WO2021061867A1 - Anti-cd47 antibodies, activatable anti-cd47 antibodies, and methods of use thereof - Google Patents

Anti-cd47 antibodies, activatable anti-cd47 antibodies, and methods of use thereof Download PDF

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Publication number
WO2021061867A1
WO2021061867A1 PCT/US2020/052332 US2020052332W WO2021061867A1 WO 2021061867 A1 WO2021061867 A1 WO 2021061867A1 US 2020052332 W US2020052332 W US 2020052332W WO 2021061867 A1 WO2021061867 A1 WO 2021061867A1
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seq
amino acid
acid sequence
antibody
sequence
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PCT/US2020/052332
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French (fr)
Inventor
Li Mei
Jeanne Grace Flandez
Dylan DANIEL
Kimberly Ann TIPTON
Daniel Robert Hostetter
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Cytomx Therapeutics, Inc.
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Priority to JP2022518171A priority Critical patent/JP2022548310A/en
Priority to CN202080076276.1A priority patent/CN114650844A/en
Priority to EP20789345.4A priority patent/EP4034171A1/en
Publication of WO2021061867A1 publication Critical patent/WO2021061867A1/en

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    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the invention relates generally to antibodies that specifically bind to CD47, activatable antibodies that specifically bind to CD47, and methods of making and using these antibodies and activatable antibodies in a variety of therapeutic, prophylactic, and diagnostic contexts.
  • prodrugs of an active chemical entity are administered in a relatively inactive (or significantly less active) form. Once administered, the prodrug is metabolized in vivo into the active compound.
  • prodrug strategies can provide for increased selectivity of the drug for its intended target and for a reduction of adverse effects.
  • BRIEF SUMMARY OF THE INVENTION Provided herein are activatable antibodies that bind to CD47 and methods of making and using these activatable antibodies in a variety of therapeutic, prophylactic, and diagnostic contexts.
  • the activatable antibodies bind human and cynomolgus monkey CD47.
  • an activatable antibody that, in an activated state, binds CD47 comprising: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to human CD47 and cynomolgus monkey CD47; (b) a masking moiety (MM) coupled to the AB, wherein the MM inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved (unactivated state) state, and wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16- 29; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
  • AB antibody or an antigen binding fragment thereof
  • MM masking moiety
  • any one of the activatable antibodies provided herein are conjugated to an agent, generating a conjugated activatable antibody.
  • the agent is a toxin or fragment thereof.
  • the agent is a microtubule inhibitor.
  • the agent is a nucleic acid damaging agent.
  • the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.
  • a conjugated activatable antibody comprising: an antibody or antigen binding fragment thereof (AB) that, in an activated state, binds CD47; and a toxin conjugated to the AB via a linker, wherein the conjugated activatable antibody comprises amino acid sequences, a linker, and a toxin selected from a single row in Table D, wherein for the given combination: (a) the AB comprises a heavy chain comprising the amino acid sequence of the heavy chain sequence or heavy chain variable domain sequence corresponding to the given combination in the single row listed in Table D; (b) the AB comprises a light chain comprising the amino acid sequence of the light chain sequence or light chain variable domain sequence corresponding to the given combination in the single row listed in Table D; and (c) the linker and the toxin comprise the linker and the toxin corresponding to the given combination in the single row listed in Table D.
  • AB antibody or antigen binding fragment thereof
  • a pharmaceutical composition comprising any of the activatable antibodies or conjugated activatable antibodies provided herein; and a carrier.
  • the pharmaceutical composition of comprises an additional agent.
  • the additional agent is a therapeutic agent.
  • an isolated nucleic acid molecule encoding any one of the activatable antibodies described herein.
  • a vector comprising the isolated nucleic acid molecule.
  • a method of producing an activatable antibody by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises the nucleic acid molecules or the vectors provided herein.
