatment with a reducing agent.
  • the ratio of reducing agent to activatable constructs, length of incubation, incubation temperature, and/or pH of the reducing reaction solution can require optimization in order to achieve partial reduction of the activatable constructs with the methods and materials described herein. Any appropriate combination of factors (e.g., ratio of reducing agent to activatable constructs, the length and temperature of incubation with reducing agent, and/or pH of reducing agent) can be used to achieve partial reduction of the activatable constructs (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).
  • An effective ratio of reducing agent to activatable constructs can be any ratio that at least partially reduces the activatable constructs in a manner that allows conjugation to an agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).
  • the ratio of reducing agent to activatable constructs will be in a range from about 20: 1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8: 1 to 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to 1:1.5, from about4:l to 1:1.5, from about 3:1 to 1:1.5, from about2:l to 1:1.5, from about 1.5:1 to 1:1.5, or
  • the ratio is in a range of from about 5:1 to 1:1. In some aspects, the ratio is in a range of from about 5:1 to 1.5:1. In some aspects, the ratio is in a range of from about 4: 1 to 1 : 1. In some aspects, the ratio is in a range from about 4:1 to 1.5:1. In some aspects, the ratio is in a range from about 8: 1 to about 1:1. In some aspects, the ratio is in a range of from about 2.5:1 to 1:1.
  • An effective incubation time and temperature for treating an activatable construct with a reducing agent can be any time and temperature that at least partially reduces the activatable constructs in a manner that allows conjugation of an agent to an activatable construct (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).
  • the incubation time and temperature for treating an activatable construct will be in a range from about 1 hour at 37 °C to about 12 hours at 37 °C (or any subranges therein).
  • An effective pH for a reduction reaction for treating an activatable construct with a reducing agent can be any pH that at least partially reduces the activatable constructs in a manner that allows conjugation to an agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).
  • the agent can conjugate to the interchain thiols in the activatable constructs.
  • An agent can be modified in a manner to include thiols using a thiol-containing reagent (e.g., cysteine or N- acetyl cysteine).
  • a thiol-containing reagent e.g., cysteine or N- acetyl cysteine.
  • the activatable constructs can be partially reduced following incubation with reducing agent (e.g., TCEP) for about 1 hour at about 37 °C at a desired ratio of reducing agent to activatable constructs.
  • An effective ratio of reducing agent to activatable constructs can be any ratio that partially reduces at least two interchain disulfide bonds located in the activatable constructs in a manner that allows conjugation of a thiol-containing agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).
  • an activatable construct is reduced by a reducing agent in a manner that avoids reducing any intrachain disulfide bonds. In some aspects, an activatable construct is reduced by a reducing agent in a manner that avoids reducing any intrachain disulfide bonds and reduces at least one interchain disulfide bond.
  • the activatable constructs can also include an agent conjugated to the activatable constructs.
  • the conjugated agent is a therapeutic agent.
  • the agent e.g., agent conjugated to an activatable construct
  • the agent is a detectable moiety such as, for example, a label or other marker.
  • the agent is or includes a radiolabeled amino acid, one or more biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and/or one or more chemiluminescent agents.
  • detectable moieties are attached by spacer molecules.
  • the agent e.g., cytotoxic agent conjugated to an activatable construct
  • the agent is linked to the activatable constructs using a carbohydrate moiety, sulfhydryl group, amino group, or carboxylate group.
  • the agent e.g., cytotoxic agent conjugated to an activatable construct
  • the agent is conjugated to the activatable constructs via a linker and/or a CM (also referred to as a cleavable sequence).
  • the agent e.g., cytotoxic agent conjugated to an activatable construct
  • the agent is conjugated to a cysteine or a lysine in the activatable constructs.
  • the agent e.g., cytotoxic agent conjugated to an activatable construct
  • the linker is a thiol-containing linker.
  • PRFKIIGG SEQ ID NO: 116
  • PRFR1IGG SEQ ID NO: 117
  • Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 119)
  • GGSIDGR SEQ ID NO: 1244
  • Gelatinase A PLGLWA (SEQ ID NO: 125)
  • Calf skin collagen (al (I) chain) GPQGIAGQ (SEQ ID NO: 126)
  • Bovine cartilage collagen (al(II) chain) GIAGQ (SEQ ID NO: 128)
  • Rat aiM EPQ ALAMS (SEQ ID NO: 134)
  • QALAMSAI (SEQ ID NO: 135)
  • Rat a2M AAYHLVSQ (SEQ ID NO: 136) MDAFLESS (SEQ ID NO: 137)
  • Rat ail3(2J) ESLPVVAV (SEQ ID NO: 138)
  • Rat ail3(27J) SAPAVESE (SEQ ID NO: 139)
  • VAQFVLTE (SEQ ID NO: 142)
  • an effective conjugation of an agent e.g., cytotoxic agent
  • an activatable construct can be accomplished by any chemical reaction that will bind the agent to the activatable constructs while also allowing the agent and the activatable constructs to retain functionality.
  • bifunctional protein-coupling agents can be used for conjugation including, without limitation, N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e g., bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (e g., bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (e g., tolyene 2,6-diisocyanate), and bisactive fluorine compounds (e.g., l,5-difhioro-2,4-dinitro), amino acids, amino acids (
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
  • a carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) chelating agent can be used to conjugate a radionucleotide to the activatable constructs.
  • MX-DTPA l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
  • Suitable linkers and CMs are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201 -208 (1984) describing use of MBS (M- maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an activatable construct by way of an oligopeptide linker.
  • MBS M- maleimidobenzoyl-N-hydroxysuccinimide ester
  • suitable linkers include: (i) EDC (1 -ethyl-3-(3- dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl- alpha-methyl-alpha-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem.
  • Sulfo-LC-SPDP sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate
  • sulfo-NHS N-hydroxy sulfo-succinimide: Pierce Chem. Co., Cat. #24510 conjugated to EDC.
  • Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.
  • linkers and CMs described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties.
  • sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.
  • NHS-ester containing linkers are less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically-hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.
  • Sulfo-NHS in particular, can enhance the stability of carbodimide couplings.
  • Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
  • an agent is conjugated to the constructs using a modified amino acid sequence included in the amino acid sequence of the activatable constructs.
  • the protein construct is designed for controlled placement and/or dosage of the conjugated agent (e.g., cytotoxic agent).
  • the conjugated agent e.g., cytotoxic agent
  • the activatable constructs can be modified to include a cysteine amino acid residue at positions on the first monomer, the second monomer, the third monomer, and/or the fourth monomer that provide reactive thiol groups and does not negatively impact protein folding and/or assembly and does not alter antigen-binding properties.
  • the activatable constructs can be modified to include one or more non-natural amino acid residues within the amino acid sequence to provide suitable sites for conjugation. In some aspects, the activatable constructs is modified to include enzymatically activatable peptide sequences.
  • the present disclosure provides a nucleic acid encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein.
  • the polypeptide comprises any one of the sequences in Table 16.
  • provided herein is an mRNA comprising the nucleic acid encoding a polypeptide of the activatable construct.
  • the mRNA molecule comprises one or more of the following elements, in the 5'-to-3' direction, (i) a 5 '-cap; (ii) a 5 '-untranslated region (UTR); (iii) an open reading frame encoding a polypeptide or interest or multiple polypeptides of interest, e.g., the activatable construct; (iv) a 3'-UTR; and (v) a poly-A tail.
  • the mRNA comprises at least one modified or non-naturally occurring nucleotide.
  • the at least one modified or non-naturally occurring nucleotide comprises at least one backbone modification, sugar modification or base modification.
  • the at least one modified or non-naturally occurring nucleotide comprises at least one base modification.
  • RNA polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • modifications of polynucleotides include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine; 2-methylthio-N6- methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6- glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6- threonylcarbamoyladenosine; l,2'-O-dimethyladenosine; 1 -methyladenosine; 2'-O- methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio
  • 1-2-ethanoic acid Pseudouracil; 4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine; 1- m ethyl- 1-deaza-pseudouri dine; 1-propynyl-uridine; 1-taurinom ethyl- 1-methyl-uri dine; 1- taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2-m ethoxy -4-thio-pseudouri dine;
  • the nucleobase is selected from the group consisting of pseudouridine or N1 -methylpseudouridine.
  • the nucleoside is not pseudouridine (T) or 5-methyl- cytidine (m5C).
  • the present invention provides polynucleotides which may be isolated and/or purified. These polynucleotides may encode one or more polypeptides of interest and comprise a sequence of n number of linked nucleosides or nucleotides comprising at least one modified nucleoside or nucleotide as compared to the chemical structure of an A, G, U or C nucleoside or nucleotide.
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • modified nucleobases in polynucleotides are selected from the group consisting of pseudouridine ( ⁇
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • modified nucleobases in polynucleotides are selected from the group consisting of 1- methyl-pseudouridine (m 1 !]/), 5-methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5 C), pseudouridine ( ⁇
  • polynucleotides includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide.
  • modifications to a nucleoside may include one or more modifications to the nucleobase, the sugar, and/or the internucleoside linkage.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of: pseudouridine-alpha-thio-TP, 1 -methyl-pseudouridine-alpha-thio-TP, 1 -ethyl-pseudouridine-TP, 1-propyl-pseudouridine-TP, l-(2,2,2-trifluoroethyl)-pseudouridine-TP, 2-amino-adenine-TP, xanthosine, 5-bromo-cytidine, 5-aminoallyl-cytidine-TP, or 2-aminopurine-riboside-TP.
  • pseudouridine-alpha-thio-TP 1 -methyl-pseudouridine-alpha-thio-TP
  • 1 -ethyl-pseudouridine-TP 1-propyl-pseudouridine-TP
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of: pseudouridine-alpha-thio-TP, 1 -methyl-pseudouridine-alpha-thio-TP, 1 -ethyl-pseudouridine-TP, 1-propyl-pseudouridine-TP, 5-bromo-cytidine, 5-aminoallyl-cytidine-TP, or 2-aminopurine- riboside-TP.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of: pseudouridine-alpha- thio-TP, 1-methyl-pseudouridine-alpha-thio-TP, or 5-bromo-cytidine.
  • the isolated polynucleotide includes at least two modified nucleosides or nucleotides.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of at least one of each of 5-bromo-cytidine-TP and 1-methyl-pseudouridine-TP.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of at least one of each of 5-bromo-cytidine-TP and pseudouridine-TP.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of: 2-thio-pseudouridine- TP, 5-trifluoromethyl-uridine-TP, 5-trifluoromethyl-cytidine-TP, 3-methyl-pseudouridine, 5- methyl-2-thio-uridine-TP, N4-methyl-cytidine-TP, 5-hydroxymethyl-cytidine-TP, 3-methyl- cytidine-TP, 5-oxyacetic acid methyl ester-uridine-TP, 5-methoxycarbonylmethyl-uridine-TP, 5- methylaminomethyl-uridine-TP, 5-methoxy-uridine-TP, N1 -methyl-guanosine-TP, 8-aza- adenine-TP, 2-thio-uridine-TP, 5-bromo-uridine-TP, 2-thio-cytidine-TP, alpha-thio
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of at least one of each of 5-trifluoromethyl-cytidine-TP and 1-methyl-pseudouridine-TP; 5 -hydroxy methyl- cytidine-TP and 1-methyl-pseudouridine-TP; 5-trifluoromethyl-cytidine-TP and pseudouridine- TP; or N4-acetyl-cytidine-TP and 5-methoxy-uridine-TP.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of 2-thio-pseudouridine- TP, 5-trifluoromethyl-cytidine-TP, 5-methyl-2-thio-uridine-TP, 5-hydroxymethyl-cytidine-TP, 5- oxyacetic acid methyl ester-uridine-TP, 5-methoxy-uridine-TP, N4-acetyl-cytidine-TP, 2-thio- uridine-TP, 5-bromo-uridine-TP, alpha-thio-cytidine-TP, 5-aminoallyl-uridine-TP, or alpha-thio- uridine-TP.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of at least one of each of 5-trifluoromethyl-cytidine-TP and 1-methyl-pseudouridine-TP or 5-hydroxymethyl- cytidine-TP and 1-methyl-pseudouridine-TP.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of 2-thio-pseudouridine- TP, 5-trifluoromethyl-cytidine-TP, 5-methyl-2-thio-uridine-TP, N4-methyl-cytidine-TP, 5- hydroxymethyl-cytidine-TP, 5-oxyacetic acid methyl ester-uridine-TP, 5- methoxycarbonylmethyl-uridine-TP, 5-methoxy-uridine-TP, 2-thio-uridine-TP, 5-bromo-uridine- TP, alpha-thio-cytidine-TP, 5-aminoallyl-uridine-TP, or alpha-thio-uridine-TP.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of 2-thio-pseudouridine- TP, 5-trifluoromethyl-cytidine-TP, 5-hydroxymethyl-cytidine-TP, or 5-methoxy-uridine-TP.
  • the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of at least one of each of N4-acetyl-cytidine-TP and 5-methoxy-uridine-TP.
  • the nucleic acid sequence encoding the first polypeptide of the activatable construct (or the protein portion of the first monomer construct) is at least 70% identical (e.g., at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84 % identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the nucleic acid sequence encoding the second polypeptide of the activatable construct (or the protein portion of the second monomer construct).
  • the first polypeptide of the activatable construct and the second polypeptide of the activatable construct comprise identical components.
  • the first and second polypeptides of the activatable construct constructs are encoded by the same polypeptide (i.e., the same amino acid sequence).
  • they are encoded by the same nucleic acid (i.e., the same nucleic acid sequence).
  • the first and second polypeptide of the activatable construct are encoded by the same nucleic acid.
  • the nucleic acid encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein comprises a vector.
  • the nucleic acid encodes a polypeptide according to any one of the sequences in Table 16.
  • the vector is an expression vector.
  • pairs of vectors that together comprise any of one of the pair of nucleic acids described herein.
  • the pair of vectors is a pair of expression vectors.
  • One skilled in the art will be capable of selecting suitable vectors or sets of vectors (e.g., expression vectors) for making any of the activatable constructs described herein, and using the vectors or sets of vectors to express any of the activatable constructs described herein.
  • suitable vectors or sets of vectors e.g., expression vectors
  • the cell in selecting a vector or a set of vectors, the cell must be considered because the vector(s) may need to be able to integrate into a chromosome of the cell and/or replicate in it.
  • Exemplary vectors that can be used to produce an activatable construct are also described below.
  • the term “vector” refers to a polynucleotide capable of inducing the expression of a recombinant protein (e.g., a first or second monomer) in a cell (e g., any of the cells described herein).
  • a “vector” is able to deliver nucleic acids and fragments thereof into a host cell, and includes regulatory sequences (e.g., promoter, enhancer, poly(A) signal). Exogenous polynucleotides may be inserted into the expression vector in order to be expressed.
  • the term “vector” also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.
  • suitable vectors that include any of the nucleic acids described herein, and suitable for transforming cells (e.g., mammalian cells) are well-known in the art. See, e.g., Sambrook et al., Eds. “Molecular Cloning: A Laboratory Manual,” 2 nd Ed., Cold Spring Harbor Press, 1989 and Ausubel et al., Eds. “Current Protocols in Molecular Biology,” Current Protocols, 1993.
  • Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors.
  • a vector can, for example, include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the activatable constructs described herein.
  • the activatable constructs are made biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.
  • the vector includes a nucleic acid encoding the first polypeptide of the activatable construct and the first polypeptide of any activatable construct described herein. In some aspects, the vector is an expression vector.
  • host cells including any of the nucleic acids, or vector or sets of vectors described herein including any of the nucleic acids described herein.
  • nucleic acids and vectors e.g., any of the vectors or any of the sets of vectors described herein
  • methods that can be used to introducing a nucleic acid into a cell include: lipofection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.
  • the introducing step includes introducing into a cell a vector (e.g., any of the vectors or sets of vectors described herein) including a nucleic acid encoding the monomers that make up any of the activatable constructs described herein.
  • a vector e.g., any of the vectors or sets of vectors described herein
  • the introducing step includes introducing into a cell a vector (e.g., any of the vectors or sets of vectors described herein) including a nucleic acid encoding the monomers that make up any of the activatable constructs described herein.
  • the activatable construct can be produced by any cell, including a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell.
  • a prokaryotic cell e.g., a bacterial cell
  • a eukaryotic cell refers to a cell having a distinct, membranebound nucleus. Such cells may include, for example, mammalian, insect, fungal, or plant cells.
  • the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae.
  • the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells.
  • Non-limiting examples of mammalian cells include a rodent cell (e.g., a mouse cell, a rat cell, a hamster cell, such as Chinese hamster ovary (CHO) cells, or a non-human primate cell, or a human cell, such as human embryonic kidney cells (e.g., HEK293 cells).
  • rodent cell e.g., a mouse cell, a rat cell, a hamster cell, such as Chinese hamster ovary (CHO) cells, or a non-human primate cell
  • a human cell such as human embryonic kidney cells (e.g., HEK293 cells).
  • the cell contains the nucleic acid encoding the first monomer and the second monomer of any one of the activatable constructs described herein. In some embodiments, the cell contains the pair of nucleic acids that together encode the first monomer and the second monomer of any of the activatable constructs described herein. In some aspects, the nucleic acid encoding the first monomer and the second monomer is integrated into the genomic DNA of the host cell.
  • any of the activatable constructs described herein that include: (a) culturing any of the recombinant host cells described herein in a liquid culture medium under conditions sufficient to produce the activatable constructs; and (b) recovering the activatable constructs from the host cell and/or the liquid culture medium.
  • Cells can be maintained in vitro under conditions that favor cell proliferation, cell differentiation and cell growth.
  • cells can be cultured by contacting a cell (e.g., any of the cells described herein) with a cell culture medium that includes the necessary growth factors and supplements sufficient to support cell viability and growth.
  • the method further includes isolating the recovered activatable constructs.
  • methods of isolation include: ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand-affinity chromatography, ion-exchange chromatography (e.g., anion or cation), and hydrophobic interaction chromatography.
  • compositions and methods described herein may involve use of non-reducing or partially-reducing conditions that allow disulfide bonds to form between the dimerization domains to form and maintain dimerization of the activatable constructs.
  • the method further includes formulating the isolated the activatable constructs into a pharmaceutical composition.
  • a pharmaceutical composition e.g., a pharmaceutical composition.
  • routes of administration e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular.
  • compositions e.g., pharmaceutical compositions
  • kits that include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.
  • the present disclosure provides a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising the activatable construct according to any of the aspects disclosed herein.
  • the subject has been identified or diagnosed as having a cancer, an autoimmune disease, or an inflammatory disorder.
  • the term “subject” refers to any mammal.
  • the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster or a guinea pig), a non-human primate (e.g., a simian (e.g., a monkey (e.g., a baboon, a marmoset), or an ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human.
  • the subject is a human.
  • the subject has been previously identified or diagnosed as having the disease (e.g., cancer (e.g., any of the cancers described herein)).
  • the disease e.g., cancer (e.g., any of the cancers described herein)
  • the term “treat” includes reducing the severity, frequency or the number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., a cancer (e.g., any of the cancers described herein)) in the subject (e.g., any of the subjects described herein).
  • a disease e.g., a cancer (e.g., any of the cancers described herein)
  • treating results in reducing cancer growth, inhibiting cancer progression, inhibiting cancer metastasis, or reducing the risk of cancer recurrence in a subject having cancer.
  • the methods and uses of the present disclosure include any route of administration including intravenous, infusion, intratumoral, subcutaneous, intraperitoneal, intradermal, oral (e.g., inhalation), intranasal, transdermal (e.g., topical), transmucosal, and/or intramuscular.
  • the disease is a cancer.
  • methods of treating a subject in need thereof e.g., any of the exemplary subjects described herein or known in the art
  • administering e.g., administering to the subject a therapeutically effective amount of any of the activatable constructs described herein or any of the compositions (e.g., pharmaceutical compositions) described herein.
  • cancer include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, a lymphoma (e.g., B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, cutaneous T-cell lymphoma), a leukemia (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL)), myelodysplastic syndromes (MDS), Kaposi sarcoma,
  • CLL chronic lymphocytic leukemia
  • AML acute myeloid leukemia
  • the cancer is a lymphoma.
  • the lymphoma is Burkitt’s lymphoma.
  • the subject has been identified or diagnosed as having familial cancer syndromes such as Li Fraumeni Syndrome, Familial Breast-Ovarian Cancer (BRCA1 or BRAC2 mutations) Syndromes, and others.
  • familial cancer syndromes such as Li Fraumeni Syndrome, Familial Breast-Ovarian Cancer (BRCA1 or BRAC2 mutations) Syndromes, and others.
  • BRCA1 or BRAC2 mutations Familial Breast-Ovarian Cancer
  • the disclosed methods are also useful in treating non-solid cancers.
  • Exemplary solid tumors include malignancies (e.g., sarcomas, adenocarcinomas, and carcinomas) of the various organ systems, such as those of lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary (e.g., renal, urothelial, or testicular tumors) tracts, pharynx, prostate, and ovary.
  • malignancies e.g., sarcomas, adenocarcinomas, and carcinomas
  • gastrointestinal e.g., colon
  • genitourinary e.g., renal, urothelial, or testicular tumors
  • Exemplary adenocarcinomas include colorectal cancers, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, and cancer of the small intestine.
  • Exemplary cancers described by the National Cancer Institute include: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymoma,
  • Lymphoma Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's, Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant
  • Oral Cancer Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood; Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer;
  • Thyroid Cancer Thyroid Cancer
  • Thyroid Cancer Childhood
  • Transitional Cell Cancer of the Renal Pelvis and Ureter Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood;
  • Urethral Cancer Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.
  • exemplary cancers include diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • Metastases of the aforementioned cancers can also be treated or prevented in accordance with the methods described herein.
  • these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the cancer in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the cancer in the subject prior to treatment).
  • the methods further include administering to a subject an additional therapeutic agent (e.g., one or more of the therapeutic agents listed in Table 3).
  • an additional therapeutic agent e.g., one or more of the therapeutic agents listed in Table 3.
  • compositions including any of the activatable constructs, or polynucleotides (e.g., mRNA) or vectors encoding the polypeptides thereof, described herein and one or more (e.g., 1, 2, 3, 4, or 5) pharmaceutically acceptable carriers (e.g., any of the pharmaceutically acceptable carriers described herein), diluents, or excipients.
  • pharmaceutically acceptable carriers e.g., any of the pharmaceutically acceptable carriers described herein
  • the present disclosure provides a composition comprising the activatable construct, or polynucleotides (e.g., mRNA) or vectors encoding the polypeptides thereof, according to any of the aspects disclosed herein.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the compositions e.g. pharmaceutical compositions
  • that include any of the activatable constructs, or polynucleotides (e g., mRNA) or vectors encoding the polypeptides thereof, described herein are formulated for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intratumoral).
  • Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the type of intended administration.
  • a variety of pharmaceutically acceptable excipients are known in the art (see, e.g., A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy,” 2nd edition,. Rowe, et al. Handbook of pharmaceutical excipients. Pharmaceutical press, 2005.
  • the compositions are formulated in a nanoparticle. In some embodiments, the compositions are formulated in a lipid nanoparticle. In some aspects, the compositions are formulated in a lipid-polycation complex, referred to as a cationic lipid nanoparticle. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and/or polyarginine. In some embodiments, the compositions are formulated in a lipid nanoparticle that includes a noncationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.
  • lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) 7Vtz . Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51 :8529- 8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).
  • compositions of nucleotides of the disclosure can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
  • pharmaceutical compositions of RNA (e.g., mRNA) compositions include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations.
  • Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unicellular vesicle
  • LUV large unilamellar vesicle
  • Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes may contain a low or a high pH in order to improve the delivery of the formulations.
  • any of the pharmaceutical compositions described herein can include one or more buffers (e.g., a neutral -buffered saline, a phosphate-buffered saline (PBS), amino acids (e.g., glycine), one or more carbohydrates (e.g., glucose, mannose, sucrose, dextran, or mannitol), one or more antioxidants, one or more chelating agents (e.g., EDTA or glutathione), one or more preservatives, and/or a pharmaceutically acceptable carrier (e.g., bacteriostatic water, PBS, or saline) or excipient.
  • buffers e.g., a neutral -buffered saline, a phosphate-buffered saline (PBS)
  • amino acids e.g., glycine
  • carbohydrates e.g., glucose, mannose, sucrose, dextran, or mannitol
  • antioxidants e.g
  • the phrase “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial agents, antimicrobial agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Suitable carriers include, but are not limited to: water, saline, ringer’s solutions, dextrose solution, and about 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • 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. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition may be formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • any of the activatable molecules described herein are prepared with carriers that protect against rapid elimination from the body, e.g., sustained and controlled release formulations, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic-co- glycolic acid, and polylactic acid. Methods for preparation of such pharmaceutical compositions and formulations are apparent to those skilled in the art.
  • the activatable molecules may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the CM- containing polypeptides, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L- glutamic acid and y ethyl-L-glutamate, non-degradable ethyl ene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • 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 dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (CAS No. 61791-12-6) (BASF, Parsippany, N.J.), which is a mixture of polyoxyethylated triglycerides, by reacting castor oil with ethylene oxide in a molar ratio of 1 : 35, that acts as a nonionic surfactant, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition may be sterile and should be fluid and of a viscosity that facilitates easy syringeability. It may be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating on the particles such as lecithin, and by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the pharmaceutical compositions may further comprise one or more antibacterial and/or antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • antibacterial and/or antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and the like, as well as salts, such as, for example, sodium chloride and the like may be included in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
  • the pharmaceutical composition may comprise a sterile injectable solution.
  • Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by fdtered sterilization.
  • dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • any of the activatable constructs described herein are prepared with carriers that protect against rapid elimination from the body, e.g., sustained and controlled release formulations, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collage, polyorthoesters, and polylactic acid.
  • kits that include one or more second therapeutic agent(s) selected from Table 3 in addition to an the activatable constructs described herein.
  • the second therapeutic agent(s) may be provided in a dosage administration form that is separate from the activatable constructs. Alternatively, the second therapeutic agent(s) may be formulated together with the activatable constructs.
  • kits described herein can include instructions for using any of the compositions (e.g., pharmaceutical compositions) and/or any of the activatable constructs described herein. In some aspects, the kits can include instructions for performing any of the methods described herein. In some aspects, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some aspects, the kits can provide a syringe for administering any of the pharmaceutical compositions described herein.
  • the present disclosure includes and finds support for and in any one or any combination of the following items:
  • FIG. 1C A schematic of a masked activatable antibody (an activatable construct) of the present disclosure is shown in FIG. 1C.
  • Expression plasmids were constructed to express a masked light chain and a masked heavy chain.
  • the expression plasmid for a masked light chain encoded a mature polypeptide consisting of a first masking moiety attached to the light chain of the antibody with a first cleavable moiety.
  • the expression plasmid for a masked heavy chain encoded a mature polypeptide consisting of a second masking moiety attached to the heavy chain of the antibody with a second cleavable moiety.
  • ProC3551 was prepared by transforming Expi293 host cells, per the manufacturer’s recommendations (Gibco ExpiFectamine 293 Transfection Kit), with two plasmids: one expressing the polynucleotide sequence of SEQ ID NO: 684 and one expressing the sequence of SEQ ID NO: 695, followed by cultivation of the resulting recombinant host cells.
  • the masked activatable antibodies were purified from the culture supernatant by protein A- affinity chromatography followed by size-exclusion chromatography (SEC), and were confirmed to have >95% monomer content by analytical SEC.
  • Example 2 ITGA3B1 Binding ELISA A solid-phase binding assay was used to demonstrate the binding of anti-human ITGA3B1 antibodies (shown in FIGs. 2A-2D). Briefly, recombinant human ITGA3B1 protein (Aero Biosystems) diluted in IX PBS, pH 7.2 was coated on ELISA plates (100 pl at 1 pg/ml) overnight at 4C. The plates were washed six times with IX PBS-T (IX PBS + 0.05% Tween-20) and blocked with IX blocking buffer (IX PBS-T + 0.5% BSA) for Ih at room temperature (RT).
  • IX PBS-T IX PBS + 0.05% Tween-20
  • IX blocking buffer IX PBS-T + 0.5% BSA
  • the blocked plates were washed and then incubated with three-fold serial dilutions of anti- ITGA3B 1 antibody (starting at 100 nM) or activatable anti-ITGA3B l antibodies (starting at 1 pM) in blocking buffer.
  • the activatable antibodies were assayed in their uncleaved form using three different masks. After washing as described above, the amount of bound antibody or bound activatable antibody was detected by incubating with 1 :25000 dilution of anti-human IgG (anti-Fc) conjugated with horseradish peroxidase (Jackson ImmunoResearch) in blocking buffer for 30min at RT.
  • the plates were washed and incubated with lOOpl of Ultra TMB-ELISA reagent (Thermo Fisher Scientific) for lOmin at RT and the reaction was stopped with IN sulfuric acid.
  • the absorbance at 450nm was measured and reported as an optical density value (A450).
  • the blank value was subtracted from the raw A450 values to analyze the binding data in GraphPad Prism (vlO.1.2).
  • the EC50 values were obtained for each antibody and activatable antibody and the masking efficiency (ME) for each test article relative to the unmasked antibody was calculated as the ratio of the EC50 of the activatable antibody to that of the original antibody.
  • the EC50s of the masked activatable antibodies having masks on both heavy and light chains were greater than that of the light chain-masked or heavy chain-masked molecules, indicating higher masking than that of either the heavy chain-masked or light chain-masked antibody molecules. This was true for all four peptide masks tested on the anti-ITGA3Bl antibody.
  • ProC3542 maskless: SEQ ID NOs: 681 and 692
  • ProC3543 MMs conjugated to light chains only: SEQ ID NOs: 681 and 694
  • ProC3547 MMs conjugated to heavy chains only: SEQ ID NOs: 683 and 692
  • ProC3551 MMs conjugated to both heavy and light chains: SEQ ID NOs: 683 and 694.
  • ProC3551 MMs conjugated to both the heavy and light chains had 83-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 6-7-fold masking efficiency.
  • ProC3542 As shown in FIG. 2B, the following constructs were tested having various configurations of the same mask (Mask 2331): ProC3542 (describe above); ProC3544 (MMs conjugated to light chains only: SEQ ID NOs: 681 and 696); ProC3548 (MMs conjugated to heavy chains only: SEQ ID NOs: 685 and 692); and ProC3552 (MMs conjugated to both heavy and light chains: SEQ ID NOs: 685 and 696).
  • ProC3552 with MMs conjugated to both the heavy and light chains had 34-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains (i.e., one mask per antigen binding domain) with 3-10-fold masking efficiency.
  • ProC3542 As shown in FIG. 2C, the following constructs were tested having various configurations of the same mask (Mask 2333): ProC3542 (described above); ProC3545 (MMs conjugated to light chains only: SEQ ID NOs: 681 and 698); ProC3549 (MMs conjugated to heavy chains only: SEQ ID NOs: 687 and 682); and ProC3553 (MMs conjugated to both heavy and light chains: SEQ ID NO: 687 and 698). As shown in Table 6, ProC3553 with MMs conjugated to both the heavy and light chains had 59-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 3-9-fold masking efficiency.
  • ProC3542 (described above); ProC3546 (MMs conjugated to light chains only: SEQ ID NOs: 681 and 700); ProC3550 (MMs conjugated to heavy chains only: SEQ ID NOs: 689 and 692); and ProC3554 (MMs conjugated to both heavy and light chains: SEQ ID NOs: 689 and 700).
  • ProC3554 MMs conjugated to both heavy and light chains: SEQ ID NOs: 689 and 700.
  • ProC3554 with MMs conjugated to both the heavy and light chains had 55-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 5-6-fold masking efficiency.
  • Table 4 ITGA3B1 Binding Activity of Activatable Anti-ITGA3B1 Antibodies with Mask 2325 (FIG. 2A).
  • Table 5 ITGA3B1 Binding Activity of Activatable Anti-ITGA3B1 Antibodies with Mask 2331 (FIG. 2B).
  • Table 6 ITGA3B1 Binding Activity of Activatable Anti-ITGA3B1 Antibodies with Mask 2333 (FIG. 2C).
  • Table 7 ITGA3B1 Binding Activity of Activatable Anti-ITGA3B1 Antibodies with Mask 2337 (FIG. 2D).
  • a solid-phase binding assay was used to assess the binding of anti-human EGFR antibodies (shown in FIGs. 3A-3D).
  • the ELISA method used was similar to the one described above but with Fc-tagged recombinant human EGFR extracellular domain (ECD) protein conjugated to an IgGl Fc domain (ProC788, EGFR-ECD-IgGl) as the coating antigen and antihuman IgG (anti-Fab) conjugated with horseradish peroxidase (Sigma Aldrich) as the detection antibody.
  • ECD extracellular domain
  • IgGl Fc domain ProC788, EGFR-ECD-IgGl
  • anti-Fab antihuman IgG conjugated with horseradish peroxidase
  • the EC50s of the masked activatable antibodies having masks on both heavy and light chains were greater than that of the light chain-masked or heavy chain-masked molecules, indicating higher masking than either the light chain-masked or heavy chain-masked molecules. This was observed with two peptide masks tested when the same mask was present on both the heavy and the light chains or when different masks were present on the heavy and the light chains.
  • ProC052 maskless: SEQ ID NOs: 703 and 706
  • ProC3539 MMs conjugated to light chains only: SEQ ID NOs: 703 and 710
  • ProC3540 MMs conjugated to heavy chains only: SEQ ID NOs: 708 and 706
  • ProC3541 MMs conjugated to both heavy and light chains: SEQ ID NOs: 708 and 710).
  • ProC3541 MMs conjugated to both the heavy and light chains had 260-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 6-8-fold masking efficiency.
  • ProC052 (described above); ProC4138 (MMs conjugated to light chains only: SEQ ID NOs: 703 and 523); ProC4139 (MMs conjugated to heavy chains only: SEQ ID NOs: 712 and 706); and ProC4140 (MMs conjugated to both heavy and light chains: SEQ ID NOs: 712 and 523).
  • ProC4140 with MMs conjugated to both the heavy and light chains had 619-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 10-18-fold masking efficiency.
  • ProC052 (described above); ProC3540 (3954 mask on heavy chains only: SEQ ID NOs: 708 and 706); ProC4138 (CF41 mask on light chains only: SEQ ID NOs: 703 and 714); and ProC4141 (Mask 3954 on heavy chains and mask CF41 on light chains: SEQ ID NOs: 708 and 714).
  • ProC4141 with Mask 3954 on heavy chains and mask CF41 on light chains had 575-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains, respectively, with 6-18-fold masking efficiency.
  • ProC052 (described above); ProC3539 (Mask 3954 on the light chains only: SEQ ID NOs: 703 and 710); ProC4139 (Mask CF41 on the heavy chains only: SEQ ID NOs: 712 and 706); and ProC4142 (Mask CF41 on the heavy chains and mask 3954 on the light chains: SEQ ID NOs: 712 and 710).
  • ProC4142 with Mask CF41 on the heavy chains and mask 3954 on the light chains had 399- fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains, respectively, with 8-10-fold masking efficiency.
  • Table 9 EGFR Binding Activity of Activatable Anti-EGFR Antibodies with Mask 3954 (FIG. 3A).
  • Table 10 EGFR Binding Activity of Activatable Anti-EGFR Antibodies with Mask CF41 (FIG. 3B).
  • Table 11 EGFR Binding Activity of Activatable Anti-EGFR Antibodies with Mask 3954 on the heavy chain and Mask CF41 on the light chain (FIG. 3C).
  • Table 12 EGFR Binding Activity of Activatable Anti-EGFR Antibodies with Mask 3954 on the light chain and Mask CF41 on the heavy chain (FIG. 3D). Tested constructs are summarized as follows: Table 13. CM sequences
  • An activatable construct comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB 1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), wherein the HVD1 is covalently coupled directly or indirectly to a first masking moiety (MM1) via a first cleavable moiety (CM1), wherein the LVD1 is covalently coupled directly or indirectly to a second masking moiety (MM2) via a second cleavable moiety (CM2); a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2), a first dimerization domain (DD1) and a second dimerization domain (DD2), and wherein the MM1 and the MM2 inhibit binding of the AB1 to the first antigen.
  • a first antigen binding domain AB
  • activatable construct according to any one or combination of items, wherein the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide.
  • the first polypeptide comprises the MM2, the CM2, and the LVD1
  • the second polypeptide comprises the MM1 , the CM1, the HVD1, and the DD1.
  • activatable construct of any one or combination of items wherein the activatable construct further comprises a first heavy chain constant domain (HC1), wherein the HVD1 is coupled directly or indirectly to the C-terminus of the MM1 via the CM1 and directly or indirectly to the N-terminus of the DD1 via the HC1, and wherein the activatable construct further comprises a first light chain constant domain (LC1), wherein the LVD1 is coupled directly or indirectly to the C-terminus of the MM2 via the CM2, wherein the LVD1 is coupled directly or indirectly to the N-terminus of the LC1, and wherein the HC1 is coupled, directly or indirectly, to the LC 1.
  • HC1 first heavy chain constant domain
  • LC1 first light chain constant domain
  • activatable construct of any one of items 6-12 wherein the activatable construct comprises a first, a second, and a third polypeptide comprising, respectively, in an N- terminal to C-terminal direction: a) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 ; and iii) HVD2-LVD2-DD2; b) i) MM2-CM2-LVD 1 -LC 1 ; ii)MMl-CMl-HVDl-HCl-DDl; and iii) LVD2-HVD2-DD2; c) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 ; and iii) MM3-CM3-HVD2-LVD2-DD2; d) i) MM2-CM2-LV
  • the activatable construct of item 3 further comprising a fourth polypeptide, wherein the third polypeptide comprises the HVD2 and the DD2, and wherein the fourth polypeptide comprises the LVD2.
  • the fourth polypeptide comprises a third masking moiety (MM3) coupled directly or indirectly to the LVD2 via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
  • the fourth polypeptide comprises a fourth masking moiety (MM4) coupled directly or indirectly to the LVD2 via a fourth cleavable moiety (CM4), and wherein the MM4 inhibits binding of the AB2 to the second antigen.
  • MM4 masking moiety
  • activatable construct of any one of items 15-17 wherein the activatable construct further comprises a second heavy chain constant domain (HC2) and/or wherein the activatable construct further comprises a first heavy chain constant domain (HC1).
  • HC2 second heavy chain constant domain
  • HC1 first heavy chain constant domain
  • activatable construct of any one of items 15-18 wherein the activatable construct further comprises a second light chain constant domain (LC2) and/or wherein the activatable construct further comprises a first light chain constant domain (LC1).
  • LC2 second light chain constant domain
  • LC1 first light chain constant domain
  • first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) HVD2-HC2-DD2; and iv) LVD2-LC2; b) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2; and iv) LVD2-LC2; c) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) HVD2-HC2-DD2; and
  • activatable construct of any one or combination of items, wherein the activatable construct specifically binds to a single epitope.
  • AB3 antigen binding domain having specific binding affinity for a third antigen
  • the AB3 comprises a heavy chain variable domain 3 (HVD3) and a light chain variable domain 3 (LVD3)
  • HVD3 heavy chain variable domain 3
  • LLD3 light chain variable domain 3
  • MM5 fifth masking moiety
  • CM5 fifth cleavable moiety
  • the AB3 comprises a scFv, wherein the AB3 comprises: i) MM5-CM5-HVD3-LVD3-X; or ii) MM5-CM5-LVD3-HVD3-X; or wherein the AB3 comprises in an N-terminal to C-terminal direction: iii) X-LVD3-HVD3-CM5-MM5; or iv) X-HVD3-LVD3-CM5-MM5; wherein X represents attachment to the N-terminus or the C-terminus of the DD1 or the DD2, and wherein each is a direct or an indirect covalent linkage.
  • AB4 antigen binding domain having specific binding affinity for a fourth antigen
  • the AB4 comprises a heavy chain variable domain 4 (HVD4) and a light chain variable domain 4 (LVD4)
  • HVD4 heavy chain variable domain 4
  • LLD4 light chain variable domain 4
  • MM6 sixth masking moiety
  • CM6 sixth cleavable moiety
  • the AB4 comprises a scFv, wherein the AB4 comprises: i) MM6-CM6-HVD4-LVD4-Y; or ii) MM6-CM6-LVD4-HVD4- Y; or wherein the AB4 comprises in an N-terminal to C-terminal direction: iii) Y-LVD4-HVD4-CM6-MM6; or iv) Y-HVD4-LVD4-CM6-MM6; wherein Y represents attachment to the N-terminus or the C-terminus of the DD2, and wherein each is a direct or an indirect covalent linkage.
  • each of the CM1 and the CM2 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one or combination of items 5-52, wherein each of the CM1, the CM2, the and CM3 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one or combination of items 17-52, wherein each of the CM1, the CM2, the CM3, and the CM4 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one or combination of items 48-52, wherein each of the CM1, CM2, CM3, CM4, and CM5 independently comprises a substrate for the same or different protease in any combination thereof; or the activatable construct of any one or combination of items 51-52, wherein each of the CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a
  • each of the CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a total of about 3 amino acids to about 15 amino acids.
  • each CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a substrate for a protease selected from the group consisting of ADAMS, AD AMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM 15, ADAMI 7/T ACE, ADAMDEC1, AD AMTS 1, ADAMTS4, ADAMTS5, Aspartate proteases, BACE, Renin, Aspartic cathepsins, Cathepsin D, Cathepsin E, Caspases, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cysteine cathepsins, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathe
  • CM1, CM2, CM3, CM4, CM5, and CM6 each independently comprises a sequence selected from the group consisting of sequences listed in Table 13.
  • CM1, CM2, CM3, CM4, CM5, and CM6 each independently comprises a sequence selected from the group consisting of: LSGRSDNH (SEQ ID NO: 8), NTLSGRSENHSG (SEQ ID NO: 9), NTLSGRSGNHGS (SEQ ID NO: 10), TSTSGRSANPRG (SEQ ID NO: 11), TSGRSANP (SEQ ID NO: 12), VHMPLGFLGP (SEQ ID NO: 13), AVGLLAPP (SEQ ID NO: 14), AQNLLGMV (SEQ ID NO: 15), QNQALRMA (SEQ ID NO: 16), LAAPLGLL (SEQ ID NO: 17), STFPFGMF (SEQ ID NO: 18), ISSGLLSS (SEQ ID NO: 19), PAGLWLDP (SEQ ID NO: 20), VAGRSMRP (SEQ ID NO: 21), VVPEGRRS (SEQ ID NO: 22), ILPRSPAF (SEQ ID NO: 23),
  • CM1, CM2, CM3, CM4, CM5, and CM6 each independently comprises a sequence selected from the group consisting of: ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), ISSGLLSGRSDQH (SEQ ID NO: 54), SGRSDNI (SEQ ID NO: 100), ISSGLLSGRSDNI (SEQ ID NO: 68), ISSGLLSGRSDNP (SEQ ID NO: 57), and PWGLSGRS (SEQ ID NO: 157).
  • each of the MM1 and the MM2 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one or combination of items 5-59, wherein each of the MM1, the MM2, the and MM3 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one or combination of items 17-59, wherein each of the MM1, the MM2, the MM3, and the MM4 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one or combination of items 48-59, wherein each of the MM1, MM2, MM3, MM4, and MM5 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; or the activatable construct of any one or combination of items 51-59, wherein each of the MM1, MM2, MM3, MM4, MM5,
  • MM1, MM2, MM3, MM4, MM5, and MM6 each independently comprise a sequence selected from the group consisting of: XCXXRXXFEXXDCVX, where X equals any amino acid (SEQ ID NO: 716); TCPTRWHFETTDCVM (SEQ ID NO: 717); ICGSRLDFELNDCVM (SEQ ID NO: 718);
  • WCRDRSHFETGDCVM (SEQ ID NO: 719); ICTSRWEFENRDCVM (SEQ ID NO: 720);
  • VCRDRNEFEVGDCVM SEQ ID NO: 721
  • TCKNRLEFERGDCVM SEQ ID NO: 722
  • VCSSRLEFEQKDCVM SEQ ID NO: 723
  • WCRDREHFEKGDCVM SEQ ID NO: 724
  • YCANRYEFEYGDCVM (SEQ ID NO: 725); VCRTRWHFETTDCVM (SEQ ID NO:
  • VCSNRAEFEWGDCVM SEQ ID NO: 726
  • VCASRWHFENTDCVM SEQ ID NO: 727
  • NCASRWHFENEDCVM SEQ ID NO: 728
  • VCSGRLEFELGDCVM SEQ ID NO: 729
  • VCSSRWEFETNDCVM SEQ ID NO: 731
  • FCRDRLDFDTMDCVM SEQ ID NO: 731
  • VCRTRWHFETTDCVM SEQ ID NO: 739
  • ICHDPYMNIDYTCKL SEQ ID NO: 738
  • VMCYWEGWGFGRCPL SEQ ID NO: 741
  • VWYCDGGYNECATRS SEQ ID NO: 741
  • CISPRGCPDGPYVMY (SEQ ID NO: 749); CISPRGCPDGPYVM (SEQ ID NO: 750);
  • CISPRGCEPGTYVPT SEQ ID NO: 751
  • CISPRGCPGQIWHPP SEQ ID NO: 752
  • GSHCLIPINMGAPSC (SEQ ID NO: 753); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 754); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 755);
  • ADHVFWGSYGCISPRGCPG (SEQ ID NO: 756); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 757); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 758); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 759); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 760);
  • CPYYTLSYCGCISPRGCPG (SEQ ID NO: 761); CNHVYFGTCGCISPRGCPG (SEQ ID NO: 762); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 763); CHHFTLTTCGCISPRGCPG (SEQ ID NO: 764); YNPCATPMCCISPRGCPG (SEQ ID NO: 765);
  • CNHHYFYTCGCISPRGCG (SEQ ID NO: 766); CNHHYHYYCGCISPRGCG (SEQ ID NO: 767); CNHVYFGTCGCISPRGCG (SEQ ID NO: 768); CHHVYWGHCGCISPRGCG (SEQ ID NO: 769); CPHFTTTSCGCISPRGCG (SEQ ID NO: 770);
  • CNHFTLTTCGCISPRGCG (SEQ ID NO: 771); CHHFTLTTCGCISPRGCG (SEQ ID NO: 772); CPYYTLSYCGCISPRGCG (SEQ ID NO: 773); CPHVSFGSCGCISPRGCG (SEQ ID NO: 774); ADHVFWGSYGCISPRGCG (SEQ ID NO: 775); YNPCATPMCCISPRGCG (SEQ ID NO: 776); CHHVYWGHCGCISPRGCG (SEQ ID NO: 777);
  • MMYCGGNEVLCGPRV (SEQ ID NO: 792); GYRWGCEWNCGGITT (SEQ ID NO: 793); GYLWGCEWNCGGITT (SEQ ID NO: 794); MMYCGGNEIFCEPRG (SEQ ID NO: 795); GYGWGCEWNCGGSSP (SEQ ID NO: 796); and MMYCGGNEIFCGPRG (SEQ ID NO: 797).
  • MM1, MM2, MM3, MM4, MM5, and MM6 each independently comprise a sequence selected from the group consisting of: VCRDRNEFEVGDCVM (SEQ ID NO: 721); TCLSRYEFETTDCVM (SEQ ID NO: 738); VCSNRAEFEWGDCVM (SEQ ID NO: 727); VCSGRLEFELGDCVM (SEQ ID NO: 730); CISPRGCPDGPYVMY (SEQ ID NO: 749); and LSCEGWAMNREQCRA (SEQ ID NO: 783).
  • DD1 and DD2 are human IgGl Fc domains or a mutant or truncation variant thereof.
  • each of DD1 and DD2 each independently comprises a sequence that is at least 80% identical or at least 90% identical to a sequence selected from: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGX (SEQ ID NO: 655), wherein X is lysine or absent;
  • each of DD1 and DD2 independently comprises a sequence selected from:
  • each linker is independently selected from the group consisting of: GSSGGSGGSGG (SEQ ID NO:623); GGGS (SEQ ID NO: 622); GGGSGGGS (SEQ ID NO: 624); GGGSGGGSGGGS (SEQ ID NO: 625); GGGGSGGGGSGGGGS (SEQ ID NO: 626); GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 627); GGGGSGGGGS (SEQ ID NO: 628); GGGGS (SEQ ID NO: 629); GS; GGGGSGS (SEQ ID NO: 630); GGGGSGGGGSGGGGSGS (SEQ ID NO: 631); GGSLDPKGGGGS (SEQ ID NO: 632); PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 633); SKYGPPCPPCPAPEFLG (SEQ ID NO: 634); GKSSGSGSESKS (SEQ ID NO: 634); GKSSGSGSESKS (SEQ ID NO:
  • HVD2 comprises a sequence selected from: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSV KYNEGFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQ GTTVTVSS (SEQ ID NO: 680); or
  • LVD1, the LVD2, or both comprises a sequence selected from:
  • DIQMTQ SPS SLS AS VGDRVTITC S AS S SIS SNYLHWYQQKPGK VPKLLIYRTSNL ASGVP S RFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIK (SEQ ID NO: 691); or
  • LVD3 comprises a sequence selected from: DIQMTQ SPS SLS AS VGDRVTITC S AS S SIS SNYLHWYQQKPGK VPKLLIYRTSNL ASGVP S RFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIK (SEQ ID NO: 691); or QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 705).
  • the activatable construct of item 50 wherein the LVD3, the LVD4, or both comprises a sequence selected from: DIQMTQSPSSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPS RFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIK (SEQ ID NO: 691); or QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 705).
  • DIQMTQ SPS SLS AS VGDRVTITC S AS S SIS SNYLHWYQQKPGK VPKLLIYRTSNL ASGVP S RFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 692); or QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDE QLKSGTAS VVCLLNNF YPREAKVQWKVDNALQ SGNSQE S VTEQD SKD ST Y
  • QGQSGQVCRDRNEF EVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
  • QGQSGQVCRDRNEF EVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
  • QGQSGQVCRDRNEF EVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
  • the first polypeptide comprises an amino acid sequence of: QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694); and wherein the second polypeptide comprising an amino acid sequence of:
  • the fourth polypeptide comprises an amino acid sequence of: QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694); and wherein the third polypeptide comprising an amino acid sequence of:
  • activatable construct of any one or combination of items, wherein the activatable construct is a multi-specific activatable antibody.
  • a composition comprising the activatable construct of any one or combination of items 1-99.
  • a container, vial, syringe, injector pen, or kit comprising at least one dose of the composition of any one or combination of items 100-101.
  • a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of the activatable construct of any one or combination of items 1-98 or the composition of any one or combination of items 100- 101.
  • the second therapeutic agent is a checkpoint inhibitor, a cytokine, or a cytotoxic agent.
  • a vector comprising the nucleic acid of item 107.
  • the cell of item 110 wherein the cell is a mammalian cell.
  • a method of producing the activatable construct of any one or combination of items 1-99 comprising culturing the cell of any one or combination of items 110-112 in a liquid culture medium to produce the activatable construct; and recovering the activatable construct from the liquid culture medium.
  • the method of item 113 further comprising: purifying the activatable construct recovered from the liquid culture medium to obtain a purified activatable construct.
  • the method of item 114 further comprising: combining the purified activatable construct with a carrier to form a pharmaceutical composition.
  • An activatable construct produced by the method of any one or combination of items 114-115.
  • An activatable construct comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB 1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), a first light chain constant domain (LC1), a first heavy chain constant domain (HC1); a first masking moiety (MM1) coupled directly or indirectly to a first cleavable moiety (CM1), wherein the MM1 is coupled directly or indirectly to the HVD1, the LVD1, the HC1, or the LC1 via the CM1; a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2); a second masking moiety (MM2) coupled directly or indirectly to a second cleavable moiety (CM2), wherein the MM2 is coupled directly or indirectly to the HVD2 or the
  • activatable construct of item 117 wherein the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide.
  • CM3 third cleavable moiety
  • MM3 third masking moiety
  • activatable construct of items 117-119 wherein the activatable construct further comprises a fourth cleavable moiety (CM4) and a fourth masking moiety (MM3).
  • CM4 cleavable moiety
  • MM3 fourth masking moiety
  • the activatable construct of items 117-120 wherein the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, the second polypeptide comprises the LVD1, LC1, CM3, and the MM3, and the third polypeptide comprises the DD2.
  • the activatable construct of items 117-120 wherein the first polypeptide comprises the MM1, CM1, LVD1, and the LC1, the second polypeptide comprises the MM4, CM4, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, and the third polypeptide comprises the MM3, CM3, and the DD2.
  • the activatable construct of items 117-120 wherein the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, the second polypeptide comprises the MM4, CM4, LVD1, LC1, CM3, and the MM3, and the third polypeptide comprises the DD2.
  • the MM4 inhibits binding of the AB1 to the first antigen.
  • the activatable construct of items 117-120 wherein the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, the second polypeptide comprises the MM3, CM3, LVD1, and LC1, and the third polypeptide comprises the DD2.
  • 145. The activatable construct of any one of items 141-145, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; ii) MM3-CM3-LVD1-LC1; and iii) DD2; or b) i) MM1-CM1-HVD1-HC1-LVD2-HVD2 -CM2 -MM2 -DD 1 ; ii) MM3-CM3-LVD1-LC1; and iii) DD2; wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are
  • An mRNA comprising the nucleic acid of item 107.
  • composition comprising the nucleic acid of item 107, the mRNA of any one of items 146-148, or the vector of any one of items 108-109.
  • composition of item 149 further comprising a pharmaceutically acceptable carrier or excipient.
  • the first polypeptide comprises an amino acid sequence of:
  • the first polypeptide comprises an amino acid sequence of:
  • a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of the nucleic acid of item 107, the mRNA of any one of items 146-148, or the vector of any one of items 108-109.
  • the second therapeutic agent is a checkpoint inhibitor, a cytokine, or a cytotoxic agent.

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Abstract

Activatable constructs and methods of making and treating using the same, comprising a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), wherein the HVD1 is covalently coupled directly or indirectly to a first masking moiety (MM1) via a first cleavable moiety (CM1), wherein the LVD1 is covalently coupled directly or indirectly to a second masking moiety (MM2) via a second cleavable moiety (CM2), a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2), a first dimerization domain (DD1) and a second dimerization domain (DD2).

Description

ACTIVATABLE CONSTRUCTS, COMPOSITIONS AND METHODS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the priority benefit of U.S. provisional application no. 63/647,169 filed May 14, 2024, the contents of which are incorporated herein by reference in their entireties.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 14, 2025, is named “4862-148WOl-SeqListing.xml” and is 748,980 bytes in size.
TECHNICAL FIELD
The present disclosure relates to the field of biotechnology, and more specifically, to activatable constructs, and to methods of making and using the polypeptides and activatable constructs in a variety of therapeutic, diagnostic, and prophylactic applications.
BACKGROUND
Antibody-based therapies have emerged as treatments for a variety of disorders, but in some cases, toxicities due to broad target expression have limited their therapeutic effectiveness. In addition, antibody -based therapeutics have exhibited other limitations such as rapid clearance from the circulation following administration.
Accordingly, there is a continuing need for antibody-based molecules that enable targeting of multiple targets and/or multiple epitopes with a single molecule and have fewer of the undesired adverse effects of existing antibody-based therapies.
SUMMARY OF THE INVENTION
In one aspect, the present disclosure provides an activatable construct including a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB1 includes a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), wherein the HVD1 is covalently coupled directly or indirectly to a first masking moiety (MM1) via a first cleavable moiety (CM1) , wherein the LVD1 is covalently coupled directly or indirectly to a second masking moiety (MM2) via a second cleavable moiety (CM2), a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 includes a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2), and a first dimerization domain (DD1) and a second dimerization domain (DD2). In some aspects, the AB1 is coupled directly or indirectly to the DD1. In some aspects, the AB2 is coupled directly or indirectly to the DD2. In some aspects, the MM1 and the MM2 inhibit binding of the AB1 to the first antigen.
In some aspects, the AB2 is not coupled to a MM. In some aspects, the AB2 is coupled directly or indirectly to a third masking moiety (MM3) via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
In some aspects, the AB2 is further coupled directly or indirectly to a fourth masking moiety (MM4) via a fourth cleavable moiety (CM4), wherein the MM3 and MM4 inhibit binding of the AB2 to the second antigen.
In one aspect, the present disclosure provides a composition containing the activatable construct according to any of the aspects disclosed herein. In one aspect, the present disclosure provides a container, vial, syringe, injector pen, or kit comprising at least one dose of a composition comprising the activatable construct according to any of the aspects disclosed herein.
In one aspect, the present disclosure provides a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising the activatable construct according to any of the aspects disclosed herein. In some aspects, the subject has been identified or diagnosed as having a cancer, an autoimmune disease, or an inflammatory disorder.
In one aspect, the present disclosure provides a nucleic acid encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein. In some aspects, the nucleic acid encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein comprises a vector. In some aspects, the nucleic acid encodes a polypeptide according to any one of the sequences in Table 16.
In one aspect, the present disclosure provides a cell transfected with the nucleic acid or vector encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein. In some aspects, the cell is a mammalian cell.
In one aspect, the present disclosure provides a method of producing the activatable construct according to any of the aspects disclosed herein. In some aspects, the method comprises culturing a cell comprising a nucleic acid or vector disclosed herein in a liquid culture medium to produce the activatable construct according to any of the aspects disclosed herein and recovering the activatable construct from the liquid culture medium. In some aspects, the method comprises purifying the activatable construct recovered from the liquid culture medium to obtain a purified activatable construct. In some aspects, the method comprises combining the purified activatable construct with a carrier to form a pharmaceutical composition.
These and other aspects of the invention will be apparent upon reference to the following detailed description, claims, aspects, procedures, compounds, and/or compositions and associated background information and references, which are hereby incorporated in their entirety.
BRIEF DESCRIPTION OF DRAWINGS
FIGs. 1A-1D provide schematics of antibody constructs. FIG. 1A is a schematic of an activatable construct with the masking moiety attached to the light chain with a cleavable moiety. FIG. IB is a schematic of an activatable construct with the masking moiety attached to the heavy chain with a cleavable moiety. FIG. 1C is a schematic of an activatable construct with masking moi eties attached to each light chain with a cleavable moiety and masking moi eties attached to each heavy chain with a cleavable moiety (wherein the masking moieties and cleavable moieties may be the same or different). FIG. ID is a schematic of an activatable construct with a masking moiety attached to the light chain with a cleavable moiety and another masking moiety (the same or different from the first masking moiety) attached to the heavy chain with a cleavable moiety (the same or different from the first cleavable moiety), and another masking moiety attached to a scFv with a cleavable moiety.
FIGs. 2A-2D show results of an ELISA binding assay to determine the shift in ability of the molecules to bind human Integrin alpha 3 beta 1 (ITGA3B1) antigen coated on a plate. FIG. 2A provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 2325 to bind human ITGA3B1 antigen coated on a plate. FIG. 2B provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 2331 to bind human ITGA3B1 antigen coated on a plate. FIG. 2C provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 2333 to bind human ITGA3B1 antigen coated on a plate. FIG. 2D provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 2337 to bind human ITGA3B1 antigen coated on a plate.
FIGs. 3A-3D provide the results of an ELISA binding assay to determine the shift in ability of the molecules to bind epidermal growth factor receptor (EGFR) antigen coated on a plate. FIG. 3A provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 3954 to bind EGFR antigen coated on a plate. FIG. 3B provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask CF41 to bind EGFR antigen coated on a plate. FIG. 3C provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 3954 on the heavy chain and Mask CF41 on the light chain to bind EGFR antigen coated on a plate. FIG. 3D provides the results of an ELISA binding assay to determine the shift in ability of the molecules with Mask 3954 on the light chain and Mask CF41 on the heavy chain to bind EGFR antigen coated on a plate.
FIGs. 4A-4B are schematics of activatable constructs comprising a single masked Fab domain and a dual masked scFv domain, including an additional masking moiety attached to an Fc domain (FIG. 4A) or an additional masking moiety attached to a light chain constant region (FIG. 4B)
FIGs. 5A-5C are schematics of activatable constructs comprising a dual masked Fab domain and a dual masked scFv domain, including an additional masking moiety attached to an Fc domain (FIG. 5A) or an additional masking moiety attached to a light chain constant region (FIG. 5B). A single masked scFv is depicted in FIG. 5C.
FIGs. 6-9 show configurations of exemplary activatable molecules. The molecules are designed such that the activated molecules resulting from the activation of the activatable molecules do not comprise half-life extending moieties and thus have a shorter half-life than counterpart molecules that are the same as the activated molecules but comprising the half-life extending moieties.
DETAILED DESCRIPTION
While aspects of the subject matter of the present disclosure may be embodied in a variety of forms, the following description is merely intended to disclose some of these forms as specific examples of the subject matter encompassed by the present disclosure. Accordingly, the subject matter of this disclosure is not intended to be limited to the forms or aspects so described. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
The term “a” and “an” refers to one or more (i.e., at least one) of the grammatical object of the article. By way of example, “a cell” encompasses one or more cells.
As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art. For example, ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value where appropriate.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 0.01 to 2.0” should be interpreted to include not only the explicitly recited values of about 0.01 to about 2.0, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.5, 0.7, and 1.5, and sub-ranges such as from 0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described. Additionally, it is noted that all percentages are in weight, unless specified otherwise.
In understanding the scope of the present disclosure, the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of,” as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel character! stic(s) of features, elements, components, groups, integers, and/or steps. It is understood that reference to any one of these transition terms (i.e. “comprising,” “consisting,” or “consisting essentially”) provides direct support for replacement to any of the other transition term not specifically used. For example, amending a term from “comprising” to “consisting essentially of’ or “consisting of’ would find direct support due to this definition for any elements disclosed throughout this disclosure. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.
As used herein, an “activatable construct” may refer to a polypeptide, multiple polypeptides or a nucleic acid, e.g., polynucleotide, or mRNA encoding the polypeptide or multiple polypeptides.
As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.
Furthermore, certain molecules, constructs, compositions, elements, moieties, excipients, disorders, conditions, properties, steps, or the like may be discussed in the context of one specific embodiment or aspect or in a separate paragraph or section of this disclosure. It is understood that this is merely for convenience and brevity, and any such disclosure is equally applicable to and intended to be combined with any other embodiments or aspects found anywhere in the present disclosure and claims, which all form the application and claimed invention at the filing date. For example, a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, composition, or method is intended to and does find direct support for embodiments related to constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.
Unless otherwise specified, the phrase a “nucleic acid sequence encoding” includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.
Unless otherwise specified, a “polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, RNA, mRNA, or synthetic origin or some combination thereof, which by virtue of its origin the “polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, and/or (3) does not occur in nature as part of a larger sequence. In some embodiments, polynucleotides include the nucleic acid molecules encoding heavy chain immunoglobulin molecules, and nucleic acid molecules encoding light chain immunoglobulin molecules.
Unless otherwise specified, the terms “polypeptide” and “recombinant polypeptide” are used interchangeably herein to refer to a polymer of amino acids that exists in a form that is not found in nature. In some aspects, a “polypeptide” includes an expressed polymer of amino acids with or without post-translational modifications. In some aspects, the polypeptide comprises one or more glycosyl groups attached thereto. Polypeptides employed herein may be encoded by cDNA, recombinant RNA, recombinant DNA, messenger RNA, or a polynucleotide of synthetic origin or some combination thereof. By virtue of its origin, or source of derivation, the “polypeptide” (1) is not in a naturally occurring organism (e.g., is not an endogenous polypeptide of a naturally occurring organism) and (2) is present in a form not found in nature.
The terms “cleavable moiety” and “CM” are used interchangeably herein to refer to a peptide, the amino acid sequence of which encodes a substrate for a sequence-specific protease. Cleavable moieties that are suitable for use as a CM include any of the protease substrates that are known the art. Exemplary cleavable moieties are described in more detail below.
The terms “masking moiety” and “MM” are used interchangeably herein to refer to a polypeptide that reduces or inhibits one or more activities of an antigen binding domain. The phrase “N-terminally positioned” when referring to a position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence means that the first domain or sequence is located closer to the N-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some aspects, there may be additional sequences and/or domains between the first domain or sequence and the second domain or sequence.
The phrase “C-terminally positioned” when referring to a position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence means that the first domain or sequence is located closer to the C-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some aspects, there may be additional sequences and/or domains between the first domain or sequence and the second domain or sequence.
Unless otherwise specified, the phrase “N-terminal to C-terminal direction” refers to the N-terminus to the C-terminus of the polypeptide elements disclosed herein in the left to right direction.
The term “exogenous” refers to any material introduced from or originating from outside a cell, a tissue, or an organism that is not produced by or does not originate from the same cell, tissue, or organism in which it is being introduced.
The term “transduced,” “transfected,” or “transformed” refers to a process by which an exogenous nucleic acid is introduced or transferred into a cell. A “transduced,” “transfected,” or “transformed” cell (e g., mammalian cell) is one that has been transduced, transfected, or transformed with exogenous nucleic acid (e.g., a vector) that includes an exogenous nucleic acid encoding any of the activatable constructs described herein.
The term “nucleic acid” refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some aspects of any of the nucleic acids described herein, the nucleic acid is DNA. In some aspects of any of the nucleic acids described herein, the nucleic acid is RNA. As used herein the phrase “specifically binds,” or “immunoreacts with” means that the activatable construct reacts with one or more antigenic determinants of the desired target antigen and does not react with other polypeptides, or binds at much lower affinity or potency, e.g., a KD or ECso about or greater than 10’6 M.
The term “treatment” refers to ameliorating at least one symptom of a disorder. In some aspects, the disorder being treated is a cancer. In some aspects, the disorder being treated is an autoimmune disease. In some aspects, the disorder being treated is an inflammatory disorder.
As used herein, the term “linker” refers to a peptide, the amino acid sequence of which is not a substrate for a protease. Exemplary linkers are described in more detail below.
As used herein, the term “linking region” or “LR” refers to the stretch of amino acid residues between the C-terminus of the antigen binding domain and the amino acid residue that is N-terminally adjacent to the proximal point of interaction between the dimerization domains (i.e., the linking region does not include the C-terminal amino acid of the antigen binding domain or the N-terminal amino acid of the DD that forms the proximal point of interaction to the DD of the corresponding second monomer). For example, when the DDs are a pair of Fc domains, the linking region is the stretch of amino acid residues between the C-terminus of the antigen binding domain and the first N-terminal cysteine residue of the Fc that participates in the disulfide linkage with the second Fc domain (e.g., Cysteine 226 of an IgGl or IgG4 Fc domain, according to EU numbering). In some aspects, when the DDs are a pair of Fc domains, the linking region is the stretch of amino acid residues between the C-terminus of the heavy chain constant domain (e.g., a CHI domain) and the first N-terminal cysteine residue of the Fc that participates in the disulfide linkage with the second Fc domain When the dimerization domain is not a polypeptide, then the linking region is the stretch of amino acid residues following the C- terminus of the antigen binding domain until the last amino acid. For example, when the DDs are a biotin-streptavidin pair, the linking region of the biotin-containing monomer is the stretch of amino acid residues between the C-terminus of the antigen binding domain and the biotin molecule, and the linking region of the streptavidin-containing monomer is the stretch of amino acid residues between the C-terminus of the antigen binding domain and the streptavidin molecule.
As used herein, the term “mask linking region” or “MLR” refers to the stretch of amino acid residues between a MM and an antigen binding domain (AB). The MLR spans from the N- terminus of a AB to the C-terminus of a AB . Thus, the MLR may include a MM, a MM and a linker, or a MM and two linkers. Tn some aspects, the MLR spans 15 to 22 amino acids. In some aspects, the MLR spans 16 to 21 amino acids. In some aspects, the MLR spans 17 to 20 amino acids. In some aspects, the MLR spans 18 to 20 amino acids. In some aspects, the MLR spans 15, 16, 17, 18, 18, 20, 21, or 22 amino acids.
As used herein, the term “masking efficiency” refers to the activity (e.g., EC50) of the activatable construct (i.e., intact and uncleaved) divided by the activity of a control construct, wherein the control antibody or antigen binding domain may be either cleavage product of the activatable construct or the antibody or antigen binding domain used as the activatable construct, for example corresponding activatable antibody that lacks MMs.
As used herein, the term light chain variable domain (LVD) may be used interchangeably with variable light (VL) domain. As used herein, the term heavy chain variable domain (HVD) may be used interchangeably with variable heavy (VH) domain. As used herein, the term light chain constant region (LC) may also be referred to as CL and may be used interchangeably. As used herein, the term heavy chain constant region (HC) may also be referred to as CH and may be used interchangeably.
The present disclosure includes single chain polypeptides capable of forming dimer complexes as well as dimerized complexes of a first monomer construct and a second monomer construct. Dimerization of the monomeric components is facilitated by a dimerization domain capable of forming a dimer or a first dimerization domain and a second dimerization domain capable of forming a dimer. Thus, the present disclosure includes homodimers, e g., wherein each of the first and second monomer constructs are identical, as well as heterodimers, where the first and second monomer constructs different from each other.
As used herein, a polypeptide, such as an antibody (including, for example, a light chain, a heavy chain, an scFv, and the like), antibody fragment, or an Fc domain, may be a wild-type polypeptide (e.g., a naturally-existing polypeptide) or a variant of the wild-type polypeptide. A variant may be a polypeptide modified by substitution, insertion, deletion and/or addition of one or more amino acids of the wild-type polypeptide, provided that the variant retains the basic function or activity of the wild-type polypeptide. In some examples, a variant may have altered (e.g., increased or decreased) function or activity comparing with the wild-type polypeptide. Activatable Construct
The present disclosure provides activatable antigen binding domain constructs having a masking format that significantly inhibits binding of the antigen binding domain to its target, yet allows for substantial recovery of binding activity following activation. For example, activatable constructs according to any one of the aspects described herein have significantly greater masking efficiency than a corresponding activatable construct having only a single masking moiety associated with an antigen binding domain, i.e., only a single masking moiety per antigen binding domain. In certain aspects, activatable constructs according to the present disclosure have greater masking efficiency than a corresponding activatable construct having a masking moiety coupled directly or indirectly to either a heavy chain variable domain or a light chain variable domain, but not a masking moiety coupled directly or indirectly to both a heavy chain variable domain and a light chain variable domain of the same antigen binding domain.
More specifically, in one aspect, the present disclosure provides an activatable construct, comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), wherein the HVD1 is covalently coupled directly or indirectly to a first masking moiety (MM1) via a first cleavable moiety (CM1), wherein the LVD1 is covalently coupled directly or indirectly to a second masking moiety (MM2) via a second cleavable moiety (CM2); a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2), and a first dimerization domain (DD1) and a second dimerization domain (DD2), wherein the MM1 and the MM2 inhibit binding of the AB 1 to the first antigen.
In some aspects, the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide. In some aspects, the first polypeptide comprises the MM2, the CM2, and the LVD1. In some aspects, the LVD1 is coupled directly or indirectly to the C- terminus of the MM2 via the CM2. In some aspects, the second polypeptide comprises the MM1, the CM1, the HVD1, and the DD1 In some aspects, the HVD1 is coupled directly or indirectly to the MM1 via the CM1 and directly or indirectly to the DD1.
In some aspects, the third polypeptide comprises the AB2 and the DD2. In some aspects, the AB2 is not coupled to a MM. In some aspects, the AB2 is coupled directly or indirectly to a third masking moiety (MM3) via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the third polypeptide comprises from N-terminus to the C-terminus, the LVD2, the HVD2, and the DD2. In some aspects, the third polypeptide comprises from N-terminus to the C-terminus, the HVD2, the LVD2, and the DD2
In some aspects, the third polypeptide comprises the MM3, the CM3, the LVD2, the HVD2, and the DD2. In some aspects, the HVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the LVD2. In some other aspects, the LVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the HVD2.
In some aspects, the first polypeptide comprises the MM2, the CM2, and the LVD1. In some aspects, the second polypeptide comprises the MM1, the CM1, the HVD1, and the DD1. In some aspects, the second polypeptide further comprises a third masking moiety (MM3), a third cleavable moiety (CM3), and the AB2, and the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the second polypeptide comprises in an N-terminal to C-terminal direction the MM1, the CM1, the HVD1, the HVD2, the LVD2, the CM3, the MM3, and the DD1. In some aspects, the second polypeptide comprises in an N-terminal to C-terminal direction the MM1, the CM1, the HVD1, the LVD2, the HVD2, the CM3, the MM3, and the DD1. In some aspects, the third polypeptide comprises the DD2. In some aspects, the third polypeptide does not comprise a variable domain.