  • a method of manufacturing any one of the activatable antibodies provided herein that, in an activated state, binds CD47 comprising: (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to the expression of any one of the activatable antibodies described herein, and (b) recovering the activatable antibody.
  • provided herein is a method of preventing, treating, alleviating a symptom of, delaying the progression of, or otherwise ameliorating a disorder or disease in which diseased cells express CD47 or a CD47-medidated disease or disorder comprising administering a therapeutically effective amount of any one of the activatable antibodies or conjugated activatable antibodies provided herein, or pharmaceutical compositions of the same to a subject in need thereof.
  • provided herein is a method of preventing, treating, alleviating a symptom of, delaying the progression of, or otherwise ameliorating a disorder or disease associated with cells expressing CD47 or a CD47-medidated disease or disorder comprising administering a therapeutically effective amount of any one of the activatable antibodies or conjugated activatable antibodies provided herein, or pharmaceutical compositions of the same to a subject in need thereof.
  • the disorder or disease is cancer.
  • the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2- negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non- small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell
  • a method of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing CD47 comprising administering a therapeutically effective amount of any one of activatable antibodies or conjugated activatable antibodies provided herein, or pharmaceutical compositions of the same to a subject in need thereof.
  • the expression and/or activity of the CD47 is aberrant.
  • a method of inhibiting, blocking, or preventing the binding of a natural ligand to CD47 comprising administering a therapeutically effective amount of any one of the activatable antibodies or conjugated activatable antibodies provided herein, or pharmaceutical compositions of the same to a subject in need thereof.
  • the expression and/or activity of the CD47 is aberrant.
  • activatable antibodies, conjugated activatable antibody, pharmaceutical composition thereof, and methods of use thereof wherein the activatable antibodies or conjugated activatable when bound to CD47, blocks the interaction of CD47 with SIRPa.
  • FIGS.1A and 1B are graphs depicting exemplary in vitro studies of the ability of anti-human CD47 activatable antibodies of the disclosure to bind human CD47 antigen.
  • FIG.2 is a graph depicting exemplary in vitro studies of the ability of anti-human CD47 activatable antibodies of the disclosure to activate antibody-dependent cell phagocytosis (ADCP).
  • FIGS.3A-3D are graphs depicting exemplary in vitro studies of the ability of anti- human CD47 antibodies and anti-CD47 activatable antibodies of the disclosure to activate ADCP.
  • FIGS.4A-4C are graphs depicting exemplary in vitro studies of the ability of anti- human CD47 antibodies of the disclosure to activate ADCP.
  • FIGS.5A and 5B are graphs depicting an exemplary study of the in vivo efficacy of anti-human CD47 antibodies in mouse xenograft models.
  • CD47 also known as integrin associated protein (IAP) is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD47 has been shown to interact with membrane integrins on other cells. CD47 has also been shown to bind the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPa). CD47 can protect cancer cells from the immune system by preventing consumption of the cells by myeloid cells, such as a macrophages. CD47 has been shown to be expressed in human cells and overexpressed on many tumor cells.
  • IAP integrin associated protein
  • Blocking of the interaction between CD47 on a target cell and the SIRPa on a macrophage or other phagocytic cell can induce phagocytosis of the target cell.
  • blocking of the interaction between CD47 on a target cell and the SIRPa on a macrophage or other phagocytic cell can induce phagocytosis of the target cell along with engagement by the Fc receptor on the phagocytic cell.
  • the Fc receptor engagement can be performed by a Fc domain on a CD47-binding antibody.
  • the present disclosure provides antibodies, activatable antibodies, conjugated antibodies, and conjugated activatable antibodies that specifically bind mammalian CD47, methods of making and use thereof.
  • the disclosure provides anti-mammalian CD47 antibodies and fragments thereof (interchangeably referred to herein as CD47 antibodies, or ABs), conjugated CD47 antibodies, activatable CD47 antibodies, and conjugated activatable CD47 antibodies that are useful in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a disease or disorder associated with cells expressing CD47.