In some aspects, the activatable construct further comprises a third polypeptide. In some aspects, the third polypeptide comprises the HVD2 and the DD2. In some aspects, the activatable construct further comprises a fourth polypeptide. In some aspects, the fourth polypeptide comprises the LVD2. In some aspects, the third polypeptide comprises the MM3 coupled directly or indirectly to the HVD2 via the CM3 and the fourth polypeptide does not comprise a MM, wherein the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the fourth polypeptide comprises the MM3 coupled directly or indirectly to the LVD2 via the CM3 and the third polypeptide does not comprise a MM, wherein the MM3 inhibits binding of the AB2 to the second antigen.
In some aspects, the fourth polypeptide comprises a fourth masking moiety (MM4) and a fourth cleavable moiety (CM4). In some aspects, the first polypeptide comprises the MM2, the CM2, and the LVD1, the second polypeptide comprises the MM1, the CM1, the HVD1, and the DD1, the third polypeptide comprises the MM3, the CM3, the HVD2, and the DD2, and the fourth polypeptide comprises the MM4, the CM4, and the LVD2.
In some aspects, the first polypeptide comprises the MM2, the CM2, the LVD1, and the LC1, the second polypeptide comprises the MM1, the CM1, the HVD1, the HC1, and the DD1, the third polypeptide comprises the MM3, the CM3, the HVD2, the HC2, and the DD2, and the fourth polypeptide comprises the MM4, the CM4, the LVD2, and the LC2.
In some aspects, the present disclosure provides an activatable construct, comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB 1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), a first light chain constant domain (LC1), a first heavy chain constant domain (HC1); a first masking moiety (MM1) coupled directly or indirectly to a first cleavable moiety (CM1), wherein the MM1 is coupled directly or indirectly to the HVD1, the LVD1, the HC1, or the LC1 via the CM1; a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2); a second masking moiety (MM2) coupled directly or indirectly to a second cleavable moiety (CM2), wherein the MM2 is coupled directly or indirectly to the HVD2 or the LVD2 via the CM2; a first dimerization domain (DD1) and a second dimerization domain (DD2), and wherein the MM1 inhibits binding of the AB1 to the first antigen, and wherein the MM2 inhibits binding of the AB2 to the first antigen.
In some aspects, the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide. In some aspects, the activatable construct further comprises a third cleavable moiety (CM3) and a third masking moiety (MM3). In some aspects, the activatable construct further comprises a fourth cleavable moiety (CM4) and a fourth masking moiety (MM3).
In some aspects, the first polypeptide comprises the MM1, CM1, LVD1, and the LC1. In some aspects, the second polypeptide comprises the HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1. In some aspects, the third polypeptide comprises the MM3, CM3, and the DD2. In some aspects, the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the MM2 and the MM3 inhibit binding of the AB2 to the second antigen. In some aspects, the MM1 inhibits binding of the AB1 to the first antigen. An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 4A.
In some aspects, the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1. In some aspects, the second polypeptide comprises the LVD1, LC1, CM3, and the MM3. In some aspects, the third polypeptide comprises the DD2. In some aspects, the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the MM2 and the MM3 inhibit binding of the AB2 to the second antigen. In some aspects, the MM1 inhibits binding of the AB1 to the first antigen. An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 4B.
In some aspects, the first polypeptide comprises the MM1, CM1, LVD1, and the LC1. In some aspects, the second polypeptide comprises the MM4, CM4, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1. In some aspects, the third polypeptide comprises the MM3, CM3, and the DD2. In some aspects, the MM4 inhibits binding of the AB1 to a first antigen. In some aspects, the MM1 and the MM4 inhibit binding of the AB 1 to the first antigen. In some aspects, the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the MM2 and the MM3 inhibit binding of the AB2 to the second antigen. An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 5A.
In some aspects, the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1. In some aspects, the second polypeptide comprises the MM4, CM4, LVD1, LC1, CM3, and the MM3. In some aspects, the third polypeptide comprises the DD2. In some aspects, the MM4 inhibits binding of the AB1 to the first antigen. In some aspects, the MM1 and the MM4 inhibit binding of the AB 1 to the first antigen. In some aspects, the MM3 inhibits binding of the AB2 to the second antigen. In some aspects, the MM2 and the MM3 inhibit binding of the AB2 to the second antigen. An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 5B.
In some aspects, the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1. In some aspects, the second polypeptide comprises the MM3, CM3, LVD1, and the LC1. In some aspects, the third polypeptide comprises the DD2. In some aspects, the MM3 inhibits binding of the AB1 to the first antigen. In some aspects, the MM1 and the MM3 inhibit binding of the AB 1 to the first antigen. In some aspects, the MM2 inhibits binding of the AB2 to the second antigen. An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 5C.
FIG. 6 shows an exemplary activatable protein 100 comprising three polypeptides. The first polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM1 101, an optional linker 102, the CM1 103, an optional linker 104, and the ABl's light chain fragment 105. The second polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM3 151, an optional linker 152, the CM3 153, an optional linker 154, and the ABl's heavy chain fragment 121, a linker 122, the AB2's heavy chain variable region 123, a linker 124, the AB2's light chain variable region 125, a linker 126, the CM2 127, an optional linker 128, the MM2 129, a linker 130, and the first Fc domain 131. The third polypeptide comprises the second Fc domain 141. In an alternative exemplary configuration, in FIG. 6, 105 is the ABl's heavy chain fragment and 121 is the ABl's light chain fragment. In an alternative exemplary configuration, in FIG. 6, 123 is the AB2's light chain variable region and 125 is the AB2's heavy chain variable region. In an alternative exemplary configuration, in FIG. 6, the first Fc domain 131 is an Fc domain hole mutant and the second Fc domain 141 is an Fc domain knob mutant. In an alternative exemplary configuration, in FIG. 6, the first Fc domain 131 is an Fc domain knob mutant and the second Fc domain 141 is an Fc domain hole mutant.
FIG. 7 shows another exemplary activatable protein 200 comprising three polypeptides. The first polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM1 201, an optional linker 202, the CM1 203, an optional linker 204, the ABl's light chain fragment 205, a linker 206, the AB2's heavy chain variable region 207, a linker 208, and the AB2's light chain variable region 209. The second polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM3 251, an optional linker 252, the CM1 253, an optional linker 254, the ABl's heavy chain fragment 221, an optional linker 222, the CM2 223, an optional linker 224, the MM2 225, a linker 226, and the first Fc domain 227. The third polypeptide comprises the second Fc domain 241. In an alternative exemplary configuration, in FIG. 7, 205 is the ABl's heavy chain fragment and 221 is the ABl's light chain fragment. In an alternative exemplary configuration example, in FIG. 7, 207 is the AB2's light chain variable region and 209 is the AB2's heavy chain variable region. In an alternative exemplary configuration example, in FIG. 7, the first Fc domain 227 is an Fc domain hole mutant and the second Fc domain 241 is an Fc domain knob mutant. In an alternative exemplary configuration example, in FIG. 7, the first Fc domain 227 is an Fc domain knob mutant and the second Fc domain 241 is an Fc domain hole mutant.
FIG. 8 shows another exemplary activatable protein 300 comprising three polypeptides. The first polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM1 301, an optional linker 302, the CM1 303, an optional linker 304, the ABl's light chain fragment 305, a linker 306, the AB2's heavy chain variable region 307, a linker 308, and the AB2's light chain variable region 309. The second polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM3 351, an optional linker 352, the CM3 353, an optional linker 354, the ABl's heavy chain fragment 321, an optional linker 322, the CM2 323, an optional linker 324, and the first Fc domain 325. The third polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the second Fc domain 341, a linker 342, and the MM2 343. In an alternative exemplary configuration, in FIG. 8, 305 is the ABl's heavy chain fragment and 321 is the ABl's light chain fragment. In an alternative exemplary configuration, in FIG. 8, 307 is the AB2's light chain variable region and 309 is the AB2's heavy chain variable region. In an alternative exemplary configuration, in FIG. 8, the first Fc domain 325 is an Fc domain hole mutant and the second Fc domain 341 is an Fc domain knob mutant. In an alternative exemplary configuration, in FIG. 8, the first Fc domain 325 is an Fc domain knob mutant and the second Fc domain 341 is an Fc domain hole mutant.
FIG. 9 shows another exemplary activatable protein 400 comprising three polypeptides. The first polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM1 401, an optional linker 402, the CM1 403, an optional linker 404, the ABl's light chain fragment 405, a linker 406, the AB2's heavy chain variable region 407, a linker 408, and the AB2's light chain variable region 409. The second polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM3 451 , an optional linker 452, the CM3 453, an optional linker 454, the ABTs heavy chain fragment 421, an optional linker 422, the CM2 423, an optional linker 424, and the first Fc domain 425. The third polypeptide, in order from an amino (N) terminal region to carboxyl (C) terminal region, comprises the MM2441, a linker 442, and the second Fc domain 443. In an alternative exemplary configuration, in FIG. 9, 405 is the ABFs heavy chain fragment and 421 is the ABFs light chain fragment. In an alternative exemplary configuration, in FIG. 9, 407 is the AB2's light chain variable region and 409 is the AB2's heavy chain variable region. In an alternative exemplary configuration, in FIG. 9, the first Fc domain 425 is an Fc domain hole mutant and the second Fc domain 443 is an Fc domain knob mutant. In an alternative exemplary configuration, in FIG. 9, the first Fc domain 425 is an Fc domain knob mutant and the second Fc domain 443 is an Fc domain hole mutant.
While the activatable constructs of the present disclosure are described in terms of polypeptide structures or multiple polypeptide compositions or complexes, the present disclosure also includes a nucleic acid, e.g., polynucleotide, or mRNA encoding the polypeptide or multiple polypeptides, for providing activatable constructs.
Antigen Binding Domains
The antigen binding domains employed herein are formed by combination of a heavy chain variable domain and a light chain variable domains. For example, HVD1 and LVD1 together form AB 1. Similarly, HVD2 and LVD2 together form AB2. As described in more detail herein below, in some aspects, AB1 and AB2 bind to the same antigen (i.e., the first antigen and the second antigen are the same), In certain of these aspects, HVD1 and HVD2 are identical, and LVD1 and LVD2 are identical. In other of these aspects, HVD1 is different from HVD2 and/or LVD1 is different from LVD2. In other aspects, AB1 and AB2 bind different antigens (i.e., the first antigen and the second antigen are different). In certain of these aspects, the set of HVD1 and LVD1 is different from the set of HVD2 and LVD2. In some aspects, the activatable construct further comprises a first heavy chain constant domain (HC1) coupled to the
C-terminus of the HVD1. In some aspects, the HVD1 is coupled directly or indirectly to the C- terminus of the MM1 via the CM1 and directly or indirectly to the N-terminus of the DD1 via the HC1 . In some aspects, the activatable construct further comprises a first light chain constant domain (LC1) coupled to the C-terminus of the LVD1. In some aspects, the LVD1 is coupled directly or indirectly to the C-terminus of the MM2 via the CM2, wherein the LVD1 is coupled directly or indirectly to the N-terminus of the LC1, and wherein the HC1 is coupled, directly or indirectly, to the LC1.
In some aspects, the AB1, the AB2, or both comprise an antibody fragment or an antigen binding fragment. In some aspects, the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. Additional examples of the antigen-binding fragments include a VH domain, a VHH domain, a VNAR domain, and a single chain fragment variable (scFv), BiTE or a component thereof, a (scFv)2, a NANOBODY®, a nanobody -HS A, VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab’)2, a diabody, a CROSSMAB®, a DAF (two-in-one), a DAE (four-in-one), a DUTAMAB®, a DT- IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a FcAb, a kl-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)- V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, ZYBODY™, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH , Fab-scFv, a F(ab’)2-scFv2, a scFv-KfH, a Fab-scFv-Fc, a tetravaient HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, a VHH-Fc, a tandem VHH-Fc, a LTTH-Fc KiH, a Fab- VHH-Fc, an Intrabody, a dock and lock, an ImmTAC® (immune- mobilizing monoclonal TCRs (T cell receptors) against cancer), an IgG-IgG conjugate, a Cov-X- Body, a scFvl- PEG-scFv2, an Adnectin, a DARPin®, a fibronectin, an IgG, an IgM, an IgA, an IgE, an IgD, a DEP conjugate, TMEAbodyTM, SAFEbody®, TRITAC® , or SHIELD antibody.
In some aspects, the activatable construct further comprises a second heavy chain constant domain (HC2) coupled to the C-terminus of the HVD2. In some aspects, the activatable construct further comprises a second light chain constant domain (LC2) coupled to the C- terminus of the LVD2. In some aspects, the HVD2 is coupled directly or indirectly to the C- terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the HC2. In some aspects, the LVD2 is coupled directly or indirectly to the C-terminus of the MM4 via the CM4. In some aspects, the LVD2 is coupled directly or indirectly to the N-terminus of the LC2 and the HC2 is coupled directly or indirectly to the LC2. In some aspects, the HVD1 together with the HC1 and the LVD1 together with the LC1 comprise an Fab domain, wherein the HVD1-HC1 is coupled directly or indirectly to the DD1 . In some aspects, the HVD2 and LVD2 form a scFv domain coupled directly or indirectly to the DD2. In some aspects, the HVD2 and LVD2 are not coupled to a MM. In some aspects, at least one of the HVD2 or the LVD2 in the scFv domain is coupled directly or indirectly to the MM3 via the CM3 and the other HVD2 or LVD2 is coupled directly or indirectly to the DD2.
In some aspects, the activatable construct comprises an antibody. In some aspects, an activatable construct of the present disclosure is a homodimer comprising two identical polypeptide chains. In some aspects, an activatable construct is a heterodimer comprising two different polypeptide chains. In some aspects, an activatable construct is a bispecific activatable antibody. In some aspects, an activatable construct is a multi-specific activatable antibody. Antibodies to any of a variety of targets may be employed, such as those described herein and also in, for example, WO2019/075405, WO2018/165619, WO2018/222949, WO2018/085555, WO20 17/011580, WO2016/179257, WO2016/179285, WO2016/179335, WO2016/ 149201, WO20 16/ 118629, WO2021/061867, WO2016/014974, W02020/092881, W02020/086665, WO2013/163631, WO2014197612, WO2014/052462, WO2013/192550, WO2020/252358, WO20 19/046652, WO2015/066279, WO2019/173771, WO2020/252349, WO2019/13374, W02024/015830, WO2023/183888, WO2023/192606, WO2023/064955, which are incorporated herein by reference in their entireties.
Examples of activatable constructs include those having the following structures in an N- terminal to C-terminal direction: a) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 ; and iii) HVD2-LVD2-DD2; b) i) MM2-CM2-LVD 1 -LC 1 ; ii)MMl-CMl-HVDl-HCl-DDl; and iii) LVD2-HVD2-DD2; c) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-DD1; and iii) MM3- CM3-HVD2-LVD2-DD2; d) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; and iii) MM3- CM3- LVD2-HVD2-DD2; e) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -HVD2-LVD2-CM3 -MM3 -DD 1 ; and iii) DD2; f) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-LVD2-HVD2-CM3-MM3-DD1; and iii) DD2; g) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-DD1; iii) HVD2-HC2-DD2; and iv) LVD2-LC2; h) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2; and iv) LVD2-LC2; i) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-DD1; iii) HVD2-HC2-DD2; and iv) MM3-CM3-LVD2-LC2; or j) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2; and iv) MM4-CM4-LVD2-LC2; wherein the HC1 is coupled directly or indirectly to the LC1.
Additional examples of activatable constructs include those having the following structures in an N-terminal to C-terminal direction: k) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; l) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-LVD2-HVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; m) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; ii) LVD1-LC1-CM3-MM3; and iii) DD2; n) i) MM 1 -CM 1 -HVD 1 -HC 1 -LVD2-HVD2 -CM2 -MM2 -DD 1 ; ii) LVD1-LC1-CM3-MM3; and iii) DD2; or o) i) MM1-CM1-LVD1-LC1 ii) MM4-CM4-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; p) i) MM1-CM1-LVD1-LC1 ii) MM4-CM4-HVD1-HC1-LVD2-HVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; q) i) MM 1 -CM 1 -HVD 1 -HC 1 -HVD2-L VD2-CM2-MM2-DD 1 ; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2; r) i) MM 1 -CM 1 -HVD 1 -HC 1 -L VD2-HVD2 -CM2-MM2-DD 1 ; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2; s) i) MM 1 -CM 1 -HVD 1 -HC 1 -HVD2-L VD2-CM2-MM2-DD 1 ; ii) MM3-CM3-LVD1-LC1; and iii) DD2; or t) i) MM 1 -CM 1 -HVD 1 -HC 1 -L VD2-HVD2 -CM2-MM2-DD 1 ; ii) MM3-CM3-LVD1-LC1; and iii) DD2.
As used herein, unless otherwise stated, each between elements of an activatable construct is a direct or indirect linkage (e.g., via a linker).
In some aspects, the first antigen and the second antigen are the same molecule or the same epitope. In some aspects, the activatable construct specifically binds to a single epitope. In some aspects, the MM1, the MM2, the MM3, and the MM4 comprise the same amino acid sequence. In some aspects, the MM1 and the MM4 comprise the same amino acid sequence. In some aspects, the MM2 and the MM3 comprise the same amino acid sequence. In some aspects, the second polypeptide chain and the third polypeptide chains comprise the same amino acid sequence. In some aspects, the first polypeptide chain and the fourth polypeptide chains comprise the same amino acid sequence.
In some aspects, the first antigen and the second antigen are different epitopes on the same molecule. In some aspects, the first antigen and the second antigen are different molecules or different epitopes.
In some aspects, the MM1 and the MM2 comprise the same sequence. In some aspects, the MM3 comprises a different amino acid sequence than the MM1, the MM2, or both the MM1 and the MM2. In some aspects, the MM3 comprises a different amino acid sequence than the MM1 and the MM2. In some aspects, the MM1 and the MM3 are different amino acid sequences.
In some aspects, the activatable construct is bispecific. In some aspects, the activatable construct comprises a bispecific antibody. In some aspects, the second polypeptide chain and the third polypeptide chain are different amino acid sequences. In some aspects, the first polypeptide chain and the fourth polypeptide chain are different amino acid sequences. In some aspects, the first polypeptide chain and the fourth polypeptide chain comprise the same amino acid sequence and the second polypeptide chain and third polypeptide chain comprise the same amino acid sequence. In some aspects, the first polypeptide chain and the fourth polypeptide chain comprise the same amino acid sequence and the second polypeptide chain and third polypeptide chain comprise a different amino acid sequence. In some aspects, the amino acid sequences of the HVD1-HC1 domains are different amino acid sequences than the HVD2-HC2 domains.
In some aspects, the CM1 and the CM3 comprise the same amino acid sequence. In some aspects, the CM1 and the CM3 are different amino acid sequences. In some aspects, the second polypeptide chain and the third polypeptide chain are different amino acid sequences.
In some aspects, the DD1 and the DD2 comprise the same amino acid sequence. In some aspects, the DD1 and the DD2 are different amino acid sequences. In some aspects, the activatable construct further includes a third antigen binding domain (AB3) having specific binding affinity for a third antigen. In some aspects, the AB3 comprises a heavy chain variable domain 3 (HVD3) and a light chain variable domain 3 (LVD3). In some aspects, the AB3 is coupled directly or indirectly to a fifth masking moiety (MM5) via a fifth cleavable moiety (CM5) and the MM5 inhibits binding of the AB3 to the third antigen.
In some aspects, the AB1 is coupled directly or indirectly to the N-terminus of the DD1. In some aspects, the AB2 is coupled directly or indirectly to the N-terminus of the DD2. In some aspects, the AB3 is coupled directly or indirectly to the C-terminus of the DD1 or the DD2.
In some aspects, the AB3 comprises a scFv, wherein the AB3 comprises: i) MM5-CM5-HVD3-LVD3-X; or ii) MM5-CM5-LVD3-HVD3-X; or wherein X represents attachment to the N-terminus or the C-terminus of the DD1 or the DD2, and each is a direct or an indirect covalent linkage. In one aspect, X represents attachment to the N-terminus of the DD2. In one aspect, X represents attachment to the C- terminus of the DD2. In one aspect, X represents attachment to the N-terminus of the DD 1. In one aspect, X represents attachment to the C-terminus of the DD1.
In some aspects, the C-terminus of the DD1 or the DD2 is attached to the N-terminus of: iii) LVD3-HVD3-CM5-MM5; or iv) HVD3-LVD3-CM5-MM5;
These constructs may also be depicted as X-LVD3-HVD3-CM5-MM5 or X-HVD3- LVD3-CM5-MM5, wherein X represents attachment to the C-terminus of the DD1 or the DD2. In some aspects, a masked AB 3 is attached to the N-terminus or the C-terminus of one of the DD1 or DD2 of any of the activatable constructs disclosed herein. Examples of activatable constructs with an AB3 include those having the following structures in an N-terminal to C- terminal direction: a) i) MM2-CM2-LVD1-LC1; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 -LVD3 -HVD3 -CM5 -MM5 ; iii) MM3-CM3-HVD2-HC2-DD2; and iv) MM4-CM4-LVD2-LC2; b) i)MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2-LVD3-HVD3-CM5-MM5; and iv) MM4-CM4-LVD2-LC2; c) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 -HVD 3 -L VD3 -CM5 -MM5 ; iii) MM3-CM3-HVD2-HC2-DD2; and iv) MM4-CM4-LVD2-LC2; d) i)MM2-CM2-LVDl-LCl; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2-HVD3-LVD3-CM5-MM5; and iv) MM4-CM4-LVD2-LC2; e) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5-MM5; and iii) MM3-CM3-DD2; f) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2-LVD3-HVD3-CM5-MM5; g) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5-
MM5; ii) LVD1-LC1-CM3-MM3; and iii) DD2; h) i) MM 1 -CM 1 -HVD 1 -HC 1 -HVD2-L VD2-CM2-MM2-DD 1 ; ii) LVD1-LC1-CM3-MM3; and iii) DD2-LVD3-HVD3-CM5-MM5; i) i) MM1-CM1-LVD1-LC1 ii) MM4-CM4-HVD 1 -HC 1 -HVD2-L VD2-CM2-MM2-DD 1 -L VD3 -HVD 3 -CM5 -
MM5; and iii) MM3-CM3-DD2; j) i) MM1-CM1-LVD1-LC1 ii) MM4-CM4-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2-LVD3-HVD3-CM5-MM5; k) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5- MM5; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2; l) i) MM 1 -CM 1 -HVD 1 -HC 1 -HVD2-L VD2-CM2-MM2-DD 1 ; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2-LVD3-HVD3-CM5-MM5; h) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5- MM5; ii) MM3-CM3-LVD1-LC1; and iii) DD2; or i) i) MM 1 -CM 1 -HVD 1 -HC 1 -HVD2-L VD2-CM2-MM2-DD 1 ; ii) MM3-CM3-LVD1-LC1; and iii) DD2-LVD3-HVD3-CM5-MM5; wherein the LVD2 and the HVD2 and the HVD3 and LVD3, respectively, may be in any order, and wherein each is a direct or an indirect covalent linkage.
In some aspects, the activatable construct further comprises a fourth antigen binding domain (AB4) having specific binding affinity for a fourth antigen. In some aspects, the AB4 comprises a heavy chain variable domain 4 (HVD4) and a light chain variable domain 4 (LVD4). In some aspects, the AB4 is coupled directly or indirectly to a sixth masking moiety (MM6) via a sixth cleavable moiety (CM6) and the MM6 inhibits binding of the AB4 to the fourth antigen. In some aspects, the AB3 is coupled directly or indirectly to the C-terminus of the DD1 and the AB4 is coupled directly or indirectly to the C-terminus of the DD2.
In some aspects, the AB4 comprises a scFv, wherein the AB4 comprises: i) MM6-CM6-HVD4-LVD4-Y; or ii) MM6-CM6-LVD4-HVD4-Y; wherein Y represents attachment to the N-terminus or the C-terminus of the DD2, and each is a direct or an indirect covalent linkage. In one aspect, Y represents attachment to the N-terminus of the DD2. In one aspect, Y represents attachment to the C-terminus of the DD2. In one aspect, Y represents attachment to the N-terminus of the DD1. In one aspect, Y represents attachment to the C-terminus of the DD1. In some aspects, the C-terminus of the DD1 or the DD2 is attached to the N-terminus of: iii) LVD4-HVD4-CM6-MM6; or iv) HVD4-LVD4-CM6-MM6;
These constructs may also be depicted as Y- LVD4-HVD4-CM6-MM6 or Y-HVD4- LVD4-CM6-MM6, wherein Y represents attachment to the C-terminus of the DD1 or the DD2.
In some aspects, a masked AB4 is attached to the N-terminus or the C-terminus of one of the DD1 or DD2 of any of the activatable constructs disclosed herein. Examples of activatable constructs with an AB4 include those having the following structures in an N-terminal to C- terminal direction: a) i) MM2-CM2-LVD1-LC1; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 -LVD3 -HVD3 -CM5 -MM5 ; iii) MM3-CM3-HVD2-HC2-DD2-LVD4-HVD4-CM6-MM6; and iv) MM4-CM4-LVD2-LC2; b) i)MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 -LVD4-HVD4-CM6-MM6; iii) MM3-CM3-HVD2-HC2-DD2-LVD3-HVD3-CM5-MM5; and iv) MM4-CM4-LVD2-LC2; c) i) MM2-CM2-LVD1-LC1; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 -HVD 3 -L VD3 -CM5 -MM5 ; iii) MM3-CM3-HVD2-HC2-DD2-HVD4-LVD4-CM6-MM6; and iv) MM4-CM4-LVD2-LC2; or d) i)MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM1 -HVD 1 -HC 1 -DD 1 -HVD4-LVD4-CM6-MM6; iii) MM3-CM3-HVD2-HC2-DD2-HVD3-LVD3-CM5-MM5; and iv) MM4-CM4-LVD2-LC2, e) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5-MM5; and iii) MM3-CM3-DD2-HVD4-LVD4-CM6-MM6; f) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-HVD4-LVD4-CM6-MM6; and iii) MM3-CM3-DD2-LVD3-HVD3-CM5-MM5; g) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5- MM5; ii) LVD1-LC1-CM3-MM3; and iii) DD2-HVD4-LVD4-CM6-MM6; h) i) MM 1 -CM 1 -HVD 1 -HC 1 -HVD2-L VD2-CM2-MM2-DD 1 -HVD4-L VD4-CM6- MM6; ii) LVD1-LC1-CM3-MM3; and iii) DD2-LVD3-HVD3-CM5-MM5; i) i) MM1-CM1-LVD1-LC1 ii) MM4-CM4-HVD 1 -HC 1 -HVD2-LVD2-CM2-MM2-DD 1 -LVD3-HVD3 -CM5-
MM5; and iii) MM3-CM3-DD2-HVD4-LVD4-CM6-MM6; j) i) MM1-CM1-LVD1-LC1 ii) MM4-CM4-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2-LVD3-HVD3-CM5-MM5; k) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5- MM5; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2; l) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2-LVD3-HVD3-CM5-MM5; m) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-LVD3-HVD3-CM5- MM5; ii) MM3-CM3-LVD1-LC1; and iii) DD2-HVD4-LVD4-CM6-MM6; or n) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1-HVD4-LVD4-CM6- MM6; ii) MM3-CM3-LVD1-LC1; and iii) DD2-LVD3-HVD3-CM5-MM5; wherein the LVD2 and the HVD2, the HVD3 and LVD3, and the HVD4 and LVD4, respectively, may be in any order, and wherein each is a direct or an indirect covalent linkage.
Exemplary Targets
[0100] The activatable constructs of the present disclosure may bind to one or more of a variety of targets. Antigen binding domains that bind to specific binding partners are disclosure herein as well as in the relevant literature.
[0101] In some non-limiting embodiments, at least one of the AB1, the AB2, the AB3, or the AB4 in the activatable construct is a specific binding partner for any target (antigen) listed in Table 1.
[0102] In some non-limiting embodiments, at least one of the AB1, the AB2, the AB3, or the AB4 in the activatable construct is a specific binding partner for one of CD3, ITGA3B1, or EGFR, and at least one other ABs is a specific binding partner for any target listed in Table 1. [0103] In some embodiments, the activatable constructs engage immune effector cells and a tumor associated antigen. In some embodiments, a first antigen or first antigen binding domain target or epitope is a tumor associated antigen. In some embodiments, a second antigen or second antigen binding domain target or epitope is a surface antigen on an immune effector cells, such as, for example, a leukocyte, such as a T-cell, a natural killer (NK) cell, a mononuclear effector cell (such as, for example, a myeloid mononuclear cell), a macrophage, and the like. In some examples, the activatable construct may comprise an immune effector cell engaging bispecific activatable antibody, which crosslinks an immune effector cell with another cell (e.g., a cell associated with a disease such as cancer or infection). The activatable construct may comprise a leukocyte cell-engaging bispecific activatable antibody, a T cell engaging bispecific activatable antibody, a NK cell-engaging bispecific activatable antibody, a macrophage cellengaging bispecific activatable antibody, a mononuclear cell-engaging bispecific activatable antibody, or a myeloid mononuclear cell-engaging bispecific activatable antibody. In one example, the activatable antibody may comprise a T cell engaging bispecific antibody.
[0104] In some aspects, the AB 1 and the AB2 in the activatable construct are a specific binding partner for any target listed in Table 1, wherein the AB1 and AB2 are specific binding partners for the same target. In some aspects, the AB1 and the AB2 in the activatable construct are specific binding partners for any target listed in Table 1, wherein the AB1 and AB2 are specific binding partners for different targets. In an exemplary embodiment, AB1 of the activatable construct is specific binding partner for one of CD3, ITGA3B1, or EGFR and AB2 is a specific binding partner for any target listed in Table 1. In another exemplary embodiment, AB2 of the activatable construct is specific binding partner for one of CD3, ITGA3B1, or EGFR and AB l is a specific binding partner for any target listed in Table 1.
[0105]
Table 1: Exemplary Targets
Exemplary Antigen Binding Domains
A variety of antigen binding domains may be used in activatable constructs disclosed herein. The antigen binding domain may be an antibody or a fragment thereof, e.g., a monoclonal antibody, single chain antibody, Fab fragment, F(ab')2 fragment, single-chain variable fragment (scFv), diabody (a noncovalent dimer of scFv), single chain antibody (scab), a VHH, a domain antibody (dAb) or single domain antibody (nanobody, e.g., single domain heavy chain antibody, single domain light chain antibody). A single domain antibody may be an antibody fragment that is a single monomeric variable antibody domain. A single domain antibody may have similar affinity to antigens as a corresponding full-length antibody. Some exemplary antibodies are disclosed in Table 3. Other antibodies may be identified or generated using known methods, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art. Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes and Pluckthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol. 17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel et al. Nucl. Acids Research 21 : 1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A (1992), and U.S. Patent No. 5,733,743.
The present disclosure includes activatable constructs having an engineered dimerization domain for modified effector functions, e.g., so as to enhance or reduce such function to improve the effectiveness of the antibody, antibody domain, or fragment thereof. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibodydependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). In some embodiments, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti -Cancer Drug Design, 3: 219-230 (1989). In some embodiments, Fc mutations are made to remove glycosylation sites, thereby reducing Fc function.
In some embodiments, the antigen binding domains may be a scFv. In some embodiments, the antigen binding domains may be a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody. The present disclosure includes structures having combinations of one or more polypeptides comprising any of the domains listed above, e.g., one or more of SDA, Fv, ScFv, Fab, scFab, VHH, and dAb, with one or more selected from SDA, Fv, scFv, Fab, VHH, scFab, and dAb. The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigenbinding domains that specifically bind to an antigen or epitope. The term “antibody” specifically includes, e.g., intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific, and multi-specific antibodies. One example of an antibody is an antigen-binding domain formed by a VH -VL dimer. Additional examples of an antibody are described herein. Additional examples of an antibody are known in the art. There are five main heavy-chain classes or isotypes, some of which have several subtypes, and these determine the functional activity of an antibody molecule. The five major classes of immunoglobulin are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). IgG is by far the most abundant immunoglobulin and has several subclasses (IgGl, 2, 3, and 4 in humans).
Illustrative antibodies include any one or combination of the HVD1, the HVD2, the HVD3, or HVD4 comprising a sequence selected from:
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPE SGSVKYNEGFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDY WGQGTTVTVSS (SEQ ID NO: 680); or QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVT VSS (SEQ ID NO: 702).
In some aspects, any one or combination of the LVD1, the LVD2, the LVD3, or the LVD4 comprises a sequence selected from:
DIQMTQSPSSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLA SGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIK (SEQ ID NO: 691); or
QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGI PSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 705).
In some aspects, the HVD1-HC1, the HVD2-HC2, or both comprises a sequence selected from:
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPE SGSVKYNEGFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQ VYTLPP SREEMTKNQ VSLTCL VKGF YP SDIAVEWESNGQPENNYKTTPP VLD S DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 681); or
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSG GNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF P AVLQ S SGL YSL S S VVTVP S S SLGTQT YICNVNHKP SNTK VDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 703).
In some aspects, the LVD1-LC1, the LVD2-LC2, or both comprises a sequence selected from:
DIQMTQ SPS SLS AS VGDRVTITC S AS S SIS SNYLHW YQQKPGKVPKLLIYRT SNL A SGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 692); or
QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGI PSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 706).
Masking Moieties
Activatable constructs of the present disclosure may employ a variety of masking moieties (MMs). Suitable MMs inhibit interaction of the antigen binding domain with its antigen binding partner. The MM may bind to the AB 1 and/or AB2 and limit the interaction of the antigen binding domain with its antigen binding partner. In some aspects, the MMs contemplated by this disclosure may be from 1-50 amino acids in length (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, or 40 amino acids, or no greater than 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids). In some examples, the MMs may be from 8 to 15 amino acids in length. In some aspects, any of the MMs are no more than 40 amino acids in length. In preferred embodiments, the MM is no more than 20 amino acids in length. In some aspects, the MM is no more than 19, 18, 17, 16, or 15 amino acids in length. For example, the MM is not a latency associated peptide.
For example, the MM is not a cytokine. For example, the MM is not a receptor for a cytokine. For example, the MM is not an albumin. For example, in some aspects, the MM excludes proteins or polypeptides having more than 50 amino acids, In some aspects, the MM excludes proteins or polypeptides having more than 25 amino acids, In some aspects, the MM excludes proteins or polypeptides having more than 20 amino acids, In some aspects, the MM excludes proteins or polypeptides having more than 15 amino acids.
In some aspects, a MM may be a receptor for AB1 or AB2 or a fragment of a receptor therefor. In some aspects, a MM may be a scFv that binds selectively to AB 1 or to AB2.
The MMs may contain genetically encoded or genetically non-encoded amino acids.
Examples of genetically non-encoded amino acids are but not limited to D-amino acids, P-amino acids, and y-amino acids. In specific embodiments, the MMs contain no more than 50%, 40%, 30%, 20%, 15%, 10%, 5% or 1% of genetically non-encoded amino acids.
In some aspects, any one or combination of MMs described herein (e.g., MM1 and MM2; MM1, MM2, and MM3; MM1, MM2, MM3, and MM4; MM1, MM2, MM3, MM4, and MM5; or MM1, MM2, MM3, MM4, MM5, and MM6) each independently is from 2 to 50 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40 or 50 amino acids). In some aspects, any one or combination of MMs described herein (e.g., MM1 and MM2; MM1, MM2, and MM3; MM1, MM2, MM3, and MM4; MM1, MM2, MM3, MM4, and MM5; or the MM1, MM2,
MM3, MM4, MM5, and MM6) may each independently comprise a sequence selected from the group consisting of: XCXXRXXFEXXDCVX, where X equals any amino acid (SEQ ID NO: 716); TCPTRWHFETTDCVM (SEQ ID NO: 717); ICGSRLDFELNDCVM (SEQ ID NO: 718); WCRDRSHFETGDCVM (SEQ ID NO: 719); ICTSRWEFENRDCVM (SEQ ID NO: 720); VCRDRNEFEVGDCVM (SEQ ID NO: 721); TCKNRLEFERGDCVM (SEQ ID NO: 722); VCSSRLEFEQKDCVM (SEQ ID NO: 723); WCRDREHFEKGDCVM (SEQ ID NO: 724); YCANRYEFEYGDCVM (SEQ ID NO: 725); VCRTRWHFETTDCVM (SEQ ID
NO: 726); VCSNRAEFEWGDCVM (SEQ ID NO: 727); VCASRWHFENTDCVM (SEQ ID NO: 728); NCASRWHFENEDCVM (SEQ ID NO: 729); VCSGRLEFELGDCVM (SEQ ID NO: 730); VCSSRWEFETNDCVM (SEQ ID NO: 731); FCRDRLDFDTMDCVM (SEQ ID NO: 732); CCMDRLEFERGDCVM (SEQ ID NO: 733); VCGSRNEFETGDCVM (SEQ ID NO: 734); MCSGRLEFETGDCVM (SEQ ID NO: 735); ECKTRQDFEMHDCVY (SEQ ID NO: 736); QCMSRFAFEIGDCVM (SEQ ID NO: 737); TCLSRYEFETTDCVM (SEQ ID NO: 738); VCRTRWHFETTDCVM (SEQ ID NO: 739); ICHDPYMNIDYTCKL (SEQ ID NO: 740); VMCYWEGWGFGRCPL (SEQ ID NO: 741); VWYCDGGYNECATRS (SEQ ID NO: 742); AVWCDAYNKNMCWST (SEQ ID NO: 743); KCHDPYINIDYTCNN (SEQ ID NO: 744); LITCEMLMLKNCEKN (SEQ ID NO: 745); LGCKKQHHTNNTCDR (SEQ ID NO: 746); CISPRG (SEQ ID NO: 747); CISPRGCG (SEQ ID NO: 748);
CISPRGCPDGPYVMY (SEQ ID NO: 749); CISPRGCPDGPYVM (SEQ ID NO: 750); CISPRGCEPGTYVPT (SEQ ID NO: 751); CISPRGCPGQIWHPP (SEQ ID NO: 752); GSHCLIPINMGAPSC (SEQ ID NO: 753); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 754); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 755);
ADHVFWGSYGCISPRGCPG (SEQ ID NO: 756); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 757); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 758); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 759); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 760);
CPYYTLSYCGCISPRGCPG (SEQ ID NO: 761); CNHVYFGTCGCISPRGCPG (SEQ ID NO: 762); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 763); CHHFTLTTCGCISPRGCPG (SEQ ID NO: 764); YNPCATPMCCISPRGCPG (SEQ ID NO: 765);
CNHHYFYTCGCISPRGCG (SEQ ID NO: 766); CNHHYHYYCGCISPRGCG (SEQ ID NO: 767); CNHVYFGTCGCISPRGCG (SEQ ID NO: 768); CHHVYWGHCGCISPRGCG (SEQ ID NO: 769); CPHFTTTSCGCISPRGCG (SEQ ID NO: 770);
CNHFTLTTCGCISPRGCG (SEQ ID NO: 771); CHHFTLTTCGCISPRGCG (SEQ ID NO: 772); CPYYTLSYCGCISPRGCG (SEQ ID NO: 773); CPHVSFGSCGCISPRGCG (SEQ ID NO: 774); ADHVFWGSYGCISPRGCG (SEQ ID NO: 775); YNPCATPMCCISPRGCG (SEQ ID NO: 776); CHHVYWGHCGCISPRGCG (SEQ ID NO: 777);
C(N/P)H(H/V/F)(Y/T)(F/W/T/L) (SEQ ID NO: 778); (Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID NO: 779); CISPRGCGQPIPSVK (SEQ ID NO: 780); CISPRGCTQPYHVSR (SEQ ID NO: 781); CISPRGCNAVSGLGS (SEQ ID NO: 782); LSCEGWAMNREQCRA (SEQ ID NO: 783); PPLECNTKSMCSKHD (SEQ ID NO: 784); DRDCRGRRARCQQEG (SEQ ID NO: 785); FTCEGWAMNREQCRT (SEQ ID NO: 786); GRCPPSRDIRFCTYM (SEQ ID NO: 787); FSCEGWAMNRSQCRT (SEQ ID NO: 788); FTCEGWAMNRDQCRT (SEQ ID NO: 789); CISPRGCPDGPYVM (SEQ ID NO: 790); CISPRGC (SEQ ID NO: 791);
MMYCGGNEVLCGPRV (SEQ ID NO: 792); GYRWGCEWNCGGITT (SEQ ID NO: 793); GYLWGCEWNCGGITT (SEQ ID NO: 794); MMYCGGNEIFCEPRG (SEQ ID NO: 795); GYGWGCEWNCGGSSP (SEQ ID NO: 796); and MMYCGGNEIFCGPRG (SEQ ID NO: 797).
In some aspects, any one or combination of MMs described herein (e g., MM1 and MM2; MM1, MM2, and MM3; MM1, MM2, MM3, and MM4; MM1, MM2, MM3, MM4, and MM5; or MM1, MM2, MM3, MM4, MM5, and MM6) each independently comprise a sequence selected from the group consisting of: VCRDRNEFEVGDCVM (SEQ ID NO: 721); TCLSRYEFETTDCVM (SEQ ID NO: 738); VCSNRAEFEWGDCVM (SEQ ID NO: 727); VCSGRLEFELGDCVM (SEQ ID NO: 730); CISPRGCPDGPYVMY (SEQ ID NO: 749); or LSCEGWAMNREQCRA (SEQ ID NO: 783).
Illustrative MMs include those provided as SEQ ID NOs: 84-108 (for use in an anti-PDL- 1 activatable antibody) disclosed in WO 2019/165143A1, as well as those disclosed in WO 2019/075405A1, WO 2009/025846, WO 2010/096838, WO 2010/081173, WO 2013/163631, WO 2013/192546, WO 2013/192550, WO 2014/026136, WO 2014/052462, WO 2014/107599, WO 2014/197612, WO 2015/013671, WO 2015/048329, WO 2015/066279, WO 2015/116933, WO 2016/014974, WO 2016/118629, WO 2016/149201, WO 2016/179285, WO 2016/179257, WO 2016/179335, WO 2017/011580, PCT/US2017/059740, US Provisional Application Serial Numbers 62/469,429, 62/572,467, and 62/613,358, each of which is incorporated herein by reference in its entirety.
The MMs may be identified through a screening procedure from a library of candidate activatable constructs having variable MMs. For example, a CM can be selected to provide for a desired enzyme/target combination, and the amino acid sequence of the MM can be identified by the screening procedure described below to identify a MM that provides for a switchable phenotype. For example, a random peptide library (e.g., of peptides comprising about 2 to about 40 amino acids or more) may be used in the screening methods disclosed herein to identify a suitable MMs. In specific embodiments, MMs with specific binding affinity for an antigen binding domain can be identified through a screening procedure that includes providing a library of peptide scaffolds consisting of candidate MMs wherein each scaffold is made up of a transmembrane protein and the candidate MM. The library may then be contacted with an entire or portion of a protein such as a full length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing a protein (also capable of binding the binding partner of interest), and identifying one or more candidate MMs having detectably bound protein. The screening may be performed by one more rounds of magnetic-activated sorting (MACS) or fluorescence-activated sorting (FACS), as well as determination of the binding affinity of MM towards the antigen binding domain and subsequent determination of the masking efficiency, e.g., as described in US20200308243A1, which is incorporated herein by reference in its entirety. Methods for screening MMs to obtain MMs unique for the antigen binding domain and those that specifically and/or selectively bind a binding domain of a binding partner/target are provided herein and can include protein display methods.
Cleavable Moieties
Any of a variety of protease cleavable substrates may be employed in the activatable constructs of the present disclosure. Suitable substrates may be readily identified by a number of known methods, including those described in Sandersjbd, et al. "Protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting." Biotechnology Journal 12.1 (2017): 1600365; Li, Qing, et al. "Profiling protease specificity: Combining yeast ER sequestration screening (YESS) with next generation sequencing." ACS chemical biology 12.2 (2017): 510-518; and Zhou, Jie, et al. "Deep profiling of protease substrate specificity enabled by dual random and scanned human proteome substrate phage libraries." Proceedings of the National Academy of Sciences 117.41 (2020): 25464- 25475).
Illustrative cleavable moieties that are suitable for use in the activatable constructs described herein include, for example any of the sequences disclosed in WO 2010/081173; WO 2015/048329; WO 2015/116933 ; WO 2016/118629; WO 2020/118109; WO 2024/030843 ; WO 2024/030845; WO 2024/030847; WO 2024/030858; and WO 2024/030850, the disclosures of which are incorporated herein by reference in their entireties. In some aspects, any one or combination of CMs described herein (e g., CM1 and CM2; CM1, CM2, and CM3; CM1, CM2, CM3, and CM4; CM1, CM2, CM3, CM4, and CM5; or CM1, CM2, CM3, CM4, CM5, and CM6) each independently comprises a substrate for the same or different protease.
In some aspects, any one or combination of CMs described herein (e.g., CM1 and CM2; CM1, CM2, and CM3; CM1, CM2, CM3, and CM4; CM1, CM2, CM3, CM4, and CM5; or CM1, CM2, CM3, CM4, CM5, and CM6) independently comprise a total of about 3 amino acids to about 25 amino acids. In some aspects, the CM may comprise a total of about 3 amino acids to about 25 amino acids, about 3 amino acids to about 20 amino acids, about 3 amino acids to about 15 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 5 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, or about 20 amino acids to about 25 amino acids. In some aspects, each of the CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a total of about 3 amino acids to about 15 amino acids.
In some aspects, any one or combination of CMs described herein (e.g., CM1 and CM2; CM1, CM2, and CM3; CM1, CM2, CM3, and CM4; CM1, CM2, CM3, CM4, and CM5; or CM1, CM2, CM3, CM4, CM5, and CM6) independently comprises a substrate for a protease selected from the group consisting of ADAMS, AD AMTS, ADAM8, ADAM9, ADAMI 0, ADAM12, ADAM15, ADAM17/TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, Aspartate proteases, BACE, Renin, Aspartic cathepsins, Cathepsin D, Cathepsin E, Caspases, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cysteine cathepsins, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin VZL2, Cathepsin X/Z/P, Cysteine constructases, Cruzipain, Legumain, Otubain-2, KLKs, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Metallo proteinases, Meprin, Neprilysin, PSMA, BMP-1, MMPs, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, Serine proteases, activated protein C , Cathepsin A, Cathepsin G, Chymase, coagulation factor proteases, FVIIa, FIXa, FXa, FXIa, FXIIa, Elastase, Granzyme B, Guanidinobenzoatase, HtrAl, Human Neutrophil Elastase, Lactoferrin, Marapsin, NS3/4A, PACE4, Plasmin, PSA, tPA, Thrombin, Tryptase, uPA, Type II Transmembrane, Serine Proteases, TTSPs, DESCI, DPP-4, FAP, Hepsin, Matriptase-2, MT-SPl/Matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.
Increased levels of proteases having known substrates have been reported in a number of cancers. See, e.g., La Roca et al., British J. Cancer 90(7): 1414-1421, 2004. Substrates suitable for use in the CMs components employed herein include those which are more prevalently found in cancerous cells and tissue. Thus, in certain embodiments, CMs each independently comprise a substrate for a protease that is more prevalently found in diseased tissue associated with a cancer. In some aspects, the cancer is selected from the group of: gastric cancer, breast cancer, osteosarcoma, and esophageal cancer. In some aspects, the cancer is breast cancer. In some aspects, the cancer is a HER2-positive cancer. In some aspects, the cancer is Kaposi sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell cancer (RCC), melanoma, neuroblastoma, basal cell carcinoma, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, breast cancer, ovarian cancer, bladder cancer, BCG-resistant non-muscle invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck carcinoma, uterine cancer, cervical cancer, or testicular cancer, and the like. In some of the above-described embodiments, the CM components comprise substrates for protease(s) that is/are more prevalent in tumor tissue
In some aspects, any one or combination of CMs described herein (e.g., CM1 and CM2; CM1, CM2, and CM3; CM1, CM2, CM3, and CM4; CM1, CM2, CM3, CM4, and CM5; or CM1, CM2, CM3, CM4, CM5, and CM6) each independently comprises a sequence selected from the group consisting of sequences listed in Table 13.
In some aspects, any one or combination of CMs described herein (e.g., CM1 and CM2; CM1, CM2, and CM3; CM1, CM2, CM3, and CM4; CM1, CM2, CM3, CM4, and CM5; or CM1, CM2, CM3, CM4, CM5, and CM6) each independently comprises a sequence selected from the group consisting of: LSGRSDNH (SEQ ID NO: 5), TGRGPSWV (SEQ ID NO: 6), PLTGRSGG (SEQ ID NO: 7), TARGPSFK (SEQ ID NO: 8), NTLSGRSENHSG (SEQ ID NO: 9), NTLSGRSGNHGS (SEQ ID NO: 10), TSTSGRSANPRG (SEQ ID NO: 11), TSGRSANP (SEQ ID NO: 12), VHMPLGFLGP (SEQ ID NO: 13), AVGLLAPP (SEQ ID NO: 14), AQNLLGMV (SEQ ID NO: 15), QNQALRMA (SEQ ID NO: 16), LAAPLGLL (SEQ ID NO: 17), STFPFGMF (SEQ ID NO: 18), ISSGLLSS (SEQ ID NO: 19), PAGLWLDP (SEQ ID NO: 20), VAGRSMRP (SEQ ID NO: 21), VVPEGRRS (SEQ ID NO: 22), ILPRSPAF (SEQ ID NO: 23), MVLGRSLL (SEQ ID NO: 24), QGRAITFI (SEQ ID NO: 25), SPRSIMLA (SEQ ID NO: 26), SMLRSMPL (SEQ ID NO: 27), ISSGLLSGRSDNH (SEQ ID NO: 28), AVGLLAPPGGLSGRSDNH (SEQ ID NO: 29), ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 30), LSGRSGNH (SEQ ID NO: 31), SGRSANPRG (SEQ ID NO: 32), LSGRSDDH (SEQ ID NO: 33), LSGRSDIH (SEQ ID NO: 34), LSGRSDQH (SEQ ID NO: 35), LSGRSDTH (SEQ ID NO: 36), LSGRSDYH (SEQ ID NO: 37), LSGRSDNP (SEQ ID NO: 38), LSGRSANP (SEQ ID NO: 39), LSGRSANI (SEQ ID NO: 40), LSGRSDNI (SEQ ID NO: 41), MIAPVAYR (SEQ ID NO: 42), RPSPMWAY (SEQ ID NO: 43), WATPRPMR (SEQ ID NO: 44), FRLLDWQW (SEQ ID NO: 45), ISSGL (SEQ ID NO: 46), ISSGLLS (SEQ ID NO: 47), ISSGLL (SEQ ID NO: 48), ISSGLLSGRSANPRG (SEQ ID NO: 49), AVGLLAPPTSGRSANPRG (SEQ ID NO: 50), AVGLLAPPSGRSANPRG (SEQ ID NO: 51), ISSGLLSGRSDDH (SEQ ID NO: 52), ISSGLLSGRSDIH (SEQ ID NO: 53), ISSGLLSGRSDQH (SEQ ID NO: 54), ISSGLLSGRSDTH (SEQ ID NO: 55), ISSGLLSGRSDYH (SEQ ID NO: 56), ISSGLLSGRSDNP (SEQ ID NO: 57), ISSGLLSGRSANP (SEQ ID NO: 58), ISSGLLSGRSANI (SEQ ID NO: 59), AVGLLAPPGGLSGRSDDH (SEQ ID NO: 60), AVGLLAPPGGLSGRSDIH (SEQ ID NO: 61), AVGLLAPPGGLSGRSDQH (SEQ ID NO: 62), AVGLLAPPGGLSGRSDTH (SEQ ID NO: 63), AVGLLAPPGGLSGRSDYH (SEQ ID NO: 64), AVGLLAPPGGLSGRSDNP (SEQ ID NO: 65), AVGLLAPPGGLSGRSANP (SEQ ID NO: 66), AVGLLAPPGGLSGRSANI (SEQ ID NO: 67), ISSGLLSGRSDNI (SEQ ID NO: 68), AVGLLAPPGGLSGRSDNI (SEQ ID NO: 69), GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 70), GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 71), LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 72), ISSGLSS (SEQ ID NO: 73), PVGYTSSL (SEQ ID NO: 74), DWLYWPGI (SEQ ID NO: 75), LKAAPRWA (SEQ ID NO: 76), GPSHLVLT (SEQ ID NO: 77), LPGGLSPW (SEQ ID NO: 78), MGLFSEAG (SEQ ID NO: 79), SPLPLRVP (SEQ ID NO: 80), RMHLRSLG (SEQ ID NO: 81), LLAPSHRA (SEQ ID NO: 82), GPRSFGL (SEQ ID NO: 83), GPRSFG (SEQ ID NO: 84), SARGPSRW (SEQ ID NO: 85), GGWHTGRN (SEQ ID NO: 86), HTGRSGAL (SEQ ID NO: 87), AARGPAIH (SEQ ID NO: 88), RGPAFNPM (SEQ ID NO: 89), SSRGPAYL (SEQ ID NO: 90), RGPATPIM (SEQ ID NO: 91), RGPA (SEQ ID NO: 92), GGQPSGMWGW (SEQ ID NO: 93), FPRPLGITGL (SEQ ID NO: 94), SPLTGRSG (SEQ ID NO: 95), SAGFSLPA (SEQ ID NO: 96), LAPLGLQRR (SEQ ID NO: 97), SGGPLGVR (SEQ ID NO: 98), PLGL (SEQ ID NO: 99), and SGRSDNI (SEQ ID NO: 100).
In some aspects, any one or combination of CMs described herein (e.g., CM1 and CM2; CM1, CM2, and CM3; CM1, CM2, CM3, and CM4; CM1, CM2, CM3, CM4, and CM5; or CM1, CM2, CM3, CM4, CM5, and CM6) each independently comprises a sequence selected from the group consisting of: ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), ISSGLLSGRSDQH (SEQ ID NO: 54), SGRSDNI (SEQ ID NO: 100), ISSGLLSGRSDNI (SEQ ID NO: 68), ISSGLLSGRSDNP (SEQ ID NO: 57), and PWGLSGRS (SEQ ID NO: 157). Cleavable moieties that are suitable for use in the constructs described herein include truncation variants of any one of the aforementioned amino acid sequences, which retain the recognition site for the corresponding protease. These include C-terminal and/or N-terminal truncation variants comprising at least 3 contiguous amino acids of the above-described amino acid sequences, or at least 4, or at least 5, or at least 6, or at least 7 amino acids of the foregoing amino acid sequences that retain a recognition site for a protease. In certain embodiments, the truncation variant of the above-described amino acid sequences is an amino acid sequence corresponding to any of the above, but that is C- and/or N-terminally truncated by 1 to about 10 amino acids, 1 to about 9 amino acids, 1 to about 8 amino acids, 1 to about 7 amino acids, 1 to about 6 amino acids, 1 to about 5 amino acids, 1 to about 4 amino acids, or 1 to about 3 amino acids, and which: (1) has at least three amino acid residues; and (2) retains a recognition site for a protease. In some of the foregoing embodiments, the truncated CM is an N-terminally truncated CM. In some aspects, the truncated CM is a C-terminally truncated CM. In some aspects, the truncated C is a C- and an N-terminally truncated CM.
In some aspects, the protease(s) is/are those corresponding to upregulated protease activity, e.g., in a tumor microenvironment.
Dimerization Domains
Activatable constructs of the present disclosure may employ any of a variety of dimerization domains. Suitable DDs include both polymeric (e.g., a synthetic polymer, a polypeptide, a polynucleotide, and the like) and small molecule (non-polymeric moieties having a molecular weight of less than about 1 kilodalton, and sometimes less than about 800 daltons) types of moieties. The pair of DDs may be any pair of moieties that are known in the art to bind to each other. Illustrative DDs that are suitable for use in the activatable constructs described herein include, for example any of the DDs disclosed in WO2021207669A1, WO2022197764A2, or WO2024216194A1, which are incorporated by reference herein in their entireties.
In some aspects, the DD1 and the DD2 are members of a pair, or the DD is a pair, selected from the group of: a sushi domain from an alpha chain of human IL- 15 receptor (IL15Ra) and a soluble IL-15; barnase and barnstar; a protein kinase A (PKA) and an A-kinase anchoring protein (AKAP); adapter/docking tag molecules based on mutated RNase I fragments; a pair of antigen-binding domains (e.g., a pair of single domain antibodies); soluble N-ethyl- mal eimide sensitive factor attachment protein receptors (SNARE); modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25; a single domain antibody (sdAb) and corresponding epitope; an antigen-binding domain (e.g., a single chain antibody such as a single chain variable fragment (scFv), a single domain antibody, and the like) and a corresponding epitope; coiled coil polypeptide structure (e g., Fos-Jun coiled coil structures, acid/base coiled-coil helices, Glu-Lys coiled coil helices, leucine zipper structures), small molecule binding pairs such as biotin and avidin or streptavidin, amine/aldehyde, lectin/carbohydrate; a pair of polymers that can bind each other, such as, for example, a pair of sulfur- or thiol -containing polymers (e.g., a pair of Fc domains, a pair of thiolized-human serum albumin polypeptides, and the like); and the like.
In some aspects, the DD, or the DD1 and DD2, are non-polypeptide polymers. The nonpolypeptide polymers may covalently bound to each other. In some examples, the non- polypeptide polymer is a sulfur-containing polymer, e.g., sulfur-containing polyethylene glycol. In such cases, the DD, or the DD1 and DD2, may be covalently bound to each other via one or more disulfide bonds.
When the DD, or the pair of DD1 and DD2, are members of a pair of epitope and antigen-binding domain, the epitope may be a naturally or non-naturally occurring epitope. Exemplary non-naturally occurring epitopes include, for example, a non-naturally occurring peptide, such as, for example, a poly-His peptide (e.g., a His tag, and the like).
In certain specific embodiments, the DD, or the DD1 and the DD2, are a pair of Fc domains. As used herein, an “Fc domain” refers to a contiguous amino acid sequence of a single heavy chain of an immunoglobulin, e.g., the CH2-CH3 domains of IgG, IgA, or IgD, or the CH2-CH3-CH4 domains of IgE or IgM. A pair of Fc domains associate together to form an Fc region of an immunoglobulin.
In some aspects, the pair of Fc domains is a pair of human Fc domains (e.g., a pair of wildtype human Fc domains). In some aspects, the human Fc domains are human IgGl Fc domains (e.g., wildtype human IgGl Fc domains), human IgG2 Fc domains (e.g., wildtype human IgG2 Fc domains), human IgG3 Fc domains (e.g., wildtype human IgG3 Fc domains), or human IgG4 Fc domains (e.g., wildtype human IgG4 Fc domains). In some aspects, the human Fc domains comprise a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 661 . In some aspects, the human Fc domains comprise SEQ ID NO: 661.
In some aspects, the pair of Fc domains comprise a knob mutant and a hole mutant of an Fc domain. The knob and hole mutants may interact with each other to facilitate the dimerization. In some aspects, the knob and hole mutants may comprise one or more amino acid modifications within the interface between two Fc domains (e.g., in the CH3 domain). In one example, the modifications comprise amino acid substitution T366W and optionally the amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other one of the antibody heavy chains (numbering according to EU index of Kabat numbering system). Examples of the knob and hole mutants include Fc mutants of SEQ ID NOs: 663 and 664, as well as those described in U.S. Pat. Nos. 5,731,168; 7,695,936; and 10,683,368, which are incorporated herein by reference in their entireties. In some aspects, the dimerization domains comprise a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NOs: 663 and 664, respectively.
In some aspects, the DD is a homodimer. In some aspects, the DD is a heterodimer. In some aspects, the DD may comprise SEQ ID NOs: 663 and 664. In some aspects, the DD1 and the DD2 may comprise SEQ ID NOs: 663 and 664, respectively. In some aspects, the DD1 and the DD2 are the same. In some aspects, the human Fc domains include mutations to eliminate glycosylation and/or to reduce Fc-gamma receptor binding. In some aspects, the human Fc domains comprise the mutation N297Q, N297A, or N297G; In some aspects the human Fc domains comprise a mutation at position 234 and/or 235, for example L235E, or L234A and L235A (in IgGl), or F234A and L235A (in IgG4); In some aspects the human Fc domains are IgG2 Fc domains that comprise the mutations V234A, G237A, P238S, H268Q/A, V309L, A330S, or P331 S, or a combination thereof (all according to EU numbering).
Additional examples of engineered human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids in which mutations in at least one amino acid leads to reduced Fc function include, but are not limited to, mutations in amino acid 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330, and 331 of the heavy constant region (according to EU numbering). Examples of combinations of mutated amino acids are also known in the art, such as, but not limited to a combination of mutations in amino acids 234, 235, and 331, such as L234F, L235E, and P331S or a combination of amino acids 318, 320, and 322, such as E318A, K320A, and K322A.
Further examples of engineered Fc domains include F243L/R292P/Y300L/V305I/P396 IgGl; S239D/I332E IgGl; S239D/I332E/A330L IgGl; S298A/E333A/K334A; in one heavy chain, L234Y/L235Q/G236W/S239M/H268D/D270E/S298A IgGl, and in the opposing heavy chain, D270E/K326D, A330M/K334E IgG; G236A/S239D/I332E IgGl; K326W/E333S IgGl; S267E/H268F/S324T IgGl; E345R/E430G/S440Y IgGl; N297A or N297Q or N297G IgGl; L235E IgGl; L234A/L235A IgGl; F234A/L235A IgG4; H268Q/V309L/A330S/P331S IgG2; V234A/G237A/P238S/H268A/V309L/A330S/P331S IgG2; M252Y/S254T/T256E IgGl; M428L/N434S IgGl; S267E/L328F IgGl; N325S/L328F IgGl, and the like. In some aspects, the engineered Fc domain comprises one or more substitutions selected from the group consisting of N297A IgGl, N297Q IgGl, and S228P IgG4.
According to any of the aspects disclosed herein, the C-terminal residue of an IgG Fc domain may be a lysine. In some aspects, the C-terminal lysine residue of an IgG Fc domain may be absent.
In some aspects, the DD1 comprises an antigen-binding domain and the DD2 comprises a corresponding epitope. In some aspects, the antigen-binding domain is an anti-His tag antigenbinding domain and wherein the DD2 comprises a His tag. In some aspects, the antigen-binding domain is a single chain variable fragment (scFv). In some aspects, the antigen-binding domain is a single domain antibody (sdAb). In some aspects, at least one of the DD1 and the DD2 comprises a dimerization domain substituent selected from the group consisting of a nonpolypeptide polymer and a small molecule. In some aspects, the DD1 and the DD2 comprise non-polypeptide polymers covalently bound to each other. In some aspects, the non-polypeptide polymer is a sulfur-containing polyethylene glycol, and wherein the DD1 and the DD2 are covalently bound to each other via one or more disulfide bonds. In some aspects, at least one of the DD1 and the DD2 comprises a small molecule. In some aspects, the small molecule is biotin. In some aspects, the DD1 comprises biotin and the DD2 comprises an avidin.
In some aspects, the DD, or the DD1 and/or DD2, can further include a serum half-life extending moiety (e.g., polypeptides that bind serum proteins, such as immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA)). Examples of half-life extending moieties include hexa-hat GST (glutathione S-transferase) glutathione affinity, Calmodulin- binding peptide (CBP), Strep-tag, Cellulose Binding Domain, Maltose Binding Protein, S- Peptide Tag, Chitin Binding Tag, Immuno-reactive Epitopes, Epitope Tags, E2Tag, HA Epitope Tag, Myc Epitope, FLAG Epitope, AU1 and AU5 Epitopes, Glu-Glu Epitope, KT3 Epitope, IRS Epitope, Btag Epitope, Protein Kinase-C Epitope, and VSV Epitope.
In some aspects, the DD, or the DD1 and/or DD2, each include a total of about 5 amino acids to about 250 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids, about 20 amino acids to about 250 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 40 amino acids, about 40 amino acids to about 250 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 250 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 250 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, about 120 amino acids to about 250 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 140 amino acids, about 140 amino acids to about 250 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 160 amino acids, about 160 amino acids to about 250 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 180 amino acids, about 180 amino acids to about 250 amino acids, about 180 amino acids to about 200 amino acids, or about 200 amino acids to about 250 amino acids. In some aspects, DD1 and DD2 are each an Fc domain that comprises a portion of the hinge region that includes two cysteine residues, a CH2 domain, and a CH3 domain. In some aspects, DD1 and DD2 are each an Fc domain whose N-terminus is the first cysteine residue (reading in the N- to C- direction) in the hinge region that participates in a disulfide linkage with a second Fc domain (e.g., Cysteine 226 of human IgGl or IgG4, using EU numbering).
In some aspects, the dimerization domain is an IgG Fc region. In some aspects, the dimerization domain is an IgG Fc region wherein the upper hinge residues have been deleted. For example, the Fc is a variant wherein N-terminal sequences EPKSCDKTHT (SEQ ID NO: 1), ERK, ELKTPLGDTTHT (SEQ ID NO: 2), or ESKYGPP (SEQ ID NO: 3) have been deleted. In some aspects, DD1 is an Fc domain hole mutant and DD2 is an Fc domain knob mutant. In some aspects, DD1 is an Fc domain knob mutant and DD2 is an Fc domain hole mutant. In some aspects, the DD1 and the DD2 are selected from an IgGl Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a mutant or truncation variant thereof. In some aspects, the DD1 and the DD2 are human IgGl Fc domains or a mutant or truncation variant thereof. In some aspects, the N-terminus of the Fc region is the first cysteine residue (reading in the N- to C- direction) in the hinge region that participates in a disulfide linkage with a second Fc domain (e.g., Cysteine 226 of human IgGl or IgG4, using EU numbering), and wherein any linker(s) interposed between the AB1 and the N-terminal cysteine of DD1 (the “linking region”) and any linker(s) interposed between the AB2 and the N-terminal cysteine of DD2 have a combined total length of no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids, preferably no more than 10 amino acids, especially preferably no more than 7 amino acids.
In some aspects, a spacer (or header) comprises one or more glutamine (Q) residues. In some aspects, residues in the spacer minimize aminopeptidase and/or exopeptidase action to prevent cleavage of N-terminal amino acids. Illustrative and non-limiting spacer amino acid sequences may comprise or consist of any of the following exemplary amino acid sequences: QGQSGS (SEQ ID NO: 668); GQSGS (SEQ ID NO: 669); QSGS (SEQ ID NO: 670); SGS; GS; S; QGQSGQG (SEQ ID NO: 671); GQSGQG (SEQ ID NO: 672); QSGQG (SEQ ID NO: 673); SGQG (SEQ ID NO: 674); GQG; QG; G; QGQSGQ (SEQ ID NO: 675); GQSGQ (SEQ ID NO: 676); QSGQ (SEQ ID NO: 677); QGQSG (SEQ ID NO: 678); QGQS (SEQ ID NO: 679); SGQ; GQ; and Q. In some aspects, spacer sequences are omitted.
In some aspects, each of DD1 and DD2 independently comprises a sequence that is at least 80% identical, at least 90% identical, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from:
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPP VLD SDGSFFLVSRLTVDKSRWQEGNVF SC S VMHEALHNHYTQKSL SL SLGX (SEQ ID NO: 655), wherein X is lysine or absent;
GSSKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGX (SEQ ID NO: 656), wherein X is lysine or absent;
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ S SGL YSLS S VVT VPS S SLGTQT YICNVNHKP SNTK VDKK VEPKSCDKTHTCPPCP AP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALP APIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX (SEQ ID NO: 657), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPV AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX (SEQ ID NO: 658), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCP EPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGF YPSDIAVEWES SGQPENNYNTTPPMLD SDGSFFLYSKLTVDKSRWQQGNIF S CSVMHEALHNRFTQKSLSLSPGX (SEQ ID NO: 659), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQ S SGL YSLS S VVTVP S S SLGTKTYTCNVDHKP SNTKVDKRVESK YGPPCP SCP APEFL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSX1X2X3 (SEQ ID NO: 660), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent;
CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX1X2X3 (SEQ ID NO: 661), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent; or
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX1X2X3 (SEQ ID NO: 662), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent.
Linkers
In some aspects, one or more linkers (e.g., flexible linkers) are introduced into the activatable construct to provide flexibility at one or more of the junctions between domains, between moi eties, between moi eties and domains, or at any other junctions where a linker would be beneficial. In some aspects, where the activatable constructs is provided as a conformationally constrained construct, a flexible linker is inserted to facilitate formation and maintenance of a structure in the uncleaved construct. Any of the linkers described herein can provide the desired flexibility to facilitate the inhibition of the binding of a target, or to facilitate cleavage of a CM by a protease. In some aspects, linkers are included in the activatable constructs that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired activatable constructs. Some linkers may include cysteine residues, which may form disulfide bonds and reduce flexibility of the construct. It has been found that reducing the length of the linkers or linking region reduces the activity of the antigen binding domain protein in activatable constructs. In most instances, linker length is determined by counting, in a N- to C- direction, the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the preceding component, to the C-terminus of the linker adjacent to the N-terminal amino acid of the following component (i.e., where the linker length does not include either the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component). In embodiments in which a linker is employed at the N-terminus of a DD that comprises an Fc domain, linker length is determined by counting the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the preceding component to C-terminus of the linker adjacent to the first cysteine of an Fc hinge region that participates in the disulfide linkage with a second Fc domain (i.e., where the linker length does not include the C-terminal amino acid of the preceding component or the first cysteine of the Fc hinge region).
In some aspects, the linker can include a total of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 24 amino acids, about 1 amino acid to about 22 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 18 amino acids, about 1 amino acid to about 16 amino acids, about 1 amino acid to about 15 amino acids, about 1 amino acid to about 14 amino acids, about 1 amino acid to about 12 amino acids, about 1 amino acid to about 10 amino acids, about 1 amino acid to about 8 amino acids, about 1 amino acid to about 6 amino acids, about 1 amino acid to about 5 amino acids, about 1 amino acid to about 4 amino acids, about 1 amino acid to about 3 amino acids, about 1 amino acid to about 2 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 2 amino acids to about 3 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 4 amino acids to about 5 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 24 amino acids, about 5 amino acids to about 22 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 18 amino acids, about 5 amino acids to about 16 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 14 amino acids, about 5 amino acids to about 12 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 6 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 15 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about 16 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 amino acids to about 22 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10 amino acids to about 16 amino acids, about 10 amino acids to about 15 amino acids, about 10 amino acids to about 14 amino acids, about 10 amino acids to about 12 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 15 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 14 amino acids to about 15 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 24 amino acids, about 15 amino acids to about 22 amino acids, about 15 amino acids to about 20 amino acids, about 15 amino acids to about 18 amino acids, about 15 amino acids to about 16 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acid to about 25 amino acids, about 22 amino acids to about 24 amino acids, or about 24 amino acid to about 25 amino acids). In some aspects, the linker includes a total of about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, or about 25 amino acids. In some aspects, activatable constructs that do not comprise any linkers.
In some aspects of any of the activatable constructs described herein, a linker is rich in glycine (Gly or G) residues. In some aspects, the linker is rich in serine (Ser or S) residues. In some aspects, the linker is rich in glycine and serine residues. In some aspects, the linker has one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs). In some aspects, the linker has one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 622) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences). In some aspects, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 629) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences). In some aspects, the linker has one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 642) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences).
In some aspects, the activatable construct further comprises one or more linkers. In some aspects, the linker is a peptide having a length of 1 to 30, 5 to 25, 10-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some aspects, the linker comprises at least 5 amino acids.
In some aspects, a linker, or each linker, is independently selected from the group consisting of: GSSGGSGGSGG (SEQ ID NO: 623); GGGS (SEQ ID NO: 622); GGGSGGGS (SEQ ID NO: 624); GGGSGGGSGGGS (SEQ ID NO: 625); GGGGSGGGGSGGGGS (SEQ ID NO: 626); GGGGS GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 627); GGGGSGGGGS (SEQ ID NO: 628); GGGGS (SEQ ID NO: 629); GS; GGGGSGS (SEQ ID NO: 630); GGGGSGGGGSGGGGSGS (SEQ ID NO: 631); GGSLDPKGGGGS (SEQ ID NO: 632); PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 633); SKYGPPCPPCPAPEFLG (SEQ ID NO: 634); GKSSGSGSESKS (SEQ ID NO: 635); GSTSGSGKSSEGKG (SEQ ID NO: 636); GSTSGSGKSSEGSGSTKG (SEQ ID NO: 637); GSTSGSGKPGSGEGSTKG (SEQ ID NO: 638); GSTSGSGKPGSSEGST (SEQ ID NO: 639); (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 640), (GGGS)n (SEQ ID NO: 641), (GGGGS)n (SEQ ID NO: 629), wherein each n is an integer of at least one; GGSG (SEQ ID NO: 642); GGSGG (SEQ ID NO: 643); GSGSG (SEQ ID NO: 644); GSGGG (SEQ ID NO: 645); GGGSG (SEQ ID NO: 646); GSSSG (SEQ ID NO: 647); GGGGSGGGGSGGGGS (SEQ ID NO: 626); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 654); and GSTSGSGKPGSSEGST (SEQ ID NO: 639).