  • the cells are associated with normal CD47 expression and/or activity.
  • the cells are associated with aberrant CD47 expression and/or activity. In some embodiments, the cells are associated with CD47 expression and/or activity in diseased cells.
  • any of the antibodies/activatable antibodies described herein can be used in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a cancer or other neoplastic condition. Any of the antibodies/activatable antibodies described herein can also be used for detection/diagnostic applications.
  • the antibodies and activatable antibodies specifically bind human CD47 and cynomolgus monkey CD47. In some embodiments, the antibodies and activatable antibodies bind human CD47.
  • the antibodies and activatable antibodies bind cynomolgus monkey CD47. In some embodiments, the antibodies and activatable antibodies are internalized by CD47-containing cells. In some embodiments, the antibodies and activatable antibodies bind both the glycosylated and deglycosylated forms of the CD47 antigen. [00028] In some embodiments of the disclosure, the antibodies and activatable antibodies specifically bind human CD47 and cynomolgus monkey CD47 and block interaction with SIRPa. In some embodiments of the disclosure, the antibodies and activatable antibodies specifically bind human CD47 and cynomolgus monkey CD47 and activate antibody-dependent cell phagocytosis.
  • the antibodies and activatable antibodies specifically bind human CD47 and cynomolgus monkey CD47 and engage Fc receptor pathway.
  • the term “a” entity or “an” entity refers to one or more of that entity.
  • a compound refers to one or more compounds.
  • the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably.
  • singular terms shall include pluralities and plural terms shall include the singular.
  • the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin
  • Ig immunoglobulin
  • bind or “immunoreacts with” or “immunospecifically bind” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd > 10 -6 ).
  • Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab, and F(ab’) 2 fragments, scFvs, and an Fab expression library.
  • the antibodies provided herein can be of any of the IgG, IgM, IgA, IgE and IgD classes (or subclasses thereof).
  • the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
  • MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
  • the term “antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding.
  • the antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L ” ains.
  • FR refers to amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
  • the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three- dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.”
  • CDRs complementarity-determining regions
  • epitopic determinants includes any protein determinant capable of specific binding to an immunoglobulin, a scFv, or a T-cell receptor.
  • epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N-terminal or C-terminal peptides of a polypeptide.
  • an antibody is said to specifically bind an antigen when the dissociation constant is £ 1 ⁇ M; in some embodiments, £ 100 nM and in some embodiments, £ 10 nM.
  • the terms “specific binding,” “immunological binding,” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art.
  • One such method entails measuring the rates of antigen- binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
  • both the on rate constant (Kon) and the “off rate constant” (K off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
  • the ratio of K off /K on enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).
  • An antibody of the present disclosure is said to specifically bind to the target, when the binding constant (Kd) is £ 1 ⁇ M, in some embodiments £100 nM, in some embodiments ⁇ 10 nM, and in some embodiments £ 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.
  • Kd binding constant
  • isolated polynucleotide shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • polynucleotides in accordance with the disclosure include the nucleic acid molecules encoding the heavy chain immunoglobulin molecules shown herein, and nucleic acid molecules encoding the light chain immunoglobulin molecules shown herein.
  • isolated protein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of murine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • polypeptide is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the disclosure comprise the heavy chain immunoglobulin molecules shown herein, and the light chain immunoglobulin molecules shown herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof. [00039]
  • the term naturally-occurring as used herein as applied to an object refers to the fact that an object can be found in nature.
  • control sequence refers to polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated.
  • control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • polynucleotide as referred to herein means nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • oligonucleotide includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. In some embodiments, oligonucleotides are 10 to 60 bases in length and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length.
  • Oligonucleotides are usually single stranded, e.g., for probes, although oligonucleotides may be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides of the disclosure are either sense or antisense oligonucleotides.
  • the term “naturally occurring nucleotides” referred to herein includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides referred to herein includes nucleotides with modified or substituted sugar groups and the like.
  • oligonucleotide linkages includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselerloate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res.14:9081 (1986); Stec et al. J. Am. Chem. Soc.106:6077 (1984), Stein et al. Nucl. Acids Res.16:3209 (1988), Zon et al.
  • oligonucleotide can include a label for detection, if desired.
  • the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Green, Eds., Sinauer Associates, Sunderland, Mass. (1991)).
  • Stereoisomers e.g., D-amino acids of the twenty conventional amino acids, unnatural amino acids such as a-, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present disclosure.
  • Examples of unconventional amino acids include: 4 hydroxyproline, g-carboxyglutamate, e-N,N,N-trimethyllysine, e -N- acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • the left-hand end of single-stranded polynucleotide sequences is the 5’ end the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • the direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction sequence regions on the DNA strand having the same sequence as the RNA and that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”, sequence regions on the DNA strand having the same sequence as the RNA and that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences”.
  • the term substantial identity means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, in some embodiments, at least 90 percent sequence identity, in some embodiments, at least 95 percent sequence identity, and in some embodiments, at least 99 percent sequence identity. [00047] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, in some embodiments, at least 80%, 90%, 95%, and in some embodiments, 99%.
  • conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.
  • the hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine.
  • the hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine.
  • Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family.
  • Suitable amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains.
  • Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases.
  • computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three- dimensional structure are known. Bowie et al. Science 253:164 (1991).
  • Suitable amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties of such analogs.
  • Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (for example, conservative amino acid substitutions) can be made in the naturally occurring sequence (for example, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).
  • polypeptide fragment refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion and/or one or more internal deletion(s), but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, in some embodiments, at least 14 amino acids long, in some embodiments, at least 20 amino acids long, usually at least 50 amino acids long, and in some embodiments, at least 70 amino acids long.
  • analog refers to polypeptides that are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and that has specific binding to the target, under suitable binding conditions.
  • polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally occurring sequence.
  • Analogs typically are at least 20 amino acids long, in some embodiments, at least 50 amino acids long or longer, and can often be as long as a full-length naturally occurring polypeptide.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • label refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of 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). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • radioisotopes or radionuclides e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw- Hill, San Francisco (1985)).
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and in some embodiments, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
  • a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, in some embodiments, more than about 85%, 90%, 95%, and 99%.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • patient includes human and veterinary subjects.
  • CD47 Antibodies Provided herein are antibodies and antigen binding fragments thereof (ABs) that specifically bind to mammalian CD47. In some embodiments, the AB specifically binds human CD47 and cynomolgus monkey CD47.
  • the ABs provided herein that bind CD47 includes a monoclonal antibody, a domain antibody, a single chain antibody, 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.
  • such an ABs that binds CD47 is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.
  • activatable CD47 antibodies that include an antibody or antigen-binding fragment thereof (AB) that specifically binds CD47 coupled to a masking moiety (MM), such that coupling of the MM reduces the ability of the antibody or antigen- binding fragment thereof to bind CD47.
  • the MM is coupled via a sequence that includes a substrate for a protease (cleavable moiety, CM), for example, a protease that is co-localized with CD47 at a treatment site in a subject.
  • CM protease
  • the activatable CD47 antibodies of the disclosure are described in greater detail in a below section).
  • the CD47 antibodies of the disclosure specifically bind a mammalian CD47 target, such as, for example, human CD47. Also included in the disclosure are CD47 antibodies and ABs that bind to the same CD47 epitope as an antibody of the disclosure and/or an activated activatable antibody described herein. Also included in the disclosure are CD47 antibodies compete with a CD47 antibody described herein for binding to a CD47 target, e.g., human CD47. Also included in the disclosure are CD47 antibodies that cross-compete with (inhibit the binding of) a CD47 antibody and/or an activated CD47 activatable antibody described herein for binding to a CD47 target, e.g., human CD47.