Non-limiting examples of linkers can include a sequence that is at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to GGGS (SEQ ID NO: 622), GSSGGSGGSGG (SEQ ID NO: 623), GGGGSGGGGSGGGGS (SEQ ID NO: 626), GGGGSGS (SEQ ID NO: 630), GGGGSGGGGSGGGGSGS (SEQ ID NO:
631), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 654), GGSLDPKGGGGS (SEQ ID NO:
632), and GSTSGSGKPGSSEGST (SEQ ID NO: 639). In some aspects, the linker comprises a sequence of GGGS (SEQ ID NO: 622).
In some aspects, an activatable construct includes one, two, three, four, five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences of any of the exemplary linker sequences described herein or known in the art). In some aspects, a linker comprises sulfo-SIAB, SMPB, and sulfo-SMPB, wherein the linkers react with primary amines sulfhydryls.
Exemplary Activatable Construct Polypeptides
Polypeptides encoding the activatable constructs of the present disclosure can be readily constructed from the component parts as described herein. Activatable constructs may be readily designed in accordance with the structures described herein. While the present disclosure contains exemplary figures, sequences, and working examples, those examples are provided for illustrative purposes and are non-limiting. Those skilled in the art will recognize that the component parts described herein are suitable for assembly into various activatable construct structures.
In some aspects, the activatable construct has a structure as shown in Figures 1A-1C. The following are illustrative sequences:
In some aspects, the activatable construct comprises an anti-integrin antigen binding domain. In some aspects, the first polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLS AS VGDRVTITC S ASS SIS SNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRF SGSGSGTD Y TLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSS LSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700).
In some aspects, the second polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683); QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQ VSLTCL VKGF YP SDIA VEWE SNGQPENNYKTTPPVLD SDGSFFL YSKLT VDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS SAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
In some aspects, the third polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE
VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA
TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYY AMD YWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
In some aspects, the fourth polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL S AS VGDRVTITC SAS S SIS SNYLHWYQQKPGK VPKLLIYRTSNL ASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLS ASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDY TLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSS LSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SAS VGDRVTITC SAS S SIS SNYLHWYQQKPGK VPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700).
In some aspects, the light chain and the heavy chain of the first and second polypeptide, respectively, comprise the same MM. In some aspects, the first polypeptide comprises an amino acid sequence of:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694); and the second polypeptide comprising an amino acid sequence of:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683).
In some aspects, the first polypeptide comprises an amino acid sequence of:
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPS SLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGT DYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696); and the second polypeptide comprises an amino acid sequence of:
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685).
In some aspects, the first polypeptide comprises an amino acid sequence of:
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP
SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG
TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); and the second polypeptide comprises an amino acid sequence of:
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687).
In some aspects, the first polypeptide comprising an amino acid sequence of: QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLSAS VGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDYTL TISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700); and the second polypeptide comprises an amino acid sequence of:
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TIT ADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQ VSLTCL VKGF YP SDIA VEWE SNGQPENNYKTTPPVLD SDGSFFL YSKLT VDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
In some aspects, the light chain and the heavy chain of the fourth and third polypeptide, respectively, comprise the same MM. In some aspects, the fourth polypeptide comprises an amino acid sequence of: QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694); and the third polypeptide comprising an amino acid sequence of:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683).
In some aspects, the fourth polypeptide comprises an amino acid sequence of:
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPS SLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGT DYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696); and the third polypeptide comprises an amino acid sequence of:
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685). In some aspects, the fourth polypeptide comprises an amino acid sequence of:
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); and the third polypeptide comprises an amino acid sequence of:
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687).
In some aspects, the fourth polypeptide comprising an amino acid sequence of:
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700); and the third polypeptide comprises an amino acid sequence of:
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGP S VFPL AP S SK ST SGGT AALGCL VKD YFPEP VT VS WNSGALTSGVHTFP A VLQ S SGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
As a non-limiting example, the first and second polypeptide together with the third and fourth polypeptide may form an activatable construct according to Figure 1C.
In some aspects, the activatable construct comprises an anti-EGFR antigen binding domain.
In some aspects, the first polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS
VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVIL SVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLS INSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714).
In some aspects, the second polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL
VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQD WLNGKEYKCK VSNK ALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGP GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
In some aspects, the third polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL
VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQD WLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGP GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VS VLTVLHQD WLNGKEYKCK VSNKALP APIEKTISKAKGQPREPQ VYTLPP SREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
In some aspects, the fourth polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVIL
SVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLS INSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714).
In some aspects, the light chain and the heavy chain of the first and second polypeptide, respectively, comprise the same MM. In some aspects, the first polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); and the second polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708).
In some aspects, the first polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVS PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and the second polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQD WLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
In some aspects, the first polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSP VILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDF TLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and the second polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQD WLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708).
In some aspects, the first polypeptide comprises an amino acid sequence of: QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVSP GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSV ESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); and the second polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV
QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQD WLNGKEYKCK VSNK ALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
In some aspects, the light chain and the heavy chain of the fourth and third polypeptide, respectively, comprise the same MM. In some aspects, the fourth polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); and the third polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQD WLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708).
In some aspects, the fourth polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVS PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNF YPRE AK VQWKVDNALQ SGNSQE S VTEQD SKD STYSL S STLTL SKAD YEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and the third polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
In some aspects, the fourth polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSP VILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDF TLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and the third polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VL TVLHQD WLNGKE YKCKV SNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708).
In some aspects, the fourth polypeptide comprising an amino acid sequence of: QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVSP GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSV ESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAI<VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEI<HI<VYACE VTHQGLSSPVTKSFNRGEC (SEQ TD NO: 710); and the third polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSN ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
As a non-limiting example, the first and second polypeptide together with the third and fourth polypeptide may form an activatable construct according to Fig. 1C.
In some aspects, the activatable construct has a structure as shown in Figs. 4-5. In some aspects, the activatable construct comprises an anti-HER2 antigen binding domain (trastuzamab). The following are illustrative sequences:
In some aspects, the first polypeptide comprises an amino acid sequence of:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGS
EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNN YATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAY WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTS NYVNWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYC VLWYSNRWVFGGGTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGS
QGQSGSGYLWGCEWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG (SEQ ID NO: 800); the second polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 804); and the third polypeptide comprises an amino acid sequence of:
QGQSGQGYLWGCEWNCGGITTGGGSSGGSLSGRSDNIGGGGSGASGSSGASGT GTAGGTGSGSGTGSGDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPASIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVE WE SNGQPENNYKTTPP VLD SDGSFFL YSKLTVDKSRWQQGNVF SC S VMHEALHNHYT QKSLSLSPG (SEQ ID NO: 802). An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 4A.
In some aspects, the first polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNT AYLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTL VT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWW A YWGQGTL VTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYVNWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCVLWYSNRWVFGG GTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGSQGQSGSGYLWGC EWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 806); the second polypeptide comprises an amino acid sequence of: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGGGSLSGRSDNIGGGSSGGSQG QSGSGYLWGCEWNCGGITT (SEQ ID NO: 810); and the third polypeptide comprises an amino acid sequence of:
DSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 808). An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 4B.
In some aspects, the first polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNT A YLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTL VT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYVNWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCVLWYSNRWVFGG GTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGSQGQSGSGYLWGC EWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 806); the second polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 804) ; and the third polypeptide comprises an amino acid sequence of: QGQSGQGYLWGCEWNCGGITTGGGSSGGSLSGRSDNIGGGGSGASGSSGASGTGTAGG TGSGSGTGSGDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPASIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G (SEQ ID NO: 802). An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 5A.
In some aspects, the first polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNTAYLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTLVT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYVNWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCVLWYSNRWVFGG GTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGSQGQSGSGYLWGC EWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 806); the second polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECGSGGGSLSGRSDNIGGGSSGGSQGQSGSGYLWGCE WNCGGITT (SEQ ID NO: 812); and the third polypeptide comprises an amino acid sequence of:
DSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISK AKGQPREPQ VYTLPPCRDELTKNQ VSLWCL VKGF YP SDIA VEWE SNGQPENN YKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 808). An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 5B.
In some aspects, the first polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNTA YLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTL VT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWW A YWGQGTL VTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYVNWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCVLWYSNRWVFGG GTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGSQGQSGSGYLWGC EWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 806); the second polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 804); and the third polypeptide comprises an amino acid sequence of: DSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISK AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 808). An exemplary depiction of an activatable construct formed by this aspect is provided in Fig. 5C
In some aspects, the activatable construct has reduced level of activity for at least one of AB1, AB2, AB3, AB4, or any combination thereof, for example, activity is characterized by a masking efficiency that is greater than 1.5, 2, 5, 6, 7, 8, 9, or 10. In some aspects, the activatable constructs described herein have a masking efficiency that is greater than 10, greater than 100, greater than 1000, or greater than 5000.
In some aspects, the activatable construct has greater than 10-fold, 15-fold, 20-fold, 25- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 5000-fold reduced binding activity than a corresponding antibody that lacks MMs. In some aspects, the activatable construct has a masking efficiency that is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than a corresponding activatable antibody that lacks MM3, MM4, or both MM3 and MM4.
In some aspects, the activatable construct has a masking efficiency that is greater than 3- fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, or 100-fold greater than a corresponding activatable antibody having only a single MM coupled directly or indirectly to the AB1, AB2, AB3, AB4 or any combination thereof. More specifically, in some aspects, the activatable construct has a masking efficiency that is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than a corresponding activatable construct with only a single masking moiety per antigen binding domain.
In some aspects, the at least one activity is the binding affinity of the AB1 and/or the AB2 for its antigen (i.e., binding partner) as determined using surface plasmon resonance (e.g., performed in phosphate buffered saline at 25 degrees Celsius).
In some aspects, the amino acid sequence further comprises an amino acid sequence that encodes a signal peptide, e g., MRAWIFFLLCLAGRALA (SEQ ID: NO 665); MALTFALLVALLVLSCKSSCSVG (SEQ ID NO: 666), METDTLLLWVLLLWVPGSTG (SEQ ID NO: 667), or that encodes a signal peptide known in the art.
The present disclosure further includes modifications to the amino acid sequence of the polypeptides described herein or the nucleotide sequences encoding the polypeptides described herein by techniques including but not limited to site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with acidic side chains (e.g., aspartate and glutamate), amino acids with basic side chains (e.g., lysine, arginine, and histidine), non-polar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include: aliphatic-hydroxy amino acids (e.g., serine and threonine), amide family (e.g., asparagine and glutamine), aliphatic family (e.g., alanine, valine, leucine and isoleucine), and aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).
Various exemplary aspects of these activatable constructs are described below and can be used in any combination in the methods provided herein without limitation. Exemplary aspects of the activatable constructs and methods of making activatable constructs are described herein. Conjugation to Agents
This disclosure also provides methods and materials for including additional elements in any of the activatable constructs described herein including, for example, a targeting moiety to facilitate delivery to a cell or tissue of interest, an agent (e.g., a therapeutic agent, an antineoplastic agent), a toxin, or a fragment thereof.
In some aspects of any of the activatable constructs described herein, the activatable constructs are conjugated to a cytotoxic agent, including, without limitation, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) or a radioactive isotope. Non-limiting exemplary cytotoxic agents that can be conjugated to any of the activatable constructs described herein include: dolastatins and derivatives thereof (e.g., auri statin E, AFP, monomethyl auri statin D (MMAD), monomethyl auri statin F (MMAF), monomethyl auristatin E (MMAE), desmethyl auristatin E (DMAE), auristatin F, desmethyl auristatin F (DMAE), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycin, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocomstatin, pyrrolobenzimidazoles (PBI), cibrostatin6, doxaliform, cemadotin analogue (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonase toxin A (ZZ-PE38) variant, ZJ-101, anthracycline, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted campothecin, 10, 11 -difluoromethylenedi oxy camptothecin, combretastatins, debromoaplysiatoxin, KahaMide- F, discodermolide, and Ecteinascidins.
Non-limiting exemplary enzymatically active toxins that can be conjugated to any of the activatable constructs described herein include: diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii proteins, dianfhin proteins, Phytoiaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geionin, mitogeliin, restrictocin, phenomycin, neomycin, and tricothecenes.
Non-limiting exemplary anti -neoplasties that can be conjugated to any of the activatable constructs described herein include: adriamycin, cerubidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabizine, and cytarabine.
Non-limiting exemplary antivirals that can be conjugated to any of the activatable constructs described herein include: acyclovir, vira A, and Symmetrel.
Non-limiting exemplary antifungals that can be conjugated to any of the activatable constructs described herein include: nystatin.
Non-limiting exemplary conjugatable detection reagents that can be conjugated to any of the activatable constructs described herein include: fluorescein and derivatives thereof, fluorescein isothiocyanate (FITC).
Non-limiting exemplary antibacterials that can be conjugated to any of the activatable constructs described herein include: aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin. Non-limiting exemplary 3beta, 16beta, 17alpha-trihydroxycholest-5-en-22-one 16-O-(2-O- 4-methoxybenzoyl-beta-D-xylopyranosyl)-(l — >3)-(2-O-acetyl-alpha-L-arabinopyranoside) (OSW-1) that can be conjugated to any of the activatable constructs described herein include: s- nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, toposisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrol obenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepenes, calcicheamicins, podophyiitoxins, taxanes, and vinca alkoids.
Non-limiting exemplary radiopharmaceuticals that can be conjugated to any of the activatable constructs described herein include: 123I , 89Zr, 1251, 131I, "mTc, 2O1T1, 62Cu, 18F, 68Ga, 13 N, 150, 38K, 82Rb, inIn, 133Xe, nC, and "mTc (Technetium).
Non-limiting exemplary heavy metals that can be conjugated to any of the activatable constructs described herein include: barium, gold, and platinum.
Non-limiting exemplary anti-mycoplasmals that can be conjugated to any of the activatable constructs described herein include: tylosine, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.
Those of ordinary skill in the art will recognize that a large variety of possible moieties can be conjugated to any of the activatable constructs described herein. Conjugation can include any chemical reaction that will bind the two molecules so long as the activatable constructs and the other moiety retain their respective activities. Conjugation can include many chemical mechanisms, e g., covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In some aspects, the preferred binding is covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in conjugating any of the activatable constructs described herein. For example, conjugation can include organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzenes, and hexamethylene diamines. In some aspects, the activatable construct can include, or otherwise introduce, one or more non-natural amino acid residues to provide suitable sites for conjugation.
In some aspects of any of the activatable constructs described herein, an agent and/or conjugate is attached by disulfide bonds (e.g., disulfide bonds on a cysteine molecule) to the antigen-binding domain. Since many cancers naturally release high levels of glutathione, a reducing agent, glutathione present in the cancerous tissue microenvironment can reduce the disulfide bonds, and subsequently release the agent and/or the conjugate at the site of delivery.
In some aspects of any of the activatable constructs described herein, when the conjugate binds to its target in the presence of complement within the target site (e.g., diseased tissue (e.g., cancerous tissue)), the amide or ester bond attaching the conjugate and/or agent to the linker is cleaved, resulting in the release of the conjugate and/or agent in its active form. These conjugates and/or agents when administered to a subject, will accomplish delivery and release of the conjugate and/or the agent at the target site (e.g., diseased tissue (e.g., cancerous tissue)). These conjugates and/or agents are particularly effective for the in vivo delivery of any of the conjugates and/or agents described herein.
In some aspects, the linker is not cleavable by enzymes of the complement system. For example, the conjugate and/or agent is released without complement activation since complement activation ultimately lyses the target cell. In such embodiments, the conjugate and/or agent is to be delivered to the target cell (e.g., hormones, enzymes, corticosteroids, neurotransmitters, or genes). Furthermore, the linker is mildly susceptible to cleavage by serum proteases, and the conjugate and/or agent is released slowly at the target site.
In some aspects of any of the activatable constructs described herein, the conjugate and/or agent is designed such that the conjugate and/or agent is delivered to the target site (e.g., disease tissue (e.g., cancerous tissue)) but the conjugate and/or agent is not released.
In some aspects of any of the activatable constructs described herein, the conjugate and/or agent is attached to an antigen-binding domain either directly or via a non-cleavable linker. Exemplary non-cleavable linkers include amino acids (e.g., D-amino acids), peptides, or other organic compounds that may be modified to include functional groups that can subsequently be utilized in attachment to antigen-binding domains by methods described herein.
In some aspects of any of the activatable constructs described herein, an activatable construct includes at least one point of conjugation for an agent. In some aspects, all possible points of conjugation are available for conjugation to an agent. In some aspects, the one or more points of conjugation include, without limitation, sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not sulfur atoms involved in intrachain disulfide bonds,, and/or sulfur atoms of cysteine or other amino acid residues containing a sulfur atom. In such cases, residues may occur naturally in the protein construct structure or may be incorporated into the protein construct using methods including, without limitation, site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.
This disclosure also provides methods and materials for preparing an activatable construct for conjugation. In some aspects, any of the activatable constructs described herein are modified to include one or more interchain disulfide bonds. For example, disulfide bonds in the activatable constructs can undergo reduction following exposure to a reducing agent such as, without limitation, TCEP, DTT, or P-mercaptoethanol. In some cases, the reduction of the disulfide bonds is only partial. As used herein, the term partial reduction refers to situations where an activatable construct is contacted with a reducing agent and a fraction of all possible sites of conjugation undergo reduction (e.g., not all disulfide bonds are reduced). In some aspects, an activatable construct is partially reduced following contact with a reducing agent if less than 99%, (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less than 5%) of all possible sites of conjugation are reduced. In some aspects, the activatable constructs having a reduction in one or more interchain disulfide bonds is conjugated to a drug reactive with free thiols.
This disclosure also provides methods and materials for conjugating a therapeutic agent to a particular location on an activatable construct. In some aspects, any of the activatable constructs described herein are modified so that the therapeutic agents can be conjugated to the activatable constructs at particular locations. For example, an activatable construct can be partially reduced in a manner that facilitates conjugation. In such cases, partial reduction of the activatable constructs occurs in a manner that conjugation sites in the activatable constructs are not reduced. In some aspects, the conjugation site(s) are selected to facilitate conjugation of an agent at a particular location on the protein construct. Various factors can influence the “level of reduction” of the activatable constructs upon treatment with a reducing agent. For example, without limitation, the ratio of reducing agent to activatable constructs, length of incubation, incubation temperature, and/or pH of the reducing reaction solution can require optimization in order to achieve partial reduction of the activatable constructs with the methods and materials described herein. Any appropriate combination of factors (e.g., ratio of reducing agent to activatable constructs, the length and temperature of incubation with reducing agent, and/or pH of reducing agent) can be used to achieve partial reduction of the activatable constructs (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).
An effective ratio of reducing agent to activatable constructs can be any ratio that at least partially reduces the activatable constructs in a manner that allows conjugation to an agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites). In some aspects, the ratio of reducing agent to activatable constructs will be in a range from about 20: 1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8: 1 to 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to 1:1.5, from about4:l to 1:1.5, from about 3:1 to 1:1.5, from about2:l to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some aspects, the ratio is in a range of from about 5:1 to 1:1. In some aspects, the ratio is in a range of from about 5:1 to 1.5:1. In some aspects, the ratio is in a range of from about 4: 1 to 1 : 1. In some aspects, the ratio is in a range from about 4:1 to 1.5:1. In some aspects, the ratio is in a range from about 8: 1 to about 1:1. In some aspects, the ratio is in a range of from about 2.5:1 to 1:1.
An effective incubation time and temperature for treating an activatable construct with a reducing agent can be any time and temperature that at least partially reduces the activatable constructs in a manner that allows conjugation of an agent to an activatable construct (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites). In some aspects, the incubation time and temperature for treating an activatable construct will be in a range from about 1 hour at 37 °C to about 12 hours at 37 °C (or any subranges therein).
An effective pH for a reduction reaction for treating an activatable construct with a reducing agent can be any pH that at least partially reduces the activatable constructs in a manner that allows conjugation to an agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).
When a partially-reduced activatable constructs is contacted with an agent containing thiols, the agent can conjugate to the interchain thiols in the activatable constructs. An agent can be modified in a manner to include thiols using a thiol-containing reagent (e.g., cysteine or N- acetyl cysteine). For example, the activatable constructs can be partially reduced following incubation with reducing agent (e.g., TCEP) for about 1 hour at about 37 °C at a desired ratio of reducing agent to activatable constructs. An effective ratio of reducing agent to activatable constructs can be any ratio that partially reduces at least two interchain disulfide bonds located in the activatable constructs in a manner that allows conjugation of a thiol-containing agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).
In some aspects of any of the activatable constructs described herein, an activatable construct is reduced by a reducing agent in a manner that avoids reducing any intrachain disulfide bonds. In some aspects, an activatable construct is reduced by a reducing agent in a manner that avoids reducing any intrachain disulfide bonds and reduces at least one interchain disulfide bond.
In some aspects, the activatable constructs can also include an agent conjugated to the activatable constructs. In some aspects, the conjugated agent is a therapeutic agent.
In some aspects, the agent (e.g., agent conjugated to an activatable construct) is a detectable moiety such as, for example, a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and/or one or more chemiluminescent agents. In some aspects, detectable moieties are attached by spacer molecules.
In some aspects, the agent (e.g., cytotoxic agent conjugated to an activatable construct) is linked to the activatable constructs using a carbohydrate moiety, sulfhydryl group, amino group, or carboxylate group.
In some aspects, the agent (e.g., cytotoxic agent conjugated to an activatable construct) is conjugated to the activatable constructs via a linker and/or a CM (also referred to as a cleavable sequence). In some aspects, the agent (e.g., cytotoxic agent conjugated to an activatable construct) is conjugated to a cysteine or a lysine in the activatable constructs. In some aspects, the agent (e.g., cytotoxic agent conjugated to an activatable construct) is conjugated to another residue of the activatable constructs, such as those residues disclosed herein. In some aspects, the linker is a thiol-containing linker. Some non-limiting examples of the linker and/or CMs are provided in Table 2.
Table 2. Types of Cleavable Sequences/CMs Amino Acid Sequence
Plasmin CMs
Pro-urokinase PRFKIIGG (SEQ ID NO: 116) PRFR1IGG (SEQ ID NO: 117)
TGFP SSRHRRALD (SEQ ID NO: 118)
Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 119)
Staphylokinase SSSFDKGKYKKGDDA (SEQ ID NO: 120) SSSFDKGKYKRGDDA (SEQ ID NO: 121)
Factor Xa CMs IEGR (SEQ ID NO: 122) IDGR (SEQ ID NO: 123)
GGSIDGR (SEQ ID NO: 124)
MMP CMs
Gelatinase A PLGLWA (SEQ ID NO: 125)
Collagenase CMs
Calf skin collagen (al (I) chain) GPQGIAGQ (SEQ ID NO: 126)
Calf skin collagen (a2(I) chain) GPQGLLGA (SEQ ID NO: 127)
Bovine cartilage collagen (al(II) chain) GIAGQ (SEQ ID NO: 128)
Human liver collagen (al (III) chain) GPLGIAGI (SEQ ID NO: 129)
Human a M GPEGLRVG (SEQ ID NO: 130)
Human PZP YGAGLGVV (SEQ ID NO: 131) AGLGVVER (SEQ ID NO: 132)
AGLGISST (SEQ ID NO: 133)
Rat aiM EPQ ALAMS (SEQ ID NO: 134) QALAMSAI (SEQ ID NO: 135)
Rat a2M AAYHLVSQ (SEQ ID NO: 136) MDAFLESS (SEQ ID NO: 137)
Rat ail3(2J) ESLPVVAV (SEQ ID NO: 138)
Rat ail3(27J) SAPAVESE (SEQ ID NO: 139)
Human fibroblast collagenase DVAQFVLT (SEQ ID NO: 140) (autolytic cleavages) VAQFVLT (SEQ ID NO: 141)
VAQFVLTE (SEQ ID NO: 142)
AQFVLTEG (SEQ ID NO: 143)
PVQPIGPQ (SEQ ID NO: 144)
Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the activatable constructs of the disclosure. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). In general, an effective conjugation of an agent (e.g., cytotoxic agent) to an activatable construct can be accomplished by any chemical reaction that will bind the agent to the activatable constructs while also allowing the agent and the activatable constructs to retain functionality.
In some aspects, a variety of bifunctional protein-coupling agents can be used for conjugation including, without limitation, N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e g., bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (e g., bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (e g., tolyene 2,6-diisocyanate), and bisactive fluorine compounds (e.g., l,5-difhioro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). In some aspects, a carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) chelating agent can be used to conjugate a radionucleotide to the activatable constructs. (See, e.g., WO94/11026).
Suitable linkers and CMs are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201 -208 (1984) describing use of MBS (M- maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an activatable construct by way of an oligopeptide linker. In some aspects, suitable linkers include: (i) EDC (1 -ethyl-3-(3- dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl- alpha-methyl-alpha-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat #21651 G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. 2165-G); and (v) sulfo-NHS (N-hydroxy sulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC. Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.
The linkers and CMs described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically-hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
In some aspects, an agent is conjugated to the constructs using a modified amino acid sequence included in the amino acid sequence of the activatable constructs. By inserting conjugation-enabled amino acids at specific locations within the amino acid sequence of the activatable constructs, the protein construct is designed for controlled placement and/or dosage of the conjugated agent (e.g., cytotoxic agent). For example, the activatable constructs can be modified to include a cysteine amino acid residue at positions on the first monomer, the second monomer, the third monomer, and/or the fourth monomer that provide reactive thiol groups and does not negatively impact protein folding and/or assembly and does not alter antigen-binding properties. In some aspects, the activatable constructs can be modified to include one or more non-natural amino acid residues within the amino acid sequence to provide suitable sites for conjugation. In some aspects, the activatable constructs is modified to include enzymatically activatable peptide sequences.
Nucleic Acids
In one aspect, the present disclosure provides a nucleic acid encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein. In some aspects the polypeptide comprises any one of the sequences in Table 16. In one aspect, provided herein is an mRNA comprising the nucleic acid encoding a polypeptide of the activatable construct. In some aspects, the mRNA molecule comprises one or more of the following elements, in the 5'-to-3' direction, (i) a 5 '-cap; (ii) a 5 '-untranslated region (UTR); (iii) an open reading frame encoding a polypeptide or interest or multiple polypeptides of interest, e.g., the activatable construct; (iv) a 3'-UTR; and (v) a poly-A tail. In some aspects, the mRNA comprises at least one modified or non-naturally occurring nucleotide. In a particular embodiment, the at least one modified or non-naturally occurring nucleotide comprises at least one backbone modification, sugar modification or base modification. In a particular embodiment, the at least one modified or non-naturally occurring nucleotide comprises at least one base modification.
Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) that are useful in the vaccines of the present disclosure include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine; 2-methylthio-N6- methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6- glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6- threonylcarbamoyladenosine; l,2'-O-dimethyladenosine; 1 -methyladenosine; 2'-O- methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2'-O- methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis- hydroxyisopentenyl) adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6- hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2- methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1 -methyladenosine; N6, N6 (dimethyl) adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio-adenosine; 2 (amino) adenine; 2 (aminopropyl) adenine; 2 (methylthio) N6 (isopentenyl) adenine; 2-(alkyl) adenine; 2-(aminoalkyl) adenine; 2-(aminopropyl) adenine; 2-(halo) adenine; 2-(halo) adenine; 2-(propyl) adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP; 2' -Deoxy -2' -a- aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6 (alkyl) adenine; 6 (methyl) adenine; 6- (alkyl) adenine; 6-(methyl) adenine; 7 (deaza) adenine; 8 (alkenyl) adenine; 8 (alkynyl) adenine; 8 (amino) adenine; 8 (thioalkyl) adenine; 8-(alkenyl) adenine; 8-(alkyl) adenine; 8-(alkynyl) adenine; 8-(amino) adenine; 8-(halo) adenine; 8-(hydroxyl) adenine; 8-(thioalkyl) adenine; 8- (thiol) adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl) adenine; N6- (isopentyl) adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1 -Deazaadenosine TP; 2'Fluoro- N6-Bz-deoxyadenosine TP; 2'-OMe-2-Amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'- a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2'-a- Trifluoromethyladenosine TP; 2- Azidoadenosine TP; 2'-b-Ethynyladenosine TP; 2- Bromoadenosine TP; 2'-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2',2'- difluoroadenosine TP; 2'-Deoxy-2'-a-mercaptoadenosine TP; 2'-Deoxy-2’-a- thiomethoxyadenosine TP; 2'-Deoxy-2'-b-aminoadenosine TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosine TP; 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b- fluoroadenosine TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine TP; 2'- Deoxy-2'-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2- Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-Trifluoromethyladenosine TP; 3 -Deaza-3 -bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3 -Deaza-3 -fluoroadenosine TP; 3 -Deaza-3 -iodoadenosine TP; 3 -Deazaadenosine TP; 4 '-Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5 '-Homo-adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6- diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6- diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3- methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4- acetylcytidine; 2'-O-methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'- O-methylcytidine; Lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4- methylcytidine; N4,N4-Dimethyl-2'-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; a-thio-cytidine; 2-(thio) cytosine; 2'-Amino-2'-deoxy- CTP; 2'-Azido-2'-deoxy-CTP; 2'-Deoxy-2'-a-aminocytidine TP; 2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza) cytosine; 3 (methyl) cytosine; 3-(alkyl) cytosine; 3-(deaza) 5 (aza) cytosine; 3- (methyl) cytidine; 4,2'-O-dimethylcytidine; 5 (halo) cytosine; 5 (methyl) cytosine; 5 (propynyl) cytosine; 5 (trifluoromethyl) cytosine; 5-(alkyl) cytosine; 5-(alkynyl) cytosine; 5-(halo) cytosine; 5-(propynyl) cytosine; 5-(trifluoromethyl) cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5- propynyl cytosine; 6-(azo) cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl) cytosine; 1-methyl-l-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5- methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-l-methyl- pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-l -methyl- 1-deaza-pseudoisocyti dine; 4- thio-l-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl- zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl) cytidine TP; 2,2'- anhydro-cytidine TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP; 2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP; 2'0-methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP; 2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine TP; 2'-Deoxy-2'-a-mercaptocytidine TP; 2'-Deoxy-2'-a- thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'- Deoxy-2'-b-bromocytidine TP; 2'-Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine TP; 2'-Deoxy-2'-b- thiomethoxycytidine TP; 2'-O-Methyl-5-(l-propynyl) cytidine TP; 3'-Ethynylcytidine TP; 4'- Azidocytidine TP; 4’-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(l-Propynyl) ara- cytidine TP; 5-(2-Chloro-phenyl)-2 -thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5- Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5'- Homo-cytidine TP; 5-Methoxycytidine TP; 5 -Trifluoromethyl -Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2'-O- dimethylguanosine; N2-methylguanosine; Wyosine; l,2'-O-dimethylguanosine; 1- methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O- methylguanosine; 2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7- cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O- trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O- trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; N1 -methylguanosine; a-thio-guanosine; 2 (propyl) guanine; 2-(alkyl) guanine; 2'-Amino-2'-deoxy-GTP; 2'- Azido-2'-deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP; 2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl) guanine; 6-(alkyl) guanine; 6-(methyl) guanine; 6-methyl-guanosine; 7 (alkyl) guanine;
7 (deaza) guanine; 7 (methyl) guanine; 7-(alkyl) guanine; 7-(deaza) guanine; 7-(methyl) guanine;
8 (alkyl) guanine; 8 (alkynyl) guanine; 8 (halo) guanine; 8 (thioalkyl) guanine; 8-(alkenyl) guanine; 8-(alkyl) guanine; 8-(alkynyl) guanine; 8-(amino) guanine; 8-(halo) guanine; 8- (hydroxyl) guanine; 8-(thioalkyl) guanine; 8-(thiol) guanine; aza guanine; deaza guanine; N (methyl) guanine; N-(m ethyl) guanine; l-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6- thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8- aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6- thio-guanosine; 1-Me-GTP; 2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl- guanosine TP; 2'-a-Ethynylguanosine TP; 2 '-a-Trifluorom ethylguanosine TP; 2'-b- Ethynylguanosine TP; 2'-b-Trifluoromethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'-Deoxy-2'-a-mercaptoguanosine TP; 2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b- aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine TP; 2'- Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b- iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP; 5 '-Homoguanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1- methylinosine; Inosine; l,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2'-O- methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2'-O-methyluridine; 2- thiouridine; 3 -methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5- taurinomethyl-2 -thiouridine; 5-taurinomethyluridine; Dihydrouridine; Pseudouridine; (3-(3- amino-3 -carboxypropyl) uridine; l-methyl-3-(3-amino-5-carboxypropyl) pseudouridine; 1- methylpseduouridine; 1-methyl-pseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-O-methyluridine; 3 -(3 -amino-3 -carboxypropyl) uridine; 3,2'-O- dimethyluridine; 3 -Methyl -pseudo-Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl) uridine; 5-(carboxyhydroxymethyl) uridine methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro- uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine; 5- carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5-carboxymethylaminomethyl- 2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5- Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5- methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5- methyl-2 -thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2- thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; Nl-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP; 5-(iso- Pentenylaminomethyl)-2 -thiouridine TP; 5-(iso-Pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso-Pentenylaminomethyl) uridine TP; 5-propynyl uracil; a-thio-uridine; 1 (aminoalkylamino- carbonylethylenyl)-2 (thio)-pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio) pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-4 (thio) pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonylethylenyl)-2 (thio)- pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio) pseudouracil; 1 (aminocarbonylethylenyl)-4 (thio) pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2 (thio)-pseudouracil; 1 substituted 2,4-(dithio) pseudouracil; 1 substituted 4 (thio) pseudouracil; 1 substituted pseudouracil; l-(aminoalkylamino-carbonylethylenyl)-2-(thio)- pseudouracil; l-Methyl-3 -(3 -amino-3 -carboxypropyl) pseudouridine TP; l-Methyl-3-(3-amino- 3 -carboxypropyl) pseudo-UTP; 1-Methyl-pseudo-UTP; 2 (thio) pseudouracil; 2' deoxy uridine; 2' fluorouridine; 2-(thio) uracil; 2,4-(dithio) psuedouracil; 2' methyl, 2'amino, 2'azido, 2'fluro- guanosine; 2'-Amino-2'-deoxy-UTP; 2'-Azido-2'-deoxy-UTP; 2'-Azido-deoxyuridine TP; 2'-O- methylpseudouridine; 2' deoxy uridine; 2' fluorouridine; 2'-Deoxy-2'-a-aminouridine TP; 2'- Deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl) uracil; 4 (thio) pseudouracil; 4-(thio) pseudouracil; 4-(thio) uracil; 4-thiouracil; 5 (1,3-diazole-l-alkyl) uracil; 5 (2-aminopropyl) uracil; 5 (aminoalkyl) uracil; 5 (dimethylaminoalkyl) uracil; 5 (guanidiniumalkyl) uracil; 5 (methoxycarbonylmethyl)-2-(thio) uracil; 5 (methoxycarbonylmethyl) uracil; 5 (methyl) 2 (thio) uracil; 5 (methyl) 2,4 (dithio) uracil; 5 (methyl) 4 (thio) uracil; 5 (methylaminomethyl)-2 (thio) uracil; 5 (methylaminomethyl)-2,4 (dithio) uracil; 5 (methylaminomethyl)-4 (thio) uracil; 5 (propynyl) uracil; 5 (trifluoromethyl) uracil; 5-(2- aminopropyl) uracil; 5-(alkyl)-2-(thio) pseudouracil; 5-(alkyl)-2,4 (dithio) pseudouracil; 5- (alkyl)-4 (thio) pseudouracil; 5-(alkyl) pseudouracil; 5-(alkyl) uracil; 5-(alkynyl) uracil; 5- (allylamino) uracil; 5 -(cyanoalkyl) uracil; 5-(dialkylaminoalkyl) uracil; 5-(dimethylaminoalkyl) uracil; 5-(guanidiniumalkyl) uracil; 5-(halo) uracil; 5-(l,3-diazole-l-alkyl) uracil; 5-(methoxy) uracil; 5-(methoxycarbonylmethyl)-2-(thio) uracil; 5-(methoxycarbonyl-methyl) uracil; 5- (methyl) 2 (thio) uracil; 5-(methyl) 2,4 (dithio) uracil; 5-(methyl) 4 (thio) uracil; 5-(methyl)-2- (thio) pseudouracil; 5-(methyl)-2,4 (dithio) pseudouracil; 5-(methyl)-4 (thio) pseudouracil; 5- (methyl) pseudouracil; 5-(methylaminomethyl)-2 (thio) uracil; 5-(methylaminomethyl)-2,4 (dithio) uracil; 5-(methylaminomethyl)-4-(thio) uracil; 5-(propynyl) uracil; 5 -(trifluoromethyl) uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo) uracil; 6-(azo) uracil; 6-aza-uridine; ally amino-uracil; aza uracil; deaza uracil; N3 (methyl) uracil; Pseudo-UTP -
1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine; 1- m ethyl- 1-deaza-pseudouri dine; 1-propynyl-uridine; 1-taurinom ethyl- 1-methyl-uri dine; 1- taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2-m ethoxy -4-thio-pseudouri dine;
2-thio-l -methyl- 1-deaza-pseudouridine; 2-thio-l -methyl -pseudouridine; 2-thio-5-aza-uridine; 2- thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio- pseudouridine; 4-methoxy-pseudouridine; 4-thio-l-methyl-pseudouridine; 4-thio-pseudouri dine; 5-aza-uridine; Dihydropseudouridine; (+) 1 -(2 -Hydroxypropyl) pseudouridine TP; (2R)-l-(2- Hydroxypropyl) pseudouridine TP; (2S)-l-(2-Hydroxypropyl) pseudouridine TP; (E)-5-(2- Bromo-vinyl) ara-uridine TP; (E)-5-(2-Bromo-vinyl) uridine TP; (Z)-5-(2-Bromo-vinyl) ara- uridine TP; (Z)-5-(2-Bromo-vinyl) uridine TP; l-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1- (2,2,3,3,3-Pentafluoropropyl) pseudouridine TP; l-(2,2-Diethoxyethyl) pseudouridine TP; 1- (2,4,6-Trimethylbenzyl) pseudouridine TP; l-(2,4,6-Trimethyl-benzyl) pseudo-UTP; 1 -(2,4,6- Trimethyl-phenyl) pseudo-UTP; l-(2-Amino-2-carboxy ethyl) pseudo-UTP; 1 -(2 -Amino-ethyl) pseudo-UTP; l-(2-Hydroxy ethyl) pseudouridine TP; 1 -(2 -Methoxy ethyl) pseudouridine TP; 1- (3,4-Bis-trifluoromethoxybenzyl) pseudouridine TP; l-(3,4-Dimethoxybenzyl) pseudouridine TP; 1 -(3 -Amino-3 -carboxypropyl) pseudo-UTP; 1 -(3 -Amino-propyl) pseudo-UTP; l-(3- Cyclopropyl-prop-2-ynyl) pseudouridine TP; l-(4-Amino-4-carboxybutyl) pseudo-UTP; l-(4- Amino-benzyl) pseudo-UTP; l-(4-Amino-butyl) pseudo-UTP; l-(4-Amino-phenyl) pseudo- UTP; l-(4-Azidobenzyl) pseudouridine TP; l-(4-Bromobenzyl) pseudouridine TP; l-(4- Chlorobenzyl) pseudouridine TP; l-(4-Fluorobenzyl) pseudouridine TP; l-(4-Iodobenzyl) pseudouridine TP; l-(4-Methanesulfonylbenzyl) pseudouridine TP; l-(4-Methoxybenzyl) pseudouridine TP; l-(4-Methoxy -benzyl) pseudo-UTP; l-(4-Methoxy-phenyl) pseudo-UTP; 1- (4-Methylbenzyl) pseudouridine TP; l-(4-Methyl -benzyl) pseudo-UTP; l-(4-Nitrobenzyl) pseudouridine TP; 1 -(4-Nitro-benzyl) pseudo-UTP; 1 (4-Nitro-phenyl) pseudo-UTP; l-(4- Thiomethoxybenzyl) pseudouridine TP; l-(4-Trifluoromethoxybenzyl) pseudouridine TP; l-(4- Trifluoromethylbenzyl) pseudouridine TP; l-(5-Amino-pentyl) pseudo-UTP; 1 -(6- Amino-hexyl) pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; l-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}- ethoxy)-propionyl]pseudouridine TP; l-{3-[2-(2-Aminoethoxy)-ethoxy]- propionyljpseudouridine TP; 1 -Acetylpseudouridine TP; l-Alkyl-6-(l-propynyl)-pseudo-UTP;
1 -Alkyl-6-(2-propynyl)-pseudo-UTP; 1 -Alkyl-6-allyl-pseudo-UTP; 1 -Alkyl-6-ethynyl-pseudo- UTP; l-Alkyl-6-homoallyl-pseudo-UTP; l-Alkyl-6-vinyl-pseudo-UTP; 1 -Allylpseudouridine TP; 1 -Aminomethyl-pseudo-UTP; 1 -Benzoylpseudouridine TP; 1 - Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2 -pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1 -Cyanomethylpseudouridine TP; 1- Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1 -Cycloheptylmethyl -pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl-pseudo-UTP; 1 -Cyclohexyl -pseudo-UTP; 1- Cyclooctylmethyl-pseudo-UTP; 1 -Cyclooctyl -pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; 1- Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1 -Homoallylpseudouridine TP; 1- Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP; l-Me-2-thio-pseudo-UTP; l-Me-4- thio-pseudo-UTP; 1-Me-alpha-thio-pseudo-UTP; 1 -Methanesulfonylmethylpseudouridine TP; 1- Methoxymethylpseudouridine TP; l-Methyl-6-(2,2,2-Trifluoroethyl) pseudo-UTP; l-Methyl-6- (4-morpholino)-pseudo-UTP; l-Methyl-6-(4-thiomorpholino)-pseudo-UTP; l-Methyl-6- ( substituted phenyl) pseudo-UTP; l-Methyl-6-amino-pseudo-UTP; l-Methyl-6-azido-pseudo- UTP; l-Methyl-6-bromo-pseudo-UTP; l-Methyl-6-butyl-pseudo-UTP; l-Methyl-6-chloro- pseudo-UTP; l-Methyl-6-cyano-pseudo-UTP; l-Methyl-6-dimethylamino-pseudo-UTP; 1- Methyl-6-ethoxy -pseudo-UTP; 1 -Methyl-6-ethylcarboxylate-pseudo-UTP; 1 -Methyl-6-ethyl- pseudo-UTP; l-Methyl-6-fluoro-pseudo-UTP; l-Methyl-6-formyl-pseudo-UTP; l-Methyl-6- hydroxyamino-pseudo-UTP; l-Methyl-6-hydroxy-pseudo-UTP; l-Methyl-6-iodo-pseudo-UTP; l-Methyl-6-iso-propyl-pseudo-UTP; l-Methyl-6-methoxy-pseudo-UTP; l-Methyl-6- methylamino-pseudo-UTP; l-Methyl-6-phenyl-pseudo-UTP; 1 -Methyl -6-propyl -pseudo-UTP; 1- Methyl-6-tert-butyl-pseudo-UTP; 1 -Methyl-6-trifluoromethoxy-pseudo-UTP; 1 -Methyl-6- trifluoromethyl-pseudo-UTP; 1 -Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP; 1- Phenyl-pseudo-UTP; 1 -Pivaloylpseudouridine TP; 1 -Propargylpseudouridine TP; 1-Propyl- pseudo-UTP; 1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1- Thiomethoxymethylpseudouridine TP; 1 -Thiomorpholinomethylpseudouridine TP; 1- Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2'-anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-Methyl-2'-deoxy-UTP; 2'-OMe-5- Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP; 2'-a-Trifluoromethyluridine TP; 2'-b- Ethynyluridine TP; 2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP; 2'- Deoxy-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine TP; 2 '-Deoxy -2 '-b- aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP; 2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'- b-chlorouridine TP; 2'-Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP; 2'-Deoxy-2'- b-mercaptouridine TP; 2'-Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2- methoxyuridine; 2'-O-Methyl-5-(l-propynyl) uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(l-Propynyl) ara-uridine TP; 5-(2- Furanyl) uridine TP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5 '-Homo-uridine TP; 5- iodo-2'-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6- deuterouridine TP; 5-Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2- Trifluoroethyl)-pseudo-UTP; 6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo- UTP; 6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP; 6-Azido-pseudo-UTP; 6- Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6- Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl- pseudo-UTP; 6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP; 6- Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo- UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl- pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo- UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine l-(4- methylbenzenesulfonic acid) TP; Pseudouridine l-(4-methylbenzoic acid) TP; Pseudouridine TP l-[3-(2-ethoxy)]propionic acid; Pseudouridine TP l-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)- ethoxy}] propionic acid; Pseudouridine TP l-[3-{2-(2-[2-{2 (2-ethoxy)-ethoxy}-ethoxy]-ethoxy)- ethoxy}] propionic acid; Pseudouridine TP l-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP l-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1- methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP- Nl-3-propionic acid; Pseudo-UTP-Nl-4-butanoic acid; Pseudo-UTP-Nl-5-pentanoic acid; Pseudo-UTP-Nl-6-hexanoic acid; Pseudo-UTP-Nl-7-heptanoic acid; Pseudo-UTP-Nl-methyl-p- benzoic acid; Pseudo-UTP-Nl-p-benzoic acid; Wybutosine; Hydroxy wybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino) purine; l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl: l,3-(diaza)-2-(oxo)-phenthiazin-l-yl; 1,3- (diaza)-2-(oxo)-phenoxazin-l-yl; l,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino) purine; 2,4,5- (trimethyl)phenyl; T methyl, 2'amino, 2'azido, 2'fluro-cytidine; 2' methyl, 2'amino, 2'azido,
2'fluro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6- Chloro-purine; 2-aza-inosinyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro- modified bases; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo- pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl) isocarbostyrilyl; 3- (methyl) isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4- (methyl) indolyl; 4,6-(dimethyl) indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl) isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo) thymine; 6-(methyl)-7-(aza) indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)- 3 -(aza)-phenthiazin- 1 -yl ; 7 -(aminoalkylhydroxy)- 1 -(aza)-2-(thio)-3 -(aza)-phenoxazin- 1 -y 1 ; 7- (aminoalkylhy droxy)- 1 ,3 -(diaza)-2-(oxo)-phenoxazin- 1 -yl; 7-(aminoalkylhy droxy)- 1 ,3 -(diaza)- 2-(oxo)-phenthiazin-l-yl; 7-(aminoalkylhy droxy)- l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(aza) indolyl; 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl; 7- (guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl; 7- (guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7- (guanidiniumalkylhy droxy)- l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(guanidiniumalkyl- hydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl; 7-(guanidiniumalkylhydroxy)-l,3-(diaza)-2- (oxo)-phenoxazin-l-yl; 7-(propynyl) isocarbostyrilyl; 7-(propynyl) isocarbostyrilyl, propynyl-7- (aza) indolyl; 7-deaza-inosinyl; 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7- substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho- substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine;
Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2- amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; ortho- sub stituted- 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl- pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza) indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl;
Tubercidine; Xanthine; Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2- amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'- OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl) uridine TP; and N6-(19-Amino- pentaoxanonadecyl) adenosine TP.
In some aspects, the nucleobase is selected from the group consisting of pseudouridine or N1 -methylpseudouridine. In some aspects, the nucleoside is not pseudouridine (T) or 5-methyl- cytidine (m5C). The present invention provides polynucleotides which may be isolated and/or purified. These polynucleotides may encode one or more polypeptides of interest and comprise a sequence of n number of linked nucleosides or nucleotides comprising at least one modified nucleoside or nucleotide as compared to the chemical structure of an A, G, U or C nucleoside or nucleotide.
In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of pseudouridine (\|/), N1 -methylpseudouridine (m1^), N1 -ethylpseudouridine, 2-thiouridine, 4'- thiouridine, 5 -methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l -methylpseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l -methylpseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyl uridine. In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of 1- methyl-pseudouridine (m1!]/), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (\|/), a-thio-guanosine and a-thio-adenosine. In some embodiments, polynucleotides includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
In some aspects, multiple modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase, the sugar, and/or the internucleoside linkage. In some aspects having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of: pseudouridine-alpha-thio-TP, 1 -methyl-pseudouridine-alpha-thio-TP, 1 -ethyl-pseudouridine-TP, 1-propyl-pseudouridine-TP, l-(2,2,2-trifluoroethyl)-pseudouridine-TP, 2-amino-adenine-TP, xanthosine, 5-bromo-cytidine, 5-aminoallyl-cytidine-TP, or 2-aminopurine-riboside-TP.
In certain aspects having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of: pseudouridine-alpha-thio-TP, 1 -methyl-pseudouridine-alpha-thio-TP, 1 -ethyl-pseudouridine-TP, 1-propyl-pseudouridine-TP, 5-bromo-cytidine, 5-aminoallyl-cytidine-TP, or 2-aminopurine- riboside-TP.
In other aspects having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of: pseudouridine-alpha- thio-TP, 1-methyl-pseudouridine-alpha-thio-TP, or 5-bromo-cytidine.
In other aspects, the isolated polynucleotide includes at least two modified nucleosides or nucleotides.
In certain aspects having at least two modifications, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of at least one of each of 5-bromo-cytidine-TP and 1-methyl-pseudouridine-TP.
In other aspects having at least two modifications, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of at least one of each of 5-bromo-cytidine-TP and pseudouridine-TP.
In some aspects having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of: 2-thio-pseudouridine- TP, 5-trifluoromethyl-uridine-TP, 5-trifluoromethyl-cytidine-TP, 3-methyl-pseudouridine, 5- methyl-2-thio-uridine-TP, N4-methyl-cytidine-TP, 5-hydroxymethyl-cytidine-TP, 3-methyl- cytidine-TP, 5-oxyacetic acid methyl ester-uridine-TP, 5-methoxycarbonylmethyl-uridine-TP, 5- methylaminomethyl-uridine-TP, 5-methoxy-uridine-TP, N1 -methyl-guanosine-TP, 8-aza- adenine-TP, 2-thio-uridine-TP, 5-bromo-uridine-TP, 2-thio-cytidine-TP, alpha-thio-cytidine-TP, 5-aminoallyl-uridine-TP, alpha-thio-uridine-TP, or 4-thio-uridine-TP. In other aspects having at least two modifications, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of at least one of each of 5-trifluoromethyl-cytidine-TP and 1-methyl-pseudouridine-TP; 5 -hydroxy methyl- cytidine-TP and 1-methyl-pseudouridine-TP; 5-trifluoromethyl-cytidine-TP and pseudouridine- TP; or N4-acetyl-cytidine-TP and 5-methoxy-uridine-TP.
In some aspects having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of 2-thio-pseudouridine- TP, 5-trifluoromethyl-cytidine-TP, 5-methyl-2-thio-uridine-TP, 5-hydroxymethyl-cytidine-TP, 5- oxyacetic acid methyl ester-uridine-TP, 5-methoxy-uridine-TP, N4-acetyl-cytidine-TP, 2-thio- uridine-TP, 5-bromo-uridine-TP, alpha-thio-cytidine-TP, 5-aminoallyl-uridine-TP, or alpha-thio- uridine-TP.
In other aspects having at least two modifications, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of at least one of each of 5-trifluoromethyl-cytidine-TP and 1-methyl-pseudouridine-TP or 5-hydroxymethyl- cytidine-TP and 1-methyl-pseudouridine-TP.
In some aspects having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and internucleoside linkage of 2-thio-pseudouridine- TP, 5-trifluoromethyl-cytidine-TP, 5-methyl-2-thio-uridine-TP, N4-methyl-cytidine-TP, 5- hydroxymethyl-cytidine-TP, 5-oxyacetic acid methyl ester-uridine-TP, 5- methoxycarbonylmethyl-uridine-TP, 5-methoxy-uridine-TP, 2-thio-uridine-TP, 5-bromo-uridine- TP, alpha-thio-cytidine-TP, 5-aminoallyl-uridine-TP, or alpha-thio-uridine-TP.
In some aspects having at least one modification, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of 2-thio-pseudouridine- TP, 5-trifluoromethyl-cytidine-TP, 5-hydroxymethyl-cytidine-TP, or 5-methoxy-uridine-TP.
In other aspects having at least two modifications, the polynucleotide includes a backbone moiety containing the nucleobase, sugar, and intemucleoside linkage of at least one of each of N4-acetyl-cytidine-TP and 5-methoxy-uridine-TP.
In some aspects, the nucleic acid sequence encoding the first polypeptide of the activatable construct (or the protein portion of the first monomer construct) is at least 70% identical (e.g., at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84 % identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the nucleic acid sequence encoding the second polypeptide of the activatable construct (or the protein portion of the second monomer construct).
In certain embodiments, the first polypeptide of the activatable construct and the second polypeptide of the activatable construct comprise identical components. In some aspects, the first and second polypeptides of the activatable construct constructs are encoded by the same polypeptide (i.e., the same amino acid sequence). Often, when the first and second polypeptides of the activatable construct comprise the same amino acid sequence, they are encoded by the same nucleic acid (i.e., the same nucleic acid sequence). In some of these embodiments, the first and second polypeptide of the activatable construct are encoded by the same nucleic acid.
Vectors
In some aspects, the nucleic acid encoding a polypeptide of the activatable construct according to any of the aspects disclosed herein comprises a vector. In some aspects, the nucleic acid encodes a polypeptide according to any one of the sequences in Table 16. In some aspects, the vector is an expression vector.
Also provided herein are pairs of vectors that together comprise any of one of the pair of nucleic acids described herein. In some aspects, the pair of vectors is a pair of expression vectors.
One skilled in the art will be capable of selecting suitable vectors or sets of vectors (e.g., expression vectors) for making any of the activatable constructs described herein, and using the vectors or sets of vectors to express any of the activatable constructs described herein. For example, in selecting a vector or a set of vectors, the cell must be considered because the vector(s) may need to be able to integrate into a chromosome of the cell and/or replicate in it. Exemplary vectors that can be used to produce an activatable construct are also described below.
As used herein, the term “vector” refers to a polynucleotide capable of inducing the expression of a recombinant protein (e.g., a first or second monomer) in a cell (e g., any of the cells described herein). A “vector” is able to deliver nucleic acids and fragments thereof into a host cell, and includes regulatory sequences (e.g., promoter, enhancer, poly(A) signal). Exogenous polynucleotides may be inserted into the expression vector in order to be expressed. The term “vector” also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.
Methods for constructing suitable vectors that include any of the nucleic acids described herein, and suitable for transforming cells (e.g., mammalian cells) are well-known in the art. See, e.g., Sambrook et al., Eds. “Molecular Cloning: A Laboratory Manual,” 2nd Ed., Cold Spring Harbor Press, 1989 and Ausubel et al., Eds. “Current Protocols in Molecular Biology,” Current Protocols, 1993.
Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. A vector can, for example, include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the activatable constructs described herein.
In some aspects, the activatable constructs are made biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.
In some aspects, the vector includes a nucleic acid encoding the first polypeptide of the activatable construct and the first polypeptide of any activatable construct described herein. In some aspects, the vector is an expression vector.
Cells
Also provided herein are host cells (recombinant host cells) including any of the nucleic acids, or vector or sets of vectors described herein including any of the nucleic acids described herein.
Methods of introducing nucleic acids and vectors (e.g., any of the vectors or any of the sets of vectors described herein) into a cell are known in the art. Non-limiting examples of methods that can be used to introducing a nucleic acid into a cell include: lipofection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation. In some embodiments, the introducing step includes introducing into a cell a vector (e.g., any of the vectors or sets of vectors described herein) including a nucleic acid encoding the monomers that make up any of the activatable constructs described herein.
In some embodiments of any of the methods described herein, the activatable construct can be produced by any cell, including a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membranebound nucleus. Such cells may include, for example, mammalian, insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include a rodent cell (e.g., a mouse cell, a rat cell, a hamster cell, such as Chinese hamster ovary (CHO) cells, or a non-human primate cell, or a human cell, such as human embryonic kidney cells (e.g., HEK293 cells).
In some embodiments, the cell contains the nucleic acid encoding the first monomer and the second monomer of any one of the activatable constructs described herein. In some embodiments, the cell contains the pair of nucleic acids that together encode the first monomer and the second monomer of any of the activatable constructs described herein. In some aspects, the nucleic acid encoding the first monomer and the second monomer is integrated into the genomic DNA of the host cell.
Methods of Producing Activatable Constructs
Provided herein are methods of producing any of the activatable constructs described herein that include: (a) culturing any of the recombinant host cells described herein in a liquid culture medium under conditions sufficient to produce the activatable constructs; and (b) recovering the activatable constructs from the host cell and/or the liquid culture medium.
Methods of culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor cell proliferation, cell differentiation and cell growth. For example, cells can be cultured by contacting a cell (e.g., any of the cells described herein) with a cell culture medium that includes the necessary growth factors and supplements sufficient to support cell viability and growth.
In some aspects of any of the methods described herein, the method further includes isolating the recovered activatable constructs. Non-limiting examples of methods of isolation include: ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand-affinity chromatography, ion-exchange chromatography (e.g., anion or cation), and hydrophobic interaction chromatography.
Compositions and methods described herein may involve use of non-reducing or partially-reducing conditions that allow disulfide bonds to form between the dimerization domains to form and maintain dimerization of the activatable constructs.
In some aspects of any of the methods described herein, the method further includes formulating the isolated the activatable constructs into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any of the isolated the activatable constructs described herein can be formulated for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).
Also provided herein are the activatable constructs produced by any of the methods described herein. Also provided are compositions (e.g., pharmaceutical compositions) that include any of the activatable constructs produced by any of the methods described herein. Also provided herein are kits that include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.
Methods of Treatment
In one aspect, the present disclosure provides a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising the activatable construct according to any of the aspects disclosed herein. In some aspects, the subject has been identified or diagnosed as having a cancer, an autoimmune disease, or an inflammatory disorder.
As used herein, the term “subject” refers to any mammal. In some aspects, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster or a guinea pig), a non-human primate (e.g., a simian (e.g., a monkey (e.g., a baboon, a marmoset), or an ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human. In some aspects, the subject is a human.
In some aspects, the subject has been previously identified or diagnosed as having the disease (e.g., cancer (e.g., any of the cancers described herein)).
As used herein, the term “treat” includes reducing the severity, frequency or the number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., a cancer (e.g., any of the cancers described herein)) in the subject (e.g., any of the subjects described herein). In some aspects where the disease is cancer, treating results in reducing cancer growth, inhibiting cancer progression, inhibiting cancer metastasis, or reducing the risk of cancer recurrence in a subject having cancer.
In some aspects, the methods and uses of the present disclosure include any route of administration including intravenous, infusion, intratumoral, subcutaneous, intraperitoneal, intradermal, oral (e.g., inhalation), intranasal, transdermal (e.g., topical), transmucosal, and/or intramuscular.
In some aspects of any of the methods described herein, the disease is a cancer. Also provided herein are methods of treating a subject in need thereof (e.g., any of the exemplary subjects described herein or known in the art) that include administering to the subject a therapeutically effective amount of any of the activatable constructs described herein or any of the compositions (e.g., pharmaceutical compositions) described herein.
In some aspects of these methods, the subject has been identified or diagnosed as having a cancer. Non-limiting examples of cancer include: solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, a lymphoma (e.g., B-cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, cutaneous T-cell lymphoma), a leukemia (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL)), myelodysplastic syndromes (MDS), Kaposi sarcoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer including triple negative breast cancer (TNBC), colorectal cancer, ovarian cancer, nasopharyngeal adenocarcimoa, nonsmall cell lung carcinoma (NSCLC), squamous cell head and neck carcinoma, endometrial cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some aspects, the cancer is a lymphoma. In some aspects, the lymphoma is Burkitt’s lymphoma. In some aspects, the subject has been identified or diagnosed as having familial cancer syndromes such as Li Fraumeni Syndrome, Familial Breast-Ovarian Cancer (BRCA1 or BRAC2 mutations) Syndromes, and others. The disclosed methods are also useful in treating non-solid cancers. Exemplary solid tumors include malignancies (e.g., sarcomas, adenocarcinomas, and carcinomas) of the various organ systems, such as those of lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary (e.g., renal, urothelial, or testicular tumors) tracts, pharynx, prostate, and ovary. Exemplary adenocarcinomas include colorectal cancers, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, and cancer of the small intestine.
Exemplary cancers described by the National Cancer Institute include: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas/Carcinoids, Childhood; Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma/Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer;
Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma;
Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma, Childhood Brain Stem; Glioma, Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary);
Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's, Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer;
Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood; Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma;
Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer;
Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach
(Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T- Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant;
Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood;
Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer;
Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.
Further exemplary cancers include diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).
Metastases of the aforementioned cancers can also be treated or prevented in accordance with the methods described herein.
In some aspects, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the cancer in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the cancer in the subject prior to treatment).
In some aspects of any of the methods described herein, the methods further include administering to a subject an additional therapeutic agent (e.g., one or more of the therapeutic agents listed in Table 3).
Table 3. Additional Therapeutic Agents
Compositions/Kits
Also provided herein are compositions (e.g., pharmaceutical compositions) including any of the activatable constructs, or polynucleotides (e.g., mRNA) or vectors encoding the polypeptides thereof, described herein and one or more (e.g., 1, 2, 3, 4, or 5) pharmaceutically acceptable carriers (e.g., any of the pharmaceutically acceptable carriers described herein), diluents, or excipients.
In one aspect, the present disclosure provides a composition comprising the activatable construct, or polynucleotides (e.g., mRNA) or vectors encoding the polypeptides thereof, according to any of the aspects disclosed herein. In some aspects, the composition further comprises a pharmaceutically acceptable carrier. In some aspects, the compositions (e.g. pharmaceutical compositions) that include any of the activatable constructs, or polynucleotides (e g., mRNA) or vectors encoding the polypeptides thereof, described herein are formulated for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intratumoral). Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the type of intended administration. A variety of pharmaceutically acceptable excipients are known in the art (see, e.g., A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 2nd edition,. Rowe, et al. Handbook of pharmaceutical excipients. Pharmaceutical press, 2005.
In some aspects, the compositions are formulated in a nanoparticle. In some embodiments, the compositions are formulated in a lipid nanoparticle. In some aspects, the compositions are formulated in a lipid-polycation complex, referred to as a cationic lipid nanoparticle. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and/or polyarginine. In some embodiments, the compositions are formulated in a lipid nanoparticle that includes a noncationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE). Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid. Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) 7Vtz . Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51 :8529- 8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety). Compositions of nucleotides of the disclosure can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In some embodiments, pharmaceutical compositions of RNA (e.g., mRNA) compositions include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the formulations.
In some aspects, any of the pharmaceutical compositions described herein can include one or more buffers (e.g., a neutral -buffered saline, a phosphate-buffered saline (PBS), amino acids (e.g., glycine), one or more carbohydrates (e.g., glucose, mannose, sucrose, dextran, or mannitol), one or more antioxidants, one or more chelating agents (e.g., EDTA or glutathione), one or more preservatives, and/or a pharmaceutically acceptable carrier (e.g., bacteriostatic water, PBS, or saline) or excipient.
As used herein, the phrase “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial agents, antimicrobial agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers include, but are not limited to: water, saline, ringer’s solutions, dextrose solution, and about 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. 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. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition may be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some, any of the activatable molecules described herein are prepared with carriers that protect against rapid elimination from the body, e.g., sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic-co- glycolic acid, and polylactic acid. Methods for preparation of such pharmaceutical compositions and formulations are apparent to those skilled in the art. For example, the activatable molecules may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the CM- containing polypeptides, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L- glutamic acid and y ethyl-L-glutamate, non-degradable ethyl ene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
In some embodiments, 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 dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (CAS No. 61791-12-6) (BASF, Parsippany, N.J.), which is a mixture of polyoxyethylated triglycerides, by reacting castor oil with ethylene oxide in a molar ratio of 1 : 35, that acts as a nonionic surfactant, or phosphate buffered saline (PBS). The composition may be sterile and should be fluid and of a viscosity that facilitates easy syringeability. It may be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For dispersed particulate compositions, the proper fluidity can be maintained, for example, by the use of a coating on the particles such as lecithin, and by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some embodiments, the pharmaceutical compositions may further comprise one or more antibacterial and/or antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and the like, as well as salts, such as, for example, sodium chloride and the like may be included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
In some embodiments, the pharmaceutical composition may comprise a sterile injectable solution. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by fdtered sterilization. Generally, dispersions may be prepared by incorporating the active compound into a sterile vehicle that 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, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
In some aspects of any of the pharmaceutical compositions described herein, any of the activatable constructs described herein are prepared with carriers that protect against rapid elimination from the body, e.g., sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collage, polyorthoesters, and polylactic acid.
In one aspect, the present disclosure provides a container, vial, syringe, injector pen, or kit comprising at least one dose of a composition comprising the activatable construct according to any of the aspects disclosed herein. Also provided are kits that include one or more second therapeutic agent(s) selected from Table 3 in addition to an the activatable constructs described herein. The second therapeutic agent(s) may be provided in a dosage administration form that is separate from the activatable constructs. Alternatively, the second therapeutic agent(s) may be formulated together with the activatable constructs.
Any of the kits described herein can include instructions for using any of the compositions (e.g., pharmaceutical compositions) and/or any of the activatable constructs described herein. In some aspects, the kits can include instructions for performing any of the methods described herein. In some aspects, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some aspects, the kits can provide a syringe for administering any of the pharmaceutical compositions described herein.
The present disclosure includes and finds support for and in any one or any combination of the following items:
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1: Production of Activatable Construct
A schematic of a masked activatable antibody (an activatable construct) of the present disclosure is shown in FIG. 1C. Expression plasmids were constructed to express a masked light chain and a masked heavy chain. The expression plasmid for a masked light chain encoded a mature polypeptide consisting of a first masking moiety attached to the light chain of the antibody with a first cleavable moiety. The expression plasmid for a masked heavy chain encoded a mature polypeptide consisting of a second masking moiety attached to the heavy chain of the antibody with a second cleavable moiety.
Multiple activatable antibodies were produced having the sequences described in the examples below. For example, ProC3551 was prepared by transforming Expi293 host cells, per the manufacturer’s recommendations (Gibco ExpiFectamine 293 Transfection Kit), with two plasmids: one expressing the polynucleotide sequence of SEQ ID NO: 684 and one expressing the sequence of SEQ ID NO: 695, followed by cultivation of the resulting recombinant host cells. The masked activatable antibodies were purified from the culture supernatant by protein A- affinity chromatography followed by size-exclusion chromatography (SEC), and were confirmed to have >95% monomer content by analytical SEC.
Example 2: ITGA3B1 Binding ELISA A solid-phase binding assay was used to demonstrate the binding of anti-human ITGA3B1 antibodies (shown in FIGs. 2A-2D). Briefly, recombinant human ITGA3B1 protein (Aero Biosystems) diluted in IX PBS, pH 7.2 was coated on ELISA plates (100 pl at 1 pg/ml) overnight at 4C. The plates were washed six times with IX PBS-T (IX PBS + 0.05% Tween-20) and blocked with IX blocking buffer (IX PBS-T + 0.5% BSA) for Ih at room temperature (RT). The blocked plates were washed and then incubated with three-fold serial dilutions of anti- ITGA3B 1 antibody (starting at 100 nM) or activatable anti-ITGA3B l antibodies (starting at 1 pM) in blocking buffer. The activatable antibodies were assayed in their uncleaved form using three different masks. After washing as described above, the amount of bound antibody or bound activatable antibody was detected by incubating with 1 :25000 dilution of anti-human IgG (anti-Fc) conjugated with horseradish peroxidase (Jackson ImmunoResearch) in blocking buffer for 30min at RT. The plates were washed and incubated with lOOpl of Ultra TMB-ELISA reagent (Thermo Fisher Scientific) for lOmin at RT and the reaction was stopped with IN sulfuric acid. The absorbance at 450nm was measured and reported as an optical density value (A450). The blank value was subtracted from the raw A450 values to analyze the binding data in GraphPad Prism (vlO.1.2). The EC50 values were obtained for each antibody and activatable antibody and the masking efficiency (ME) for each test article relative to the unmasked antibody was calculated as the ratio of the EC50 of the activatable antibody to that of the original antibody.
The EC50s of the masked activatable antibodies having masks on both heavy and light chains were greater than that of the light chain-masked or heavy chain-masked molecules, indicating higher masking than that of either the heavy chain-masked or light chain-masked antibody molecules. This was true for all four peptide masks tested on the anti-ITGA3Bl antibody.
As shown in FIG. 2A, the following constructs were tested having various configurations of the same mask (Mask 2325): ProC3542 (maskless: SEQ ID NOs: 681 and 692); ProC3543 (MMs conjugated to light chains only: SEQ ID NOs: 681 and 694); ProC3547 (MMs conjugated to heavy chains only: SEQ ID NOs: 683 and 692); and ProC3551 (MMs conjugated to both heavy and light chains: SEQ ID NOs: 683 and 694). As shown in Table 4, ProC3551 with MMs conjugated to both the heavy and light chains had 83-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 6-7-fold masking efficiency.
As shown in FIG. 2B, the following constructs were tested having various configurations of the same mask (Mask 2331): ProC3542 (describe above); ProC3544 (MMs conjugated to light chains only: SEQ ID NOs: 681 and 696); ProC3548 (MMs conjugated to heavy chains only: SEQ ID NOs: 685 and 692); and ProC3552 (MMs conjugated to both heavy and light chains: SEQ ID NOs: 685 and 696). As shown in Table 5, ProC3552 with MMs conjugated to both the heavy and light chains (i.e., two masks, a heavy chain mask and a light chain mask, per antigen binding domain) had 34-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains (i.e., one mask per antigen binding domain) with 3-10-fold masking efficiency.
As shown in FIG. 2C, the following constructs were tested having various configurations of the same mask (Mask 2333): ProC3542 (described above); ProC3545 (MMs conjugated to light chains only: SEQ ID NOs: 681 and 698); ProC3549 (MMs conjugated to heavy chains only: SEQ ID NOs: 687 and 682); and ProC3553 (MMs conjugated to both heavy and light chains: SEQ ID NO: 687 and 698). As shown in Table 6, ProC3553 with MMs conjugated to both the heavy and light chains had 59-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 3-9-fold masking efficiency.
As shown in FIG. 2D, the following constructs were tested having various configurations of the same mask (Mask 2337): ProC3542 (described above); ProC3546 (MMs conjugated to light chains only: SEQ ID NOs: 681 and 700); ProC3550 (MMs conjugated to heavy chains only: SEQ ID NOs: 689 and 692); and ProC3554 (MMs conjugated to both heavy and light chains: SEQ ID NOs: 689 and 700). As shown in Table 7, ProC3554 with MMs conjugated to both the heavy and light chains had 55-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 5-6-fold masking efficiency.
Table 4: ITGA3B1 Binding Activity of Activatable Anti-ITGA3B1 Antibodies with Mask 2325 (FIG. 2A).
Table 5: ITGA3B1 Binding Activity of Activatable Anti-ITGA3B1 Antibodies with Mask 2331 (FIG. 2B). Table 6: ITGA3B1 Binding Activity of Activatable Anti-ITGA3B1 Antibodies with Mask 2333 (FIG. 2C).
Table 7: ITGA3B1 Binding Activity of Activatable Anti-ITGA3B1 Antibodies with Mask 2337 (FIG. 2D).
Table 8: Tested constructs are summarized as follows:
Example 3: EGFR Binding ELISA
A solid-phase binding assay was used to assess the binding of anti-human EGFR antibodies (shown in FIGs. 3A-3D). The ELISA method used was similar to the one described above but with Fc-tagged recombinant human EGFR extracellular domain (ECD) protein conjugated to an IgGl Fc domain (ProC788, EGFR-ECD-IgGl) as the coating antigen and antihuman IgG (anti-Fab) conjugated with horseradish peroxidase (Sigma Aldrich) as the detection antibody.
The EC50s of the masked activatable antibodies having masks on both heavy and light chains were greater than that of the light chain-masked or heavy chain-masked molecules, indicating higher masking than either the light chain-masked or heavy chain-masked molecules. This was observed with two peptide masks tested when the same mask was present on both the heavy and the light chains or when different masks were present on the heavy and the light chains.
As shown in FIG. 3A, the following constructs were tested having various mask configurations of the same mask (Mask 3954): ProC052 (maskless: SEQ ID NOs: 703 and 706); ProC3539 (MMs conjugated to light chains only: SEQ ID NOs: 703 and 710); ProC3540 (MMs conjugated to heavy chains only: SEQ ID NOs: 708 and 706); and ProC3541 (MMs conjugated to both heavy and light chains: SEQ ID NOs: 708 and 710). As shown in Table 9, ProC3541 with MMs conjugated to both the heavy and light chains had 260-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 6-8-fold masking efficiency.