  • Antibodies and/or activatable antibodies of the disclosure specifically bind a mammalian CD47, e.g. human CD47 and cynomologous CD47. Also included in the disclosure are antibodies and/or activatable antibodies that bind to the same epitope as any of the antibodies and/or activatable antibodies described herein. Also included in the disclosure are antibodies and/or antibodies activatable antibodies that compete with a CD47 antibody (inhibit the binding of) and/or a CD47 activatable antibody described herein for binding to CD47, e.g., human CD47.
  • the mammalian CD47 is selected from the group consisting of a human CD47, a murine CD47, a rat CD47, and a cynomolgus monkey CD47.
  • the AB specifically binds to human CD47, murine CD47 or cynomolgus monkey CD47 with a dissociation constant of less than 1 nM.
  • the mammalian CD47 is a human CD47.
  • the AB has one or more of the following characteristics: (a) the AB specifically binds to human CD47; and (b) the AB specifically binds to human CD47 and cynomolgus monkey CD47.
  • the AB has one or more of the following characteristics: (a) the AB specifically binds human CD47 and cynomolgus monkey CD47; (b) the AB inhibits binding of one or more of the natural mammalian ligands of CD47 to mammalian CD47; (c) the AB inhibits binding of one or more of the natural human ligands of CD47 to human CD47; and (d) the AB inhibits binding of one or more of the natural cynomolgus monkey ligands of CD47 to cynomolgus monkey CD47. [00065] In some embodiments the AB binds both glycosylated and deglycosylated forms of CD47.
  • the AB blocks the ability of a natural ligand to bind to the mammalian CD47 with an EC 50 less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 500 nM, and/or less than or equal to 1000 nM.
  • the AB blocks the ability of a natural ligand to bind to the mammalian CD47 with an EC50 less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 500 nM, and/or less than or equal to 1000 nM.
  • the AB blocks the ability of a natural ligand to bind to the mammalian CD47 with an EC 50 of 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM 10 nM to 50 nM, 50 nM to 1000 nM, 50 nM to 500 nM, 50 nM to 100 nM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 1000 nM.
  • the AB blocks the ability of a natural ligand to bind to the mammalian CD47 with an EC 50 of 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM 10 nM to 50 nM, 50 nM to 1000 nM, 50 nM to 500 nM, 50 nM to 100 nM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 1000 nM.
  • the AB of the present disclosure inhibits or reduces the growth, proliferation, and/or metastasis of cells expressing mammalian CD47. Without intending to be bound by any theory, the AB of the present disclosure may inhibit or reduce the growth, proliferation, and/or metastasis of cells expressing mammalian CD47 by specifically binding to CD47 and inhibiting, blocking, and/or preventing the binding of a natural ligand to mammalian CD47. [00069] In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to mammalian CD47. In some embodiments, the AB has a dissociation constant of about 10 nM or less for binding to mammalian CD47.
  • the AB has a dissociation constant of about 5 nM or less for binding to CD47. In some embodiments, the AB has a dissociation constant of about 1 nM or less for binding to CD47. In some embodiments, the AB has a dissociation constant of about 0.5 nM or less for binding to CD47. In some embodiments, the AB has a dissociation constant of about 0.1 nM or less for binding to CD47.
  • the AB has a dissociation constant of 0.01 nM to 100 nM, 0.01 nM to 10 nM, 0.01 nM to 5 nM, 0.01 nM to 1 nM, 0.01 to 0.5 nM, 0.01 nm to 0.1 nM, 0.01 nm to 0.05 nM, 0.05 nM to 100 nM, 0.05 nM to 10 nM, 0.05 nM to 5 nM, 0.05 nM to 1 nM, 0.05 to 0.5 nM, 0.05 nm to 0.1 nM, 0.1 nM to 100 nM, 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM, 0.1 to 0.5 nM, 0.5 nM to 100 nM, 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM, 0.1 to
  • Exemplary CD47 antibodies and activatable CD47 antibodies of the invention may include a heavy chain and a light chain that are, or are derived from, the heavy chain variable and light chain variable sequences shown below (CDR sequences are shown in bold and underline): hu H4L2 H4 VH: QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDKSTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 1) hu H4L2 L2 VL: DVVMTQSPDSLAVSLGERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 2) [00071] In some embodiments, the antibody
  • the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a light chain variable region amino acid sequence of any one of SEQ ID NOs: 2 and 76-79. [00073] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1. [00074] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1.