As shown in FIG. 3B, the following constructs were tested having various mask configurations of the same mask (Mask CF41): ProC052 (described above); ProC4138 (MMs conjugated to light chains only: SEQ ID NOs: 703 and 523); ProC4139 (MMs conjugated to heavy chains only: SEQ ID NOs: 712 and 706); and ProC4140 (MMs conjugated to both heavy and light chains: SEQ ID NOs: 712 and 523). As shown in Table 10, ProC4140 with MMs conjugated to both the heavy and light chains had 619-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains with 10-18-fold masking efficiency.
As shown in FIG. 3C, the following constructs were tested having various mask configurations using two different masks (Mask 3954 and CF41): ProC052 (described above); ProC3540 (3954 mask on heavy chains only: SEQ ID NOs: 708 and 706); ProC4138 (CF41 mask on light chains only: SEQ ID NOs: 703 and 714); and ProC4141 (Mask 3954 on heavy chains and mask CF41 on light chains: SEQ ID NOs: 708 and 714). As shown in Table 11, ProC4141 with Mask 3954 on heavy chains and mask CF41 on light chains had 575-fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains, respectively, with 6-18-fold masking efficiency.
As shown in FIG. 3D, the following constructs were tested having various mask configurations using two different masks (Mask 3954 and CF41): ProC052 (described above); ProC3539 (Mask 3954 on the light chains only: SEQ ID NOs: 703 and 710); ProC4139 (Mask CF41 on the heavy chains only: SEQ ID NOs: 712 and 706); and ProC4142 (Mask CF41 on the heavy chains and mask 3954 on the light chains: SEQ ID NOs: 712 and 710). As shown in Table 12, ProC4142 with Mask CF41 on the heavy chains and mask 3954 on the light chains had 399- fold masking efficiency compared to the same mask conjugated to either both heavy chains or both light chains, respectively, with 8-10-fold masking efficiency.
Table 9: EGFR Binding Activity of Activatable Anti-EGFR Antibodies with Mask 3954 (FIG. 3A). Table 10: EGFR Binding Activity of Activatable Anti-EGFR Antibodies with Mask CF41 (FIG. 3B).
Table 11: EGFR Binding Activity of Activatable Anti-EGFR Antibodies with Mask 3954 on the heavy chain and Mask CF41 on the light chain (FIG. 3C).
Table 12: EGFR Binding Activity of Activatable Anti-EGFR Antibodies with Mask 3954 on the light chain and Mask CF41 on the heavy chain (FIG. 3D). Tested constructs are summarized as follows: Table 13. CM sequences
Table 14. Linker sequences
Table 15. Example sequences Table 16. Example sequences
Table 17. MM sequences
NUMBERED ITEMS
The following numbered aspects also form part of the instant disclosure.
1. An activatable construct, comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB 1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), wherein the HVD1 is covalently coupled directly or indirectly to a first masking moiety (MM1) via a first cleavable moiety (CM1), wherein the LVD1 is covalently coupled directly or indirectly to a second masking moiety (MM2) via a second cleavable moiety (CM2); a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2), a first dimerization domain (DD1) and a second dimerization domain (DD2), and wherein the MM1 and the MM2 inhibit binding of the AB1 to the first antigen.
2. The activatable construct according to any one or combination of items, wherein the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide. 3. The activatable construct of item 2, wherein the first polypeptide comprises the MM2, the CM2, and the LVD1, and wherein the second polypeptide comprises the MM1 , the CM1, the HVD1, and the DD1.
4. The activatable construct of item 3, wherein the third polypeptide comprises the AB2 and the DD2.
5. The activatable construct of item 4, wherein the AB2 is coupled directly or indirectly to a third masking moiety (MM3) via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
6. The activatable construct of any one or combination of items, wherein the activatable construct further comprises a first heavy chain constant domain (HC1), wherein the HVD1 is coupled directly or indirectly to the C-terminus of the MM1 via the CM1 and directly or indirectly to the N-terminus of the DD1 via the HC1, and wherein the activatable construct further comprises a first light chain constant domain (LC1), wherein the LVD1 is coupled directly or indirectly to the C-terminus of the MM2 via the CM2, wherein the LVD1 is coupled directly or indirectly to the N-terminus of the LC1, and wherein the HC1 is coupled, directly or indirectly, to the LC 1.
7. The activatable construct of item 5, wherein the third polypeptide comprises the MM3, the CM3, the LVD2, the HVD2, and the DD2, and wherein the HVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the LVD2.
8. The activatable construct of item 5, wherein the third polypeptide comprises the MM3, the CM3, the LVD2, the HVD2, and the DD2, and wherein the LVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the HVD2.
9. The activatable construct of item 3, wherein the second polypeptide further comprises a third masking moiety (MM3), a third cleavable moiety (CM3), the AB2, and the DD1, wherein the third polypeptide comprises the DD2, and wherein the MM3 inhibits binding of the AB2 to the second antigen.
10. The activatable construct of item 9, wherein the third polypeptide does not comprise a variable domain. 11. The activatable construct of any one or combination of items, wherein the AB2 comprises a scFv.
12. The activatable construct of item 6, wherein the HVD1-HC1 and the LVD1-LC1 comprise a Fab.
13. The activatable construct of any one of items 6-12, wherein the activatable construct comprises a first, a second, and a third polypeptide comprising, respectively, in an N- terminal to C-terminal direction: a) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 ; and iii) HVD2-LVD2-DD2; b) i) MM2-CM2-LVD 1 -LC 1 ; ii)MMl-CMl-HVDl-HCl-DDl; and iii) LVD2-HVD2-DD2; c) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 ; and iii) MM3-CM3-HVD2-LVD2-DD2; d) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM 1 -CM 1 -HVD 1 -HC 1 -DD 1 ; and iii) MM3-CM3-LVD2-HVD2-DD2; e) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM3-MM3-DD1; and iii) DD2; or f) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-LVD2-HVD2-CM3-MM3-DD1; and iii) DD2; wherein the HC1 is coupled directly or indirectly to the LC1, wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
14. The activatable construct of item 3, further comprising a fourth polypeptide, wherein the third polypeptide comprises the HVD2 and the DD2, and wherein the fourth polypeptide comprises the LVD2. 15. The activatable construct of item 14, wherein the fourth polypeptide comprises a third masking moiety (MM3) coupled directly or indirectly to the LVD2 via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
16. The activatable construct of item 14, wherein the third polypeptide comprises a third masking moiety (MM3) coupled directly or indirectly to the HVD2 via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
17. The activatable construct of item 16, wherein the fourth polypeptide comprises a fourth masking moiety (MM4) coupled directly or indirectly to the LVD2 via a fourth cleavable moiety (CM4), and wherein the MM4 inhibits binding of the AB2 to the second antigen.
18. The activatable construct of any one of items 15-17, wherein the activatable construct further comprises a second heavy chain constant domain (HC2) and/or wherein the activatable construct further comprises a first heavy chain constant domain (HC1).
19. The activatable construct of any one of items 15-18, wherein the activatable construct further comprises a second light chain constant domain (LC2) and/or wherein the activatable construct further comprises a first light chain constant domain (LC1).
20. The activatable construct of any one of items 18-19, wherein the HVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the HC2, and wherein the LVD2 is coupled directly or indirectly to the C-terminus of the MM4 via the CM4, and wherein the LVD2 is coupled directly or indirectly to the N-terminus of the LC2, and wherein the HC2 is coupled directly or indirectly to the LC2.
21. The activatable construct of item 20, wherein the first polypeptide comprises the MM2, the CM2, the LVD1, and the LC1, wherein the second polypeptide comprises the MM1, the CM1, the HVD1, the HC1, and the DD1, wherein the third polypeptide comprises the MM3, the CM3, the HVD2, the HC2, and the DD2, and wherein the fourth polypeptide comprises the MM4, the CM4, the LVD2, and the LC2.
22. The activatable construct of any one of items 19-21, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) HVD2-HC2-DD2; and iv) LVD2-LC2; b) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2; and iv) LVD2-LC2; c) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) HVD2-HC2-DD2; and iv) MM3-CM3-LVD2-LC2; or d) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2; and iv) MM4-CM4-LVD2-LC2; wherein the HC1 is coupled directly or indirectly to the LC1 and the HC2 is coupled directly or indirectly to the LC2, wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
23. The activatable construct of any one or combination of items, wherein the first antigen and the second antigen are the same molecule or the same epitope.
24. The activatable construct of any one or combination of items, wherein the activatable construct specifically binds to a single epitope.
25. The activatable construct of any one or combination of items 17-24, wherein the MM1, the MM2, the MM3, and the MM4 comprise the same amino acid sequence.
26. The activatable construct of any one or combination of items 17-25, wherein the MM1 and the MM4 comprise the same amino acid sequence.
27. The activatable construct of any one or combination of items 5-26, wherein the MM2 and the MM3 comprise the same amino acid sequence. 28. The activatable construct of any one or combination of items 14-27, wherein the second polypeptide chain and the third polypeptide chains comprise the same amino acid sequence.
29. The activatable construct of any one or combination of items 14-28, wherein the first polypeptide chain and the fourth polypeptide chains comprise the same amino acid sequence.
30. The activatable construct of any one or combination of items 1-22, wherein the first antigen and the second antigen are different epitopes on the same molecule.
31.The activatable construct of any one or combination of items 1-22, wherein the first antigen and the second antigen are different molecules or different epitopes.
32. The activatable construct of any or combination of items 1-29, wherein the MM1 and the MM2 comprise the same amino acid sequence.
33. The activatable construct of any one or combination of items 5-22 or 26-27, wherein the MM3 is a different sequence amino acid than the MM1, or the MM2, or both the MM1 and the MM2.
34. The activatable construct of item 33, wherein the MM3 comprises a different amino acid sequence than the MM1 and the MM2.
35. The activatable construct of any one or combination of items 1-22 or 26-34, wherein the activatable construct is bispecific.
36. The activatable construct of any one or combination of items 4-22 or 33-35, wherein the second polypeptide chain and the third polypeptide chain comprise different amino acid sequences.
37. The activatable construct of any one or combination of items 4-22 or 35-36, wherein the first polypeptide chain and the fourth polypeptide chain comprise different amino acid sequences.
38. The activatable construct of any one or combination of items 17-29, wherein the first polypeptide chain and the fourth polypeptide chain comprise the same amino acid sequence.
39. The activatable construct of item 35, wherein the amino acid sequence of the HVD1-HC1 is different than the amino acid sequence of the HVD2-HC2, and wherein each is a direct or an indirect covalent linkage.
40. The activatable construct of item 35, wherein the MM1 and the MM3 are different amino acid sequences. 41. The activatable construct of item 35, wherein the CM1 and the CM3 are different amino acid sequences.
42. The activatable construct of any one or combination of items 35-36, wherein the second polypeptide chain and the third polypeptide chain are different amino acid sequences.
43. The activatable construct of item 35, wherein the CM1 and the CM3 comprise the same amino acid sequence.
44. The activatable construct of item 35, wherein the first polypeptide chain and the fourth polypeptide chain comprise the same amino acid sequence, and wherein the second polypeptide chain and the third polypeptide chain are different amino acid sequences.
45. The activatable construct of any one or combination of items 1-35, wherein the DD1 and the DD2 comprise the same amino acid sequence.
46. The activatable construct of item 36, wherein the DD1 and the DD2 are different amino acid sequences.
47. The activatable construct of any one or combination of items, further comprising a third antigen binding domain (AB3) having specific binding affinity for a third antigen, wherein the AB3 comprises a heavy chain variable domain 3 (HVD3) and a light chain variable domain 3 (LVD3), wherein the AB3 is coupled directly or indirectly to a fifth masking moiety (MM5) via a fifth cleavable moiety (CM5), wherein the MM5 inhibits binding of the AB3 to the third antigen.
48. The activatable construct of item 47, wherein the AB1 is coupled directly or indirectly to the N-terminus of the DD1 and the AB2 is coupled directly or indirectly to the N-terminus of the DD2, and wherein the AB3 is coupled directly or indirectly to the C-terminus of the DD1 or the DD2.
49. The activatable construct of any one or combination of items 47-48, wherein the AB3 comprises a scFv, wherein the AB3 comprises: i) MM5-CM5-HVD3-LVD3-X; or ii) MM5-CM5-LVD3-HVD3-X; or wherein the AB3 comprises in an N-terminal to C-terminal direction: iii) X-LVD3-HVD3-CM5-MM5; or iv) X-HVD3-LVD3-CM5-MM5; wherein X represents attachment to the N-terminus or the C-terminus of the DD1 or the DD2, and wherein each is a direct or an indirect covalent linkage.
50. The activatable construct of any one or combination of items 47-49, further comprising a fourth antigen binding domain (AB4) having specific binding affinity for a fourth antigen, wherein the AB4 comprises a heavy chain variable domain 4 (HVD4) and a light chain variable domain 4 (LVD4); wherein the AB4 is coupled directly or indirectly to a sixth masking moiety (MM6) via a sixth cleavable moiety (CM6), wherein the MM6 inhibits binding of the AB4 to the fourth antigen.
51. The activatable construct of item 50, wherein the AB3 is coupled directly or indirectly to the C-terminus of the DD1, and wherein the AB4 is coupled directly or indirectly to the C- terminus of the DD2.
52. The activatable construct of any one or combination of items 50-51, wherein the AB4 comprises a scFv, wherein the AB4 comprises: i) MM6-CM6-HVD4-LVD4-Y; or ii) MM6-CM6-LVD4-HVD4- Y; or wherein the AB4 comprises in an N-terminal to C-terminal direction: iii) Y-LVD4-HVD4-CM6-MM6; or iv) Y-HVD4-LVD4-CM6-MM6; wherein Y represents attachment to the N-terminus or the C-terminus of the DD2, and wherein each is a direct or an indirect covalent linkage.
53. The activatable construct of any one or combination of items, wherein each of the CM1 and the CM2 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one or combination of items 5-52, wherein each of the CM1, the CM2, the and CM3 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one or combination of items 17-52, wherein each of the CM1, the CM2, the CM3, and the CM4 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one or combination of items 48-52, wherein each of the CM1, CM2, CM3, CM4, and CM5 independently comprises a substrate for the same or different protease in any combination thereof; or the activatable construct of any one or combination of items 51-52, wherein each of the CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a substrate for the same or different protease in any combination thereof.
54. The activatable construct of item 53, wherein each of the CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a total of about 3 amino acids to about 15 amino acids.
55. The activatable construct of any one or combination of items 53-54, wherein each CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a substrate for a protease selected from the group consisting of ADAMS, AD AMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM 15, ADAMI 7/T ACE, ADAMDEC1, AD AMTS 1, ADAMTS4, ADAMTS5, Aspartate proteases, BACE, Renin, Aspartic cathepsins, Cathepsin D, Cathepsin E, Caspases, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cysteine cathepsins, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P, Cysteine constructases, Cruzipain, Legumain, Otubain-2, KLKs, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Metallo proteinases, Meprin, Neprilysin, PSMA, BMP-1, MMPs, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, Serine proteases, activated protein C , Cathepsin A, Cathepsin G, Chymase, coagulation factor proteases, FVIIa, FIXa, FXa, FXIa, FXIIa, Elastase, Granzyme B, Guanidinobenzoatase, HtrAl, Human Neutrophil Elastase, Lactoferrin, Marapsin, NS3/4A, PACE4, Plasmin, PSA, tPA, Thrombin, Tryptase, uPA, Type II Transmembrane, Serine Proteases, TTSPs, DESCI, DPP-4, FAP, Hepsin, Matriptase-2, MT- SPl/Matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.
56. The activatable construct of any one or combination of items 53-55, wherein CM1, CM2, CM3, CM4, CM5, and CM6 each independently comprises a sequence selected from the group consisting of sequences listed in Table 13.
57. The activatable construct of item 56, wherein CM1, CM2, CM3, CM4, CM5, and CM6 each independently comprises a sequence selected from the group consisting of: LSGRSDNH (SEQ ID NO: 8), NTLSGRSENHSG (SEQ ID NO: 9), NTLSGRSGNHGS (SEQ ID NO: 10), TSTSGRSANPRG (SEQ ID NO: 11), TSGRSANP (SEQ ID NO: 12), VHMPLGFLGP (SEQ ID NO: 13), AVGLLAPP (SEQ ID NO: 14), AQNLLGMV (SEQ ID NO: 15), QNQALRMA (SEQ ID NO: 16), LAAPLGLL (SEQ ID NO: 17), STFPFGMF (SEQ ID NO: 18), ISSGLLSS (SEQ ID NO: 19), PAGLWLDP (SEQ ID NO: 20), VAGRSMRP (SEQ ID NO: 21), VVPEGRRS (SEQ ID NO: 22), ILPRSPAF (SEQ ID NO: 23), MVLGRSLL (SEQ ID NO: 24), QGRAITFI (SEQ ID NO: 25), SPRSIMLA (SEQ ID NO: 26), SMLRSMPL (SEQ ID NO: 27), ISSGLLSGRSDNH (SEQ ID NO: 28), AVGLLAPPGGLSGRSDNH (SEQ ID NO: 29), ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 30), LSGRSGNH (SEQ ID NO: 31), SGRSANPRG (SEQ ID NO: 32), LSGRSDDH (SEQ ID NO: 33), LSGRSDIH (SEQ ID NO: 34), LSGRSDQH (SEQ ID NO: 35), LSGRSDTH (SEQ ID NO: 36), LSGRSDYH (SEQ ID NO: 37), LSGRSDNP (SEQ ID NO: 38), LSGRSANP (SEQ ID NO: 39), LSGRSANI (SEQ ID NO: 40), LSGRSDNI (SEQ ID NO: 41), MIAPVAYR (SEQ ID NO: 42), RPSPMWAY (SEQ ID NO: 43), WATPRPMR (SEQ ID NO: 44), FRLLDWQW (SEQ ID NO: 45), ISSGL (SEQ ID NO: 46), ISSGLLS (SEQ ID NO: 47), ISSGLL (SEQ ID NO: 48), ISSGLLSGRSANPRG (SEQ ID NO: 49), A VGLL APPT SGRSANPRG (SEQ ID NO: 50), AVGLLAPPSGRSANPRG (SEQ ID NO: 51), ISSGLLSGRSDDH (SEQ ID NO: 52), ISSGLLSGRSDIH (SEQ ID NO: 53), ISSGLLSGRSDQH (SEQ ID NO: 54), ISSGLLSGRSDTH (SEQ ID NO: 55), ISSGLLSGRSDYH (SEQ ID NO: 56), ISSGLLSGRSDNP (SEQ ID NO: 57), ISSGLLSGRSANP (SEQ ID NO: 58), ISSGLLSGRSANI (SEQ ID NO: 59), AVGLLAPPGGLSGRSDDH (SEQ ID NO: 60), AVGLLAPPGGLSGRSDIH (SEQ ID NO: 61), AVGLLAPPGGLSGRSDQH (SEQ ID NO: 62), AVGLLAPPGGLSGRSDTH (SEQ ID NO: 63), AVGLLAPPGGLSGRSDYH (SEQ ID NO: 64), AVGLLAPPGGLSGRSDNP (SEQ ID NO: 65), AVGLLAPPGGLSGRSANP (SEQ ID NO: 66), AVGLLAPPGGLSGRSANI (SEQ ID NO: 67), ISSGLLSGRSDNI (SEQ ID NO: 68), AVGLLAPPGGLSGRSDNI (SEQ ID NO: 69), GLSGRSDNHGG AVGLLAPP (SEQ ID NO: 70), GLSGRSDNHGG VHMPLGFLGP (SEQ ID NO: 71), LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 72), ISSGLSS (SEQ ID NO: 73), PVGYTSSL (SEQ ID NO: 74), DWLYWPGI (SEQ ID NO: 75), LKAAPRWA (SEQ ID NO: 76), GPSHLVLT (SEQ ID NO: 77), LPGGLSPW (SEQ ID NO: 78), MGLFSEAG (SEQ ID NO: 79), SPLPLRVP (SEQ ID NO: 80), RMHLRSLG (SEQ ID NO: 81), LLAPSHRA (SEQ ID NO: 82), GPRSFGL (SEQ ID NO: 83), GPRSFG (SEQ ID NO: 84), SARGPSRW (SEQ ID NO: 85), GGWHTGRN (SEQ ID NO: 86), HTGRSGAL (SEQ ID NO: 87), AARGPAIH (SEQ ID NO: 88), RGPAFNPM (SEQ ID NO: 89), SSRGPAYL (SEQ ID NO: 90), RGPATPIM (SEQ ID NO: 91), RGPA (SEQ ID NO: 92), GGQPSGMWGW (SEQ ID NO: 93), FPRPLGITGL (SEQ ID NO: 94), SPLTGRSG (SEQ ID NO: 95), SAGFSLPA (SEQ ID NO: 96), LAPLGLQRR (SEQ ID NO: 97), SGGPLGVR (SEQ ID NO: 98), PLGL (SEQ ID NO: 99), and SGRSDNI (SEQ ID NO: 100).
58. The activatable construct of item 57, wherein CM1, CM2, CM3, CM4, CM5, and CM6 each independently comprises a sequence selected from the group consisting of: ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), ISSGLLSGRSDQH (SEQ ID NO: 54), SGRSDNI (SEQ ID NO: 100), ISSGLLSGRSDNI (SEQ ID NO: 68), ISSGLLSGRSDNP (SEQ ID NO: 57), and PWGLSGRS (SEQ ID NO: 157).
59. The activatable construct of item 55, wherein the protease(s) is/are produced by a tumor in a subject.
60. The activatable construct of any one or combination of items, wherein each of the MM1 and the MM2 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one or combination of items 5-59, wherein each of the MM1, the MM2, the and MM3 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one or combination of items 17-59, wherein each of the MM1, the MM2, the MM3, and the MM4 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one or combination of items 48-59, wherein each of the MM1, MM2, MM3, MM4, and MM5 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; or the activatable construct of any one or combination of items 51-59, wherein each of the MM1, MM2, MM3, MM4, MM5, and MM6 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof.
61. The activatable construct of item 60, wherein the MM1, MM2, MM3, MM4, MM5, and MM6 each independently comprise a sequence selected from the group consisting of: XCXXRXXFEXXDCVX, where X equals any amino acid (SEQ ID NO: 716); TCPTRWHFETTDCVM (SEQ ID NO: 717); ICGSRLDFELNDCVM (SEQ ID NO: 718);
WCRDRSHFETGDCVM (SEQ ID NO: 719); ICTSRWEFENRDCVM (SEQ ID NO: 720);
VCRDRNEFEVGDCVM (SEQ ID NO: 721); TCKNRLEFERGDCVM (SEQ ID NO: 722);
VCSSRLEFEQKDCVM (SEQ ID NO: 723); WCRDREHFEKGDCVM (SEQ ID NO: 724);
YCANRYEFEYGDCVM (SEQ ID NO: 725); VCRTRWHFETTDCVM (SEQ ID NO:
726); VCSNRAEFEWGDCVM (SEQ ID NO: 727); VCASRWHFENTDCVM (SEQ ID NO:
728); NCASRWHFENEDCVM (SEQ ID NO: 729); VCSGRLEFELGDCVM (SEQ ID NO:
730); VCSSRWEFETNDCVM (SEQ ID NO: 731); FCRDRLDFDTMDCVM (SEQ ID NO:
732); CCMDRLEFERGDCVM (SEQ ID NO: 733); VCGSRNEFETGDCVM (SEQ ID NO:
734); MCSGRLEFETGDCVM (SEQ ID NO: 735); ECKTRQDFEMHDCVY (SEQ ID NO:
736); QCMSRFAFEIGDCVM (SEQ ID NO: 737); TCLSRYEFETTDCVM (SEQ ID NO:
738); VCRTRWHFETTDCVM (SEQ ID NO: 739); ICHDPYMNIDYTCKL (SEQ ID NO:
740); VMCYWEGWGFGRCPL (SEQ ID NO: 741); VWYCDGGYNECATRS (SEQ ID
NO: 742); AVWCDAYNKNMCWST (SEQ ID NO: 743); KCHDPYINIDYTCNN (SEQ ID NO: 744); LITCEMLMLKNCEKN (SEQ ID NO: 745); LGCKKQHHTNNTCDR (SEQ ID NO: 746); CISPRG (SEQ ID NO: 747); CISPRGCG (SEQ ID NO: 748);
CISPRGCPDGPYVMY (SEQ ID NO: 749); CISPRGCPDGPYVM (SEQ ID NO: 750);
CISPRGCEPGTYVPT (SEQ ID NO: 751); CISPRGCPGQIWHPP (SEQ ID NO: 752);
GSHCLIPINMGAPSC (SEQ ID NO: 753); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 754); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 755);
ADHVFWGSYGCISPRGCPG (SEQ ID NO: 756); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 757); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 758); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 759); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 760);
CPYYTLSYCGCISPRGCPG (SEQ ID NO: 761); CNHVYFGTCGCISPRGCPG (SEQ ID NO: 762); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 763); CHHFTLTTCGCISPRGCPG (SEQ ID NO: 764); YNPCATPMCCISPRGCPG (SEQ ID NO: 765);
CNHHYFYTCGCISPRGCG (SEQ ID NO: 766); CNHHYHYYCGCISPRGCG (SEQ ID NO: 767); CNHVYFGTCGCISPRGCG (SEQ ID NO: 768); CHHVYWGHCGCISPRGCG (SEQ ID NO: 769); CPHFTTTSCGCISPRGCG (SEQ ID NO: 770);
CNHFTLTTCGCISPRGCG (SEQ ID NO: 771); CHHFTLTTCGCISPRGCG (SEQ ID NO: 772); CPYYTLSYCGCISPRGCG (SEQ ID NO: 773); CPHVSFGSCGCISPRGCG (SEQ ID NO: 774); ADHVFWGSYGCISPRGCG (SEQ ID NO: 775); YNPCATPMCCISPRGCG (SEQ ID NO: 776); CHHVYWGHCGCISPRGCG (SEQ ID NO: 777);
C(N/P)H(H/V/F)(Y/T)(F/W/T/L) (SEQ ID NO: 778); (Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID NO: 779); CISPRGCGQPIPSVK (SEQ ID NO: 780); CISPRGCTQPYHVSR (SEQ ID NO: 781); CISPRGCNAVSGLGS (SEQ ID NO: 782); LSCEGWAMNREQCRA (SEQ ID NO: 783); PPLECNTKSMCSKHD (SEQ ID NO: 784); DRDCRGRRARCQQEG (SEQ ID NO: 785); FTCEGWAMNREQCRT (SEQ ID NO: 786); GRCPPSRDIRFCTYM (SEQ ID NO: 787); FSCEGWAMNRSQCRT (SEQ ID NO: 788); FTCEGWAMNRDQCRT (SEQ ID NO: 789); CISPRGCPDGPYVM (SEQ ID NO: 790); CISPRGC (SEQ ID NO: 791);
MMYCGGNEVLCGPRV (SEQ ID NO: 792); GYRWGCEWNCGGITT (SEQ ID NO: 793); GYLWGCEWNCGGITT (SEQ ID NO: 794); MMYCGGNEIFCEPRG (SEQ ID NO: 795); GYGWGCEWNCGGSSP (SEQ ID NO: 796); and MMYCGGNEIFCGPRG (SEQ ID NO: 797).
62. The activatable construct of item 61, wherein the MM1, MM2, MM3, MM4, MM5, and MM6 each independently comprise a sequence selected from the group consisting of: VCRDRNEFEVGDCVM (SEQ ID NO: 721); TCLSRYEFETTDCVM (SEQ ID NO: 738); VCSNRAEFEWGDCVM (SEQ ID NO: 727); VCSGRLEFELGDCVM (SEQ ID NO: 730); CISPRGCPDGPYVMY (SEQ ID NO: 749); and LSCEGWAMNREQCRA (SEQ ID NO: 783).
63. The activatable construct of any one or combination of items 1-35, wherein DD1 is an Fc domain hole mutant and DD2 is an Fc domain knob mutant, or wherein DD1 is an Fc domain knob mutant and DD2 is an Fc domain hole mutant.
64. The activatable construct of any one or combination of items, wherein the DD1 and the DD2 are selected from an IgGl Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a mutant or truncation variant thereof.
65. The activatable construct of any one or combination of items, wherein DD1 and DD2 are human IgGl Fc domains or a mutant or truncation variant thereof.
66. The activatable construct of any one or combination of items, wherein each of DD1 and DD2 each independently comprises a sequence that is at least 80% identical or at least 90% identical to a sequence selected from: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGX (SEQ ID NO: 655), wherein X is lysine or absent;
GSSKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGX (SEQ ID NO: 656), wherein X is lysine or absent;
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCP APELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX (SEQ ID NO: 657), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S SNFGTQT YTCNVDHKP SNTK VDKT VERKCC VECPPCP APP VAGP SV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGX (SEQ ID NO: 658), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S S SLGTQTYTCNVNHKP SNTK VDKRVELKTPLGDTTHTCPRCPEPKSC DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMH EALHNRFTQKSLSLSPGX (SEQ ID NO: 659), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S S SLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPSCPAPEFLGGP S V FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGX1X2X3 (SEQ ID NO: 660), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent;
CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX1X2X3 (SEQ ID NO: 661), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent or
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX1X2X3 (SEQ ID NO: 662), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent.
67. The activatable construct of item 66, wherein each of DD1 and DD2 independently comprises a sequence selected from:
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGX (SEQ ID NO: 655), wherein X is lysine or absent;
GSSKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGX (SEQ ID NO: 656), wherein X is lysine or absent;
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCP APELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX (SEQ ID NO: 657), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S SNFGTQT YTCNVDHKP SNTKVDKT VERKCC VECPPCP APP VAGP SV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVSNKGEPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGX (SEQ ID NO: 658), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S S SLGTQTYTCNVNHKP SNTKVDKRVELKTPLGDTTHTCPRCPEPKSC DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMH EALHNRFTQKSLSLSPGX (SEQ ID NO: 659), wherein X is lysine or absent;
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S S SLGTKT YTCNVDHKP SNTK VDKRVESKYGPPCPSCPAPEFLGGP S V FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGX1X2X3 (SEQ ID NO: 660), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent;
CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX1X2X3 (SEQ ID NO: 661), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent; or ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX1X2X3 (SEQ ID NO: 662), wherein Xi is L or absent, X2 is G or absent, and X3 is K or absent.
68. The activatable construct of any one or combination of items, further comprising one or more linkers.
69. The activatable construct of item 68, wherein the linker is a peptide having a length of 1 to 30, 5 to 25, 10-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.
70. The activatable construct of item 69, wherein the linker comprises at least 5 amino acids.
71. The activatable construct of any one or combination of items 68-70, wherein each linker is independently selected from the group consisting of: GSSGGSGGSGG (SEQ ID NO:623); GGGS (SEQ ID NO: 622); GGGSGGGS (SEQ ID NO: 624); GGGSGGGSGGGS (SEQ ID NO: 625); GGGGSGGGGSGGGGS (SEQ ID NO: 626); GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 627); GGGGSGGGGS (SEQ ID NO: 628); GGGGS (SEQ ID NO: 629); GS; GGGGSGS (SEQ ID NO: 630); GGGGSGGGGSGGGGSGS (SEQ ID NO: 631); GGSLDPKGGGGS (SEQ ID NO: 632); PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 633); SKYGPPCPPCPAPEFLG (SEQ ID NO: 634); GKSSGSGSESKS (SEQ ID NO: 635); GSTSGSGKSSEGKG (SEQ ID NO: 636); GSTSGSGKSSEGSGSTKG (SEQ ID NO: 637); GSTSGSGKPGSGEGSTKG (SEQ ID NO: 638); GSTSGSGKPGSSEGST (SEQ ID NO: 639); (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 640), (GGGS)n (SEQ ID NO: 641), (GGGGS)n (SEQ ID NO: 629), wherein each n is an integer of at least one; GGSG (SEQ ID NO: 642); GGSGG (SEQ ID NO: 643); GSGSG (SEQ ID NO: 644); GSGGG (SEQ ID NO: 645); GGGSG (SEQ ID NO: 646); GSSSG (SEQ ID NO: 647); GGGGSGGGGSGGGGS (SEQ ID NO: 626); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 654); and GSTSGSGKPGSSEGST (SEQ ID NO: 639).
72. The activatable construct of item 71, wherein the linker comprises a sequence of GGGS (SEQ ID NO: 622).
73. The activatable construct of any one or combination of items, wherein the HVD1, the
HVD2, or both comprises a sequence selected from: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSV KYNEGFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQ GTTVTVSS (SEQ ID NO: 680); or
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVT VSS (SEQ ID NO: 702).
74. The activatable construct of item 47, wherein the HVD3 comprises a sequence selected from:
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSV
KYNEGFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQ GTTVTVSS (SEQ ID NO: 680); or QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTD
YNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVT VSS (SEQ ID NO: 702).
75. The activatable construct of item 50, wherein the HVD3, the HVD4, or both comprises a sequence selected from:
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSV KYNEGFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQ GTTVTVSS (SEQ ID NO: 680); or
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVT VSS (SEQ ID NO: 702).
76. The activatable construct of any one or combination of items, wherein the LVD1, the LVD2, or both comprises a sequence selected from:
DIQMTQ SPS SLS AS VGDRVTITC S AS S SIS SNYLHWYQQKPGK VPKLLIYRTSNL ASGVP S RFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIK (SEQ ID NO: 691); or
QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 705).
77. The activatable construct of item 47, wherein the LVD3 comprises a sequence selected from: DIQMTQ SPS SLS AS VGDRVTITC S AS S SIS SNYLHWYQQKPGK VPKLLIYRTSNL ASGVP S RFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIK (SEQ ID NO: 691); or QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 705).
78. The activatable construct of item 50, wherein the LVD3, the LVD4, or both comprises a sequence selected from: DIQMTQSPSSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPS RFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIK (SEQ ID NO: 691); or QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 705).
79. The activatable construct of any one or combination of items, wherein the HVD1, the HVD2, or both comprises a sequence selected from: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSV KYNEGFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 681); or QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVT VS S ASTKGP S VFPL AP S SKST SGGTAALGCLVKD YFPEPVT VS WNSGALT SGVHTFP AVL Q S SGL YSL S SWT VP S S SLGTQT YICNVNHKPSNTK VDKK VEPKSCDKTHTCPPCP APEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 703). 80. The activatable construct of any one or combination of items, wherein the LVD1, the LVD2, or both comprises a sequence selected from:
DIQMTQ SPS SLS AS VGDRVTITC S AS S SIS SNYLHWYQQKPGK VPKLLIYRTSNL ASGVP S RFSGSGSGTDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 692); or QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDE QLKSGTAS VVCLLNNF YPREAKVQWKVDNALQ SGNSQE S VTEQD SKD ST YSL S STLTL S KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 707).
81. The activatable construct of any one or combination of items 2-80, wherein the first polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLS AS VGDRVTITC SASS SIS SNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRF SGSGSGTDY TLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSS LSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700).
82. The activatable construct of any one or combination of items 2-81, wherein the second polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGP S VFPL AP S SK ST SGGT AALGCL VKD YFPEP VT VS WNSGALTSGVHTFP A VLQ S SGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCL VKD YFPEP VTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AS TKGPSVFPLAPSSKSTSGGTAALGCL VKD YFPEP VTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687); or QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQ VSLTCL VKGF YP SDIA VEWE SNGQPENNYKTTPPVLD SDGSFFL YSKLT VDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
83. The activatable construct of any one or combination of items 19-81, wherein the third polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQ VSLTCL VKGF YP SDIA VEWE SNGQPENNYKTTPPVLD SDGSFFL YSKLT VDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYY AMD YWGQGTTVTVS S AST KGP S VFPL AP S SK ST SGGT AALGCL VKD YFPEP VT VS WNSGALTSGVHTFP A VLQ S SGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA
EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMD YWGQGTT VTVS S AS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
84. The activatable construct of any one or combination of items 19-83, wherein fourth polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL S AS VGDRVTITC SAS S SIS SNYLHWYQQKPGK VPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLS ASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDY TLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSS LSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL S AS VGDRVTITC SAS S SIS SNYLHWYQQKPGK VPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700).
85. The activatable construct of any one or combination of items 2-84, wherein the first polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLS ASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDY TLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSS LSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); or QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLSAS VGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDYTL TISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700); and wherein the second polypeptide comprises an amino acid sequence selected from: QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQ VSLTCL VKGF YP SDIA VEWE SNGQPENNYKTTPPVLD SDGSFFL YSKLT VDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYY AMD YWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
86. The activatable construct of any one or combination of items 19-85, wherein the third polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYY MDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS SAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689); and wherein the fourth polypeptide comprises an amino acid sequence selected from:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSL SASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694);
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLS ASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDY TLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696);
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSS LSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTD YTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); or
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLSAS VGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDYTL TISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700).