  • the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 2.
  • Exemplary CD47 antibodies and activatable CD47 antibodies of the invention include a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid
  • the CD47 antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPY
  • the CD47 antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO:
  • the CD47 antibody or antigen-binding fragment thereof comprises an amino acid sequence comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10).
  • the CD47 antibody or antigen-binding fragment thereof comprises an amino acid sequence comprising the VH CDR1 sequence comprising the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10).
  • the CD47 antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, comprising a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid
  • Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 1 and 57-59, and a light chain variable region amino acid sequence of any one of SEQ ID NOs: 2 and 76-79.
  • Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a heavy chain variable region amino acid sequence SEQ ID NO: 1, and a light chain variable region amino acid sequence SEQ ID NO: 2.
  • Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQS
  • Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 1 and 57-59, and a light chain variable region amino acid sequence of any one of SEQ ID NOs: 2 and 76-79.
  • Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 to a CD47 antibody comprising a heavy chain variable region amino acid sequence SEQ ID NO: 1, and a light chain variable region amino acid sequence SEQ ID NO: 2..
  • Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 to a CD47 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO:
  • the CD47 antibody of the disclosure comprises an isolated antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47.
  • AB antigen binding fragment thereof
  • the antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence GYT
  • the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2.
  • the isolated antibody or antigen binding fragment thereof binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as an isolated antibody that comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence
  • the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2.
  • the isolated antibody or antigen binding fragment thereof cross-competes with (inhibits the binding of) an isolated antibody that comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTY
  • the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2.
  • the CD47 antibody/activatable CD47 antibody comprises or is derived from an antibody that is manufactured, secreted or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) disclosed in US Patent No.5,330,896 and deposited at ATCC under deposit number HB 8214.
  • the CD47 antibody/activatable CD47 antibody comprises or is derived from an antibody that is manufactured, secreted or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) designated BA120 as disclosed in US Patent No.7,736,647 and deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) (Institut Pasteur, Paris, France, 25, Rue du Dondel Roux, F-75724, Paris, Cedex 15) on Jun.14, 2005, under number CNCM I-3449; the hybridoma(s) disclosed in US Patent No. 7,572,895 and deposited at the ATCC under PTA-6055; the hybridoma(s) disclosed in PCT Publication No.
  • CNCM Collection Nationale de Cultures de Microorganismes
  • the CD47 antibody/activatable CD47 antibody includes a heavy chain that comprises or is derived from a heavy chain amino acid sequence shown in PCT Publication Nos. WO 2014/144060, WO 2014/189973, WO 2014/020140, in US Patent Nos.
  • the disclosure also provides methods for producing a CD47 AB of the disclosure by culturing a cell under conditions that lead to expression of the antibody or fragment thereof, wherein the cell comprises a nucleic acid molecule of the disclosure or a vector of the disclosure.
  • the CD47 antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain variable region amino acid sequence comprising an amino acid sequence of SEQ ID NO: 1.
  • the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14.
  • the CD47 antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence come rising an amino acid sequence of SEQ ID NO: 15.
  • the CD47 antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a heavy chain variable region amino acid sequence comprising an amino acid sequence of SEQ ID NO: 1.
  • the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14.
  • the CD47 antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a light chain amino acid sequence come rising an amino acid sequence of SEQ ID NO: 15.
  • Activatable CD47 Antibodies As described above, the disclosure also provides activatable antibodies that include an antibody or antigen-binding fragment thereof that specifically binds CD47 coupled to a masking moiety (MM), such that coupling of the MM reduces the ability of the antibody or antigen-binding fragment thereof to bind CD47.
  • MM masking moiety
  • the MM is coupled via a sequence that includes a substrate for a protease (CM, cleavable moiety), for example, a protease that is active in diseased tissue and/or a protease that is co-localized with CD47 at a treatment site in a subject.
  • CM protease
  • the activatable CD47 antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, e.g., healthy tissue or other tissue not targeted for treatment and/or diagnosis, and, when activated, exhibit binding to CD47 that is at least comparable to the corresponding, unmodified antibody, also referred to herein as the parental antibody.
  • the activatable CD47 antibodies described herein overcome a limitation of antibody therapeutics, particularly antibody therapeutics that are known to be toxic to at least some degree in vivo. Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies.
  • the activatable CD47 antibodies provided herein are designed to address the toxicity associated with the inhibition of the target in normal tissues by traditional therapeutic antibodies. These activatable CD47 antibodies remain masked until proteolytically activated at the site of disease. Starting with a CD47 antibody as a parental therapeutic antibody, the activatable CD47 antibodies of the invention were engineered by coupling the antibody to an inhibitory mask through a linker that incorporates a protease substrate.
  • cleaved state of the activatable antibody refers to the condition of the activatable antibodies following modification of the CM by at least one protease.
  • uncleaved state refers to the condition of the activatable antibodies in the absence of cleavage of the CM by a protease.
  • activatable antibodies is used herein to refer to an activatable antibody in both its uncleaved (native) state, as well as in its cleaved state.
  • a cleaved activatable antibody may lack an MM due to cleavage of the CM by protease, resulting in release of at least the MM (e.g., where the MM is not joined to the activatable antibodies by a covalent bond (e.g., a disulfide bond between cysteine residues).
  • a covalent bond e.g., a disulfide bond between cysteine residues
  • activatable or switchable By activatable or switchable is meant that the activatable antibody exhibits a first level of binding to a target when the activatable antibody is in a inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target in the uninhibited, unmasked and/or cleaved state (i.e., a second conformation), where the second level of target binding is greater than the first level of binding.
  • the access of target to the AB of the activatable antibody is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease, than in the absence of such a cleaving agent.
  • the CM and AB of the activatable antibodies are selected so that the AB represents a binding moiety for a given target, and the CM represents a substrate for a protease.
  • the protease is co-localized with the target at a treatment site or diagnostic site in a subject. As used herein, co-localized refers to being at the same site or relatively close nearby.
  • a protease cleaves a CM yielding an activated antibody that binds to a target located nearby the cleavage site.
  • the activatable antibodies disclosed herein find particular use where, for example, a protease capable of cleaving a site in the CM, i.e., a protease, is present at relatively higher levels in target-containing tissue of a treatment site or diagnostic site than in tissue of non-treatment sites (for example in healthy tissue).
  • a CM of the disclosure is also cleaved by one or more other proteases.
  • activatable antibodies provide for reduced toxicity and/or adverse side effects that could otherwise result from binding of the AB at non-treatment sites if the AB were not masked or otherwise inhibited from binding to the target.
  • an activatable antibody can be designed by selecting an AB of interest (such as any CD47 antibody or fragment thereof described herein) and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides for masking of the AB or reduction of binding of the AB to its target.
  • Structural design criteria can be to be taken into account to provide for this functional feature.
  • Activatable antibodies exhibiting a switchable phenotype of a desired dynamic range for target binding in an inhibited versus an uninhibited conformation are provided.
  • Dynamic range generally refers to a ratio of (a) a maximum detected level of a parameter under a first set of conditions to (b) a minimum detected value of that parameter under a second set of conditions.
  • the dynamic range refers to the ratio of (a) a maximum detected level of target protein binding to an activatable antibody in the presence of at least one protease capable of cleaving the CM of the activatable antibodies to (b) a