87. The activatable construct of any one or combination of items 2-85, wherein the first polypeptide comprises an amino acid sequence of: QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694); and wherein the second polypeptide comprising an amino acid sequence of:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683); wherein the first polypeptide comprises an amino acid sequence of:
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLS ASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGT DYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK AD YEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696); and wherein the second polypeptide comprises an amino acid sequence of:
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685); wherein the first polypeptide comprises an amino acid sequence of:
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); and wherein the second polypeptide comprises an amino acid sequence of:
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQ VSLTCL VKGF YP SDIA VEWE SNGQPENNYKTTPPVLD SDGSFFL YSKLT VDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687); or wherein the first polypeptide comprising an amino acid sequence of:
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLSAS VGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDYTL TISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700); and wherein the second polypeptide comprises an amino acid sequence of:
QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TIT ADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
88. The activatable construct of any one or combination of items 19-86, wherein the fourth polypeptide comprises an amino acid sequence of: QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 694); and wherein the third polypeptide comprising an amino acid sequence of:
QGQSGQVCRDRNEFEVGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELSSLRSEDTAVYYCARHEERDYYGYYAMDYWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 683); wherein the fourth polypeptide comprises an amino acid sequence of:
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPS SLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGT DYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 696); and wherein the third polypeptide comprises an amino acid sequence of:
QGQSGQTCLSRYEFETTDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE
VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 685); wherein the fourth polypeptide comprises an amino acid sequence of:
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSP SSLSASVGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSG TDYTLTISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 698); and wherein the third polypeptide comprises an amino acid sequence of:
QGQSGQVCSNRAEFEWGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGA EVKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGR ATITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS SAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 687); or wherein the fourth polypeptide comprising an amino acid sequence of: QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSDIQMTQSPSSLSAS VGDRVTITCSASSSISSNYLHWYQQKPGKVPKLLIYRTSNLASGVPSRFSGSGSGTDYTL TISSLQPEDVATYYCQQGSSIPRFTSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 700); and wherein the third polypeptide comprises an amino acid sequence of: QGQSGQVCSGRLEFELGDCVMGGGSSGGSISSGLLSGRSDNPGGGSQVQLVQSGAE VKKPGSSVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSVKYNEGFKGRA TITADKSTSTAYMELS SLRSEDTAVYYC ARHEERDYYGYYAMDYWGQGTTVTVS S AST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQ VSLTCL VKGF YP SDIA VEWE SNGQPENNYKTTPPVLD SDGSFFL YSKLT VDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 689).
89. The activatable construct of any one or combination of items 2-72, wherein the first polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVIL SVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLS INSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714).
90. The activatable construct of any one or combination of items 2-72, wherein the second polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLT VLHQDWLNGKEYKCK VSNKALP AP IEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGP GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
91. The activatable construct of any one or combination of items 19-72, wherein the third polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGP GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VS VLTVLHQDWLNGKEYKCK VSNKALP APIEKTISKAKGQPREPQ VYTLPP SREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712). 92. The activatable construct of any one or combination of items 19-72, wherein the fourth polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVIL SVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLS INSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714).
93. The activatable construct of any one or combination of items 2-72, wherein the first polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVIL SVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLS INSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714 ); and wherein the second polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLT VLHQDWLNGKEYKCK VSNKALP AP IEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGP GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
94. The activatable construct of any one or combination of items 19-72, wherein the fourth polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVIL SVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLS INSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and wherein the third polypeptide comprises an amino acid selected from:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); or
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGP GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
95. The activatable construct of any one or combination of items 2-72, wherein the first polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); and wherein the second polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); wherein the first polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVS PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and wherein the second polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQD WLNGKEYKCK VSNK ALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712); wherein the first polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSP VILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDF TLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and wherein the second polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); wherein the first polypeptide comprising an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVSP GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSV ESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); and wherein the second polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
96. The activatable construct of any one or combination of items 19-72 or 96, wherein the fourth polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILS VSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); and wherein the third polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); wherein the fourth polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVS PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and wherein the third polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCK VSNK ALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712); wherein the fourth polypeptide comprises an amino acid sequence of:
QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSP VILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDF TLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 714); and wherein the third polypeptide comprises an amino acid sequence of:
QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGL
VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCK VSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 708); or wherein the fourth polypeptide comprising an amino acid sequence of: QGQSGQCISPRGCPDGPYVMYGGGSSGGSISSGLLSGRSDNPGGGSQILLTQSPVILSVSP GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSV ESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 710); and wherein the third polypeptide comprises an amino acid sequence of: QGQSGQLSCEGWAMNREQCRAGGGSSGGSISSGLLSGRSDNPGGGSQVQLKQSGPGLV QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALP APIEKTI SKAKGQPREPQ VYTLPP SREEMTKNQ V SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 712).
97. The activatable construct of any one or combination of items, wherein the activatable construct is a multi-specific activatable antibody.
98. The activatable construct of any one or combination of items, wherein the activatable construct has a masking efficiency that is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than a corresponding activatable construct with only a single masking moiety per antigen binding domain.
99. The activatable construct of any one or combination of items 17-98, wherein the activatable construct has a masking efficiency that is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than a corresponding activatable construct that lacks MM3 and MM4. 100. A composition comprising the activatable construct of any one or combination of items 1-99.
101. The composition of any one or combination of items 100, further comprising a pharmaceutically acceptable carrier or excipient.
102. A container, vial, syringe, injector pen, or kit comprising at least one dose of the composition of any one or combination of items 100-101.
103. A method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of the activatable construct of any one or combination of items 1-98 or the composition of any one or combination of items 100- 101.
104. The method of item 103, wherein the subject has been identified or diagnosed as having a cancer, an autoimmune disease, or an inflammatory disorder.
105. The method of any one or combination of items 103-104, further comprising administering a second therapeutic agent.
106. The method of item 105, wherein the second therapeutic agent is a checkpoint inhibitor, a cytokine, or a cytotoxic agent.
107. A nucleic acid encoding a polypeptide of the activatable construct of any one or combination of items 1-101.
108. A vector comprising the nucleic acid of item 107.
109. The vector of item 108, wherein the vector is an expression vector.
110. A cell transfected with the nucleic acid of item 107 or the vector of any one or combination of items 108-109.
111. The cell of item 110, wherein the cell is a mammalian cell.
112. The cell of any one or combination of items 110-111, wherein the cell is a CHO cell or an HEK293 cell.
113. A method of producing the activatable construct of any one or combination of items 1-99, comprising culturing the cell of any one or combination of items 110-112 in a liquid culture medium to produce the activatable construct; and recovering the activatable construct from the liquid culture medium.
114. The method of item 113, further comprising: purifying the activatable construct recovered from the liquid culture medium to obtain a purified activatable construct. 115. The method of item 114, further comprising: combining the purified activatable construct with a carrier to form a pharmaceutical composition.
116. An activatable construct produced by the method of any one or combination of items 114-115.
117. An activatable construct, comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB 1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), a first light chain constant domain (LC1), a first heavy chain constant domain (HC1); a first masking moiety (MM1) coupled directly or indirectly to a first cleavable moiety (CM1), wherein the MM1 is coupled directly or indirectly to the HVD1, the LVD1, the HC1, or the LC1 via the CM1; a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2); a second masking moiety (MM2) coupled directly or indirectly to a second cleavable moiety (CM2), wherein the MM2 is coupled directly or indirectly to the HVD2 or the LVD2 via the CM2; a first dimerization domain (DD1) and a second dimerization domain (DD2), and wherein the MM1 inhibits binding of the AB1 to the first antigen, and wherein the MM2 inhibits binding of the AB2 to the first antigen.
118. The activatable construct of item 117, wherein the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide.
119. The activatable construct of items 117-118, wherein the activatable construct further comprises a third cleavable moiety (CM3) and a third masking moiety (MM3).
120. The activatable construct of items 117-119, wherein the activatable construct further comprises a fourth cleavable moiety (CM4) and a fourth masking moiety (MM3).
121. The activatable construct of items 117-120, wherein the first polypeptide comprises the MM1, CM1, LVD1, and the LC1, the second polypeptide comprises the HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, and the third polypeptide comprises the MM3, CM3, and the DD2. 122. The activatable construct of item 121, wherein the MM3 inhibits binding of the AB2 to the second antigen.
123. The activatable construct of items 121-122, wherein the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
124. The activatable construct of any one of items 121-123, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; or b) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-LVD2-HVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
125. The activatable construct of items 117-120, wherein the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, the second polypeptide comprises the LVD1, LC1, CM3, and the MM3, and the third polypeptide comprises the DD2.
126. The activatable construct of item 125, wherein the MM3 inhibits binding of the AB2 to the second antigen.
127. The activatable construct of items 125-126, wherein the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
128. The activatable construct of any one of items 125-127, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; ii) LVD1-LC1-CM3-MM3; and iii) DD2; or b) i) MM1-CM1-HVD1-HC1-LVD2-HVD2 -CM2-MM2-DD 1 ; ii) LVD1-LC1-CM3-MM3; and iii) DD2; wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
129. The activatable construct of items 117-120, wherein the first polypeptide comprises the MM1, CM1, LVD1, and the LC1, the second polypeptide comprises the MM4, CM4, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, and the third polypeptide comprises the MM3, CM3, and the DD2.
130. The activatable construct of item 129, wherein the MM4 inhibits binding of the AB1 to a first antigen.
131. The activatable construct of items 129-130, wherein the MM 1 and the MM4 inhibit binding of the AB1 to the first antigen.
132. The activatable construct of items 129-131, wherein the MM3 inhibits binding of the AB2 to the second antigen.
133. The activatable construct of items 129-132, wherein the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
134. The activatable construct of any one of items 129-133, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM1-CM1-LVD1-LC1 ii) MM4-CM4-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; or b) i) MM1-CM1-LVD1-LC1 ii) MM4-CM4-HVD1-HC1-LVD2-HVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
135. The activatable construct of items 117-120, wherein the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, the second polypeptide comprises the MM4, CM4, LVD1, LC1, CM3, and the MM3, and the third polypeptide comprises the DD2. 136. The activatable construct of item 135, wherein the MM4 inhibits binding of the AB1 to the first antigen.
137. The activatable construct of items 135-136, wherein the MM1 and the MM4 inhibit binding of the AB1 to the first antigen.
138. The activatable construct of items 135-137, wherein the MM3 inhibits binding of the AB2 to the second antigen.
139. The activatable construct of items 135-138, wherein the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
140. The activatable construct of any one of items 135-139, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2; or b) i) MM 1 -CM 1 -HVD 1 -HC 1 -L VD2-HVD2 -CM2-MM2-DD 1 ; ii) MM4-CM4-LVD1-LC1-CM3-MM3; and iii) DD2; wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
141. The activatable construct of items 117-120, wherein the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, the second polypeptide comprises the MM3, CM3, LVD1, and LC1, and the third polypeptide comprises the DD2.
142. The activatable construct of item 141, wherein the MM3 inhibits binding of the AB1 to the first antigen.
143. The activatable construct of items 141-142, wherein the MM1 and the MM3 inhibit binding of the AB1 to the first antigen.
144. The activatable construct of items 141-143, wherein the MM2 inhibits binding of the AB2 to the second antigen. 145. The activatable construct of any one of items 141-145, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; ii) MM3-CM3-LVD1-LC1; and iii) DD2; or b) i) MM1-CM1-HVD1-HC1-LVD2-HVD2 -CM2 -MM2 -DD 1 ; ii) MM3-CM3-LVD1-LC1; and iii) DD2; wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
146. An mRNA comprising the nucleic acid of item 107.
147. The mRNA of item 146, wherein the mRNA comprises one or more modifications.
148. The mRNA of item 147, wherein the one or more modifications are selected from a modified or non-naturally occurring nucleotide or 5’ capping structure.
149. A composition comprising the nucleic acid of item 107, the mRNA of any one of items 146-148, or the vector of any one of items 108-109.
150. The composition of item 149, further comprising a pharmaceutically acceptable carrier or excipient.
151. The activatable construct of item 118, wherein , the first polypeptide comprises an amino acid sequence of:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNN YATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTS NYVNWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYC VLWYSNRWVFGGGTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGS QGQSGSGYLWGCEWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG (SEQ ID NO: 800); the second polypeptide comprises an amino acid sequence of:
QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 804); and the third polypeptide comprises an amino acid sequence of:
QGQSGQGYLWGCEWNCGGITTGGGSSGGSLSGRSDNIGGGGSGASGSSGASGTGTAGG TGSGSGTGSGDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPASIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G (SEQ ID NO: 802); the first polypeptide comprises an amino acid sequence of:
QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNTAYLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTLVT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYVNWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCVLWYSNRWVFGG GTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGSQGQSGSGYLWGC EWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVI<FNWYVDGVEVHNAKTI<PREEQYNSTYRVVSVLTVLHQDWLNGKEYI<CI< VSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 806); the second polypeptide comprises an amino acid sequence of: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGGGSLSGRSDNIGGGSSGGSQG QSGSGYLWGCEWNCGGITT (SEQ ID NO: 810); and the third polypeptide comprises an amino acid sequence of:
DSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISK AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 808); the first polypeptide comprises an amino acid sequence of:
QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYVNWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCVLWYSNRWVFGG GTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGSQGQSGSGYLWGC EWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVI<FNWYVDGVEVHNAKTI<PREEQYNSTYRVVSVLTVLHQDWLNGKEYI<CI< VSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 806); the second polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 804) ; and the third polypeptide comprises an amino acid sequence of:
QGQSGQGYLWGCEWNCGGITTGGGSSGGSLSGRSDNIGGGGSGASGSSGASGTGTAGG TGSGSGTGSGDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPASIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G (SEQ ID NO: 802); the first polypeptide comprises an amino acid sequence of:
QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVESGGGLV QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ADT SKNT A YLQMNSLRAEDT AVYYC SRWGGDGF YAMD YWGQGTL VT VS S ASTKGP S VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESGGGLV QPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYVNWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCVLWYSNRWVFGG GTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGSQGQSGSGYLWGC EWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 806); the second polypeptide comprises an amino acid sequence of: QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECGSGGGSLSGRSDNIGGGSSGGSQGQSGSGYLWGCE WNCGGITT (SEQ ID NO: 812); and the third polypeptide comprises an amino acid sequence of: DSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISK AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 808); or the first polypeptide comprises an amino acid sequence of:
QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSEVQLVES GGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSSCGGGGSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADS VKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLV TVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYVNWVQ QKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCVLWYSNR WVFGGGTKLTVLGSSGGSLSGRSDNIGGGSGSGTGTAGGTGGSGTGSGGGSQGQSGSG YLWGCEWNCGGITTGGGGSDSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG (SEQ ID NO: 806); the second polypeptide comprises an amino acid sequence of:
QGQSGQGALICCSDVSGLCRWCGGGSSGGSISSGLLSGRSDNHGGGSDIQMTQSPSSLSA SVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 804); and the third polypeptide comprises an amino acid sequence of:
DSTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISK AKGQPREPQ VYTLPPCRDELTKNQ VSLWCL VKGF YP SDIA VEWE SNGQPENN YKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 808).
152. A method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of the nucleic acid of item 107, the mRNA of any one of items 146-148, or the vector of any one of items 108-109.
153. The method of claim 152, wherein the subject has been identified or diagnosed as having a cancer, an autoimmune disease, or an inflammatory disorder.
154. The method of any one of claims 152-153, further comprising administering a second therapeutic agent.
155. The method of claim 154, wherein the second therapeutic agent is a checkpoint inhibitor, a cytokine, or a cytotoxic agent.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. An activatable construct, comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), wherein the HVD1 is covalently coupled directly or indirectly to a first masking moiety (MM1) via a first cleavable moiety (CM1), wherein the LVD1 is covalently coupled directly or indirectly to a second masking moiety (MM2) via a second cleavable moiety (CM2); a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2), a first dimerization domain (DD1) and a second dimerization domain (DD2), and wherein the MM1 and the MM2 inhibit binding of the AB1 to the first antigen.
2. The activatable construct of claim 1, wherein the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide.
3. The activatable construct of claim 2, wherein the first polypeptide comprises the MM2, the CM2, and the LVD1, and wherein the second polypeptide comprises the MM1, the CM1, the HVD1, and the DD1.
4. The activatable construct of claim 3, wherein the third polypeptide comprises the AB2 and the DD2.
5. The activatable construct of claim 4, wherein the AB2 is coupled directly or indirectly to a third masking moiety (MM3) via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
6. The activatable construct of any preceding claim, wherein the activatable construct further comprises a first heavy chain constant domain (HC1), wherein the HVD1 is coupled directly or indirectly to the C-terminus of the MM1 via the CM1 and directly or indirectly to the N-terminus of the DD1 via the HC1, and wherein the activatable construct further comprises a first light chain constant domain (LC1), wherein the LVD1 is coupled directly or indirectly to the C-terminus of the MM2 via the CM2, wherein the LVD1 is coupled directly or indirectly to the N-terminus of the LC1, and wherein the HC1 is coupled, directly or indirectly, to the LC1.
7. The activatable construct of claim 5, wherein the third polypeptide comprises the MM3, the CM3, the LVD2, the HVD2, and the DD2, and wherein the HVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the LVD2.
8. The activatable construct of claim 5, wherein the third polypeptide comprises the MM3, the CM3, the LVD2, the HVD2, and the DD2, and wherein the LVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the HVD2.
9. The activatable construct of claim 3, wherein the second polypeptide further comprises a third masking moiety (MM3), a third cleavable moiety (CM3), the AB2, and the DD1, wherein the third polypeptide comprises the DD2, and wherein the MM3 inhibits binding of the AB2 to the second antigen.
10. The activatable construct of claim 9, wherein the third polypeptide does not comprise a variable domain.
11. The activatable construct of any preceding claim, wherein the AB2 comprises a scFv.
12. The activatable construct of claim 6, wherein the HVD1-HC1 and the LVD1-LC1 comprise a Fab.
13. The activatable construct of any one of claims 6-12, wherein the activatable construct comprises a first, a second, and a third polypeptide comprising, respectively, in an N- terminal to C-terminal direction: a) i) MM2-CM2-LVD1-LC1; ii) MMl-CMl-HVDl-HCl-DDl;and iii) HVD2-LVD2-DD2; b) i) MM2-CM2-LVD 1 -LC 1 ; ii)MMl-CMl-HVDl-HCl-DDl; and iii) LVD2-HVD2-DD2; c) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; and iii) MM3-CM3-HVD2-LVD2-DD2; d) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-DD1; and iii) MM3-CM3-LVD2-HVD2-DD2; e) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-HVD2-LVD2-CM3-MM3-DD1; and iii) DD2; or f) i) MM2-CM2-LVD 1 -LC 1 ; ii) MM1-CM1-HVD1-HC1-LVD2-HVD2-CM3-MM3-DD1; and iii) DD2; wherein the HC1 is coupled directly or indirectly to the LC1, wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
14. The activatable construct of claim 3, further comprising a fourth polypeptide, wherein the third polypeptide comprises the HVD2 and the DD2, and wherein the fourth polypeptide comprises the LVD2.
15. The activatable construct of claim 14, wherein the fourth polypeptide comprises a third masking moiety (MM3) coupled directly or indirectly to the LVD2 via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
16. The activatable construct of claim 14, wherein the third polypeptide comprises a third masking moiety (MM3) coupled directly or indirectly to the HVD2 via a third cleavable moiety (CM3), and wherein the MM3 inhibits binding of the AB2 to the second antigen.
17. The activatable construct of claim 16, wherein the fourth polypeptide comprises a fourth masking moiety (MM4) coupled directly or indirectly to the LVD2 via a fourth cleavable moiety (CM4), and wherein the MM4 inhibits binding of the AB2 to the second antigen.
18. The activatable construct of any one of claims 15-17, wherein the activatable construct further comprises a second heavy chain constant domain (HC2) and/or wherein the activatable construct further comprises a first heavy chain constant domain (HC1).
19. The activatable construct of any one of claims 15-18, wherein the activatable construct further comprises a second light chain constant domain (LC2) and/or wherein the activatable construct further comprises a first light chain constant domain (LC1).
20. The activatable construct of any one of claims 18-19, wherein the HVD2 is coupled directly or indirectly to the C-terminus of the MM3 via the CM3 and directly or indirectly to the N-terminus of the DD2 via the HC2, and wherein the LVD2 is coupled directly or indirectly to the C-terminus of the MM4 via the CM4, and wherein the LVD2 is coupled directly or indirectly to the N-terminus of the LC2, and wherein the HC2 is coupled directly or indirectly to the LC2.
21. The activatable construct of claim 20, wherein the first polypeptide comprises the MM2, the CM2, the LVD1, and the LC1, wherein the second polypeptide comprises the MM1, the CM1, the HVD1, the HC1, and the DD1, wherein the third polypeptide comprises the MM3, the CM3, the HVD2, the HC2, and the DD2, and wherein the fourth polypeptide comprises the MM4, the CM4, the LVD2, and the LC2.
22. The activatable construct of any one of claims 19-21, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: a) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) HVD2-HC2-DD2; and iv) LVD2-LC2; b) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2; and iv) LVD2-LC2; c) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) HVD2-HC2-DD2; and iv) MM3-CM3-LVD2-LC2; or d) i) MM2-CM2-LVD1-LC1; ii) MM1-CM1-HVD1-HC1-DD1; iii) MM3-CM3-HVD2-HC2-DD2; and iv) MM4-CM4-LVD2-LC2; wherein the HC1 is coupled directly or indirectly to the LC1 and the HC2 is coupled directly or indirectly to the LC2, wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
23. The activatable construct of any preceding claim, wherein the first antigen and the second antigen are the same molecule or the same epitope.
24. The activatable construct of any preceding claim, wherein the activatable construct specifically binds to a single epitope.
25. The activatable construct of any one of claims 17-24, wherein the MM1, the MM2, the MM3, and the MM4 comprise the same amino acid sequence.
26. The activatable construct of any one of claims 17-25, wherein the MM1 and the MM4 comprise the same amino acid sequence.
27. The activatable construct of any one of claims 5-26, wherein the MM2 and the MM3 comprise the same amino acid sequence.
28. The activatable construct of any one of claims 14-27, wherein the second polypeptide chain and the third polypeptide chains comprise the same amino acid sequence.
29. The activatable construct of any one of claims 14-28, wherein the first polypeptide chain and the fourth polypeptide chains comprise the same amino acid sequence.
30. The activatable construct of any one of claims 1-22, wherein the first antigen and the second antigen are different epitopes on the same molecule.
31. The activatable construct of any one of claims 1-22, wherein the first antigen and the second antigen are different molecules or different epitopes.
32. The activatable construct of any one of claims 1-29, wherein the MM1 and the MM2 comprise the same amino acid sequence.
33. The activatable construct of any one of claims 5-22 or 26-27, wherein the MM3 is a different sequence amino acid than the MM1, or the MM2, or both the MM1 and the MM2.
34. The activatable construct of claim 33, wherein the MM3 comprises a different amino acid sequence than the MM1 and the MM2.
35. The activatable construct of any one of claims 1-22 or 26-34, wherein the activatable construct is bispecific.
36. The activatable construct of any one of claims 4-22 or 33-35, wherein the second polypeptide chain and the third polypeptide chain comprise different amino acid sequences.
37. The activatable construct of any one of claims 4-22 or 35-36, wherein the first polypeptide chain and the fourth polypeptide chain comprise different amino acid sequences.
38. The activatable construct of any one of claims 17-29, wherein the first polypeptide chain and the fourth polypeptide chain comprise the same amino acid sequence.
39. The activatable construct of claim 35, wherein the amino acid sequence of the HVD1- HC1 is different than the amino acid sequence of the HVD2-HC2, and wherein each is a direct or an indirect covalent linkage.
40. The activatable construct of claim 35, wherein the MM1 and the MM3 are different amino acid sequences.
41. The activatable construct of claim 35, wherein the CM1 and the CM3 are different amino acid sequences.
42. The activatable construct of any one of claims 35-36, wherein the second polypeptide chain and the third polypeptide chain are different amino acid sequences.
43. The activatable construct of claim 35, wherein the CM1 and the CM3 comprise the same amino acid sequence.
44. The activatable construct of claim 35, wherein the first polypeptide chain and the fourth polypeptide chain comprise the same amino acid sequence, and wherein the second polypeptide chain and the third polypeptide chain are different amino acid sequences.
45. The activatable construct of any one of claims 1-35, wherein the DD1 and the DD2 comprise the same amino acid sequence.
46. The activatable construct of any one of claims 36, wherein the DD1 and the DD2 are different amino acid sequences.
47. The activatable construct of any preceding claim, further comprising a third antigen binding domain (AB3) having specific binding affinity for a third antigen, wherein the AB3 comprises a heavy chain variable domain 3 (HVD3) and a light chain variable domain 3 (LVD3), wherein the AB3 is coupled directly or indirectly to a fifth masking moiety (MM5) via a fifth cleavable moiety (CM5), wherein the MM5 inhibits binding of the AB3 to the third antigen.
48. The activatable construct of claim 47, wherein the AB1 is coupled directly or indirectly to the N-terminus of the DD1 and the AB2 is coupled directly or indirectly to the N-terminus of the DD2, and wherein the AB3 is coupled directly or indirectly to the C-terminus of the DD1 or the DD2.
49. The activatable construct of any one of claims 47-48, wherein the AB3 comprises a scFv, wherein the AB3 comprises: i) MM5-CM5-HVD3-LVD3-X; or ii) MM5-CM5-LVD3-HVD3-X; or wherein the AB3 comprises in an N-terminal to C-terminal direction: iii) X-LVD3-HVD3-CM5-MM5; or iv) X-HVD3-LVD3-CM5-MM5; wherein X represents attachment to the N-terminus or the C-terminus of the DD1 or the DD2, and wherein each is a direct or an indirect covalent linkage.
50. The activatable construct of any one of claims 47-49, further comprising a fourth antigen binding domain (AB4) having specific binding affinity for a fourth antigen, wherein the AB4 comprises a heavy chain variable domain 4 (HVD4) and a light chain variable domain 4 (LVD4); wherein the AB4 is coupled directly or indirectly to a sixth masking moiety (MM6) via a sixth cleavable moiety (CM6), wherein the MM6 inhibits binding of the AB4 to the fourth antigen.
51. The activatable construct of claim 50, wherein the AB3 is coupled directly or indirectly to the C-terminus of the DD1, and wherein the AB4 is coupled directly or indirectly to the C- terminus of the DD2.
52. The activatable construct of any one of claims 50-51, wherein the AB4 comprises a scFv, wherein the AB4 comprises: i) MM6-CM6-HVD4-LVD4-Y; or ii) MM6-CM6-LVD4-HVD4- Y; or wherein the AB4 comprises in an N-terminal to C-terminal direction: iii) Y-LVD4-HVD4-CM6-MM6; or iv) Y-HVD4-LVD4-CM6-MM6; wherein Y represents attachment to the N-terminus or the C-terminus of the DD2, and wherein each is a direct or an indirect covalent linkage.
53. An activatable construct, comprising: a first antigen binding domain (AB1) having specific binding affinity for a first antigen, wherein the AB 1 comprises a heavy chain variable domain 1 (HVD1) and a light chain variable domain 1 (LVD1), a first light chain constant domain (LC1), a first heavy chain constant domain (HC1); a first masking moiety (MM1) coupled directly or indirectly to a first cleavable moiety (CM1), wherein the MM1 is coupled directly or indirectly to the HVD1, the LVD1, the HC1, or the LC1 via the CM1; a second antigen binding domain (AB2) having specific binding affinity for a second antigen, wherein the AB2 comprises a heavy chain variable domain 2 (HVD2) and a light chain variable domain 2 (LVD2); a second masking moiety (MM2) coupled directly or indirectly to a second cleavable moiety (CM2), wherein the MM2 is coupled directly or indirectly to the HVD2 or the LVD2 via the CM2; a first dimerization domain (DD1) and a second dimerization domain (DD2), and wherein the MM1 inhibits binding of the AB1 to the first antigen, and wherein the MM2 inhibits binding of the AB2 to the first antigen.
54. The activatable construct of any one of claim 53, wherein the activatable construct comprises a first polypeptide, a second polypeptide, and a third polypeptide.
55. The activatable construct of any one of claims 53-54, wherein the activatable construct further comprises a third cleavable moiety (CM3) and a third masking moiety (MM3).
56. The activatable construct of any one of claims 53-55, wherein the activatable construct further comprises a fourth cleavable moiety (CM4) and a fourth masking moiety (MM3).
57. The activatable construct of any one of claims 53-56, wherein the first polypeptide comprises the MM1, CM1, LVD1, and the LC1, the second polypeptide comprises the HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, and the third polypeptide comprises the MM3, CM3, and the DD2.
58. The activatable construct of claim 57, wherein the MM3 inhibits binding of the AB2 to the second antigen.
59. The activatable construct of any one of claims 57-58, wherein the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
60. The activatable construct of any one of claims 57-59, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, in an N-terminal to C-terminal direction: c) i) MM1-CM1-LVD1-LC1 ii) HVD1-HC1-HVD2-LVD2-CM2-MM2-DD1; and iii) MM3-CM3-DD2; or d) i) MM1-CM1-LVD1-LC1 ii ) HVD 1 -HC 1 -LVD2-HVD2-CM2-MM2-DD 1 ; and iii) MM3-CM3-DD2; wherein each is a direct or an indirect covalent linkage, and wherein the DD1 and the DD2 are dimerized.
61. The activatable construct of any one of claims 57-60, wherein the first polypeptide comprises the MM1, CM1, HVD1, HC1, HVD2, LVD2, CM2, MM2, and the DD1, the second polypeptide comprises the LVD1, LC1, CM3, and the MM3, and the third polypeptide comprises the DD2.
62. The activatable construct of claim 61, wherein the MM3 inhibits binding of the AB2 to the second antigen.
63. The activatable construct of any one of claims 61-62, wherein the MM2 and the MM3 inhibit binding of the AB2 to the second antigen.
64. The activatable construct of any preceding claim, wherein each of the CM1 and the CM2 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one of claims 5-63, wherein each of the CM1, the CM2, the and CM3 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one of claims 17-63, wherein each of the CM1, the CM2, the CM3, and the CM4 independently comprises a substrate for the same or different protease in any combination thereof; the activatable construct of any one of claims 48-63, wherein each of the CM1, CM2, CM3, CM4, and CM5 independently comprises a substrate for the same or different protease in any combination thereof; or the activatable construct of any one of claims 51-63, wherein each of the CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a substrate for the same or different protease in any combination thereof.
65. The activatable construct of claim 64, wherein each of the CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a total of about 3 amino acids to about 15 amino acids.
66. The activatable construct of any one of claims 53-65, wherein each CM1, CM2, CM3, CM4, CM5, and CM6 independently comprises a substrate for a protease selected from the group consisting of ADAMS, AD AMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAMI 7/T ACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, Aspartate proteases, BACE, Renin, Aspartic cathepsins, Cathepsin D, Cathepsin E, Caspases, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cysteine cathepsins, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P, Cysteine constructases, Cruzipain, Legumain, Otubain-2, KLKs, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Metallo proteinases, Meprin, Neprilysin, PSMA, BMP-1, MMPs, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, Serine proteases, activated protein C , Cathepsin A, Cathepsin G, Chymase, coagulation factor proteases, FVIIa, FIXa, FXa, FXIa, FXIIa, Elastase, Granzyme B, Guanidinobenzoatase, HtrAl, Human Neutrophil Elastase, Lactoferrin, Marapsin, NS3/4A, PACE4, Plasmin, PSA, tPA, Thrombin, Tryptase, uPA, Type II Transmembrane, Serine Proteases, TTSPs, DESCI, DPP-4, FAP, Hepsin, Matriptase-2, MT- SPl/Matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.
67. The activatable construct of claim 66, wherein the protease(s) is/are produced by a tumor in a subject.
68. The activatable construct of any preceding claim, wherein each of the MM1 and the MM2 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one of claims 5-67, wherein each of the MM1, the MM2, the and MM3 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one of claims 17-67, wherein each of the MM1, the MM2, the MM3, and the MM4 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; the activatable construct of any one of claims 48-67, wherein each of the MM1, MM2, MM3, MM4, and MM5 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof; or the activatable construct of any one of claims 51-67, wherein each of the MM1, MM2, MM3, MM4, MM5, and MM6 independently comprises a peptide of from 2 to 40 amino acids in length in any combination thereof.
69. The activatable construct of any one of claims 1-35 and 46-68, wherein DD1 is an Fc domain hole mutant and DD2 is an Fc domain knob mutant, or wherein DD1 is an Fc domain knob mutant and DD2 is an Fc domain hole mutant.
70. The activatable construct of any preceding claim, wherein the DD1 and the DD2 are selected from an IgGl Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a mutant or truncation variant thereof.
71. The activatable construct of any preceding claim, wherein DD1 and DD2 are human IgGl Fc domains or a mutant or truncation variant thereof.
72. The activatable construct of any preceding claim, further comprising one or more linkers.
73. The activatable construct of claim 72, wherein the linker is a peptide having a length of 1 to 30, 5 to 25, 10-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.
74. The activatable construct of claim 73, wherein the linker comprises at least 5 amino acids.
75. The activatable construct of any preceding claim, wherein the activatable construct is a multi-specific activatable antibody.
76. The activatable construct of any preceding claim, wherein the activatable construct has a masking efficiency that is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than a corresponding activatable construct with only a single masking moiety per antigen binding domain.
77. The activatable construct of any one of claims 17-76, wherein the activatable construct has a masking efficiency that is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than a corresponding activatable construct that lacks MM3 and MM4.
78. A composition comprising the activatable construct of any one of claims 1-77.
79. The composition of claim 78, further comprising a pharmaceutically acceptable carrier or excipient.
80. A container, vial, syringe, injector pen, or kit comprising at least one dose of the composition of any one of claims 78-79.
81. A method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of the activatable construct of any of claims 1-77 or the composition of any one of claims 78-79.
82. The method of claim 81, wherein the subject has been identified or diagnosed as having a cancer, an autoimmune disease, or an inflammatory disorder.
83. The method of any one of claims 81-82, further comprising administering a second therapeutic agent.
84. The method of claim 83, wherein the second therapeutic agent is a checkpoint inhibitor, a cytokine, or a cytotoxic agent.
85. A nucleic acid encoding a polypeptide of the activatable construct of any one of claims 1-77.
86. An mRNA comprising the nucleic acid of claim 85.
87. The mRNA of claim 86, wherein the mRNA comprises one or more modifications.
88. The mRNA of claim 87, wherein the one or more modifications are selected from a modified or non-naturally occurring nucleotide or 5’ capping structure.
89. A vector comprising the nucleic acid of claim 85.
90. The vector of claim 89, wherein the vector is an expression vector.
91. A composition comprising the nucleic acid of claim 85, the mRNA of any one of claims 86-88, or the vector of any one of claims 89-90.
92. The composition of claim 91, further comprising a pharmaceutically acceptable carrier or excipient.
93. A cell transfected with the nucleic acid of claim 85 or the vector of any one of claims 89- 90.
94. The cell of claim 93, wherein the cell is a mammalian cell.
95. The cell of any one of claims 93-94, wherein the cell is a CHO cell or an HEK293 cell.
96. A method of producing the activatable construct of any one of claims 1-77, comprising culturing the cell of any one of claims 93-95 in a liquid culture medium to produce the activatable construct; and recovering the activatable construct from the liquid culture medium.
97. The method of claim 96, further comprising: purifying the activatable construct recovered from the liquid culture medium to obtain a purified activatable construct.
98. The method of claim 97, further comprising: combining the purified activatable construct with a carrier to form a pharmaceutical composition.
99. An activatable construct produced by the method of any one of claims 96-98.
100. A method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of the nucleic acid of claim 85, the mRNA of any one of claims 86-88, or the vector of any one of claims 89-90.
101. The method of claim 100, wherein the subject has been identified or diagnosed as having a cancer, an autoimmune disease, or an inflammatory disorder.
102. The method of any one of claims 100-101, further comprising administering a second therapeutic agent.
103. The method of claim 102, wherein the second therapeutic agent is a checkpoint inhibitor, a cytokine, or a cytotoxic agent.
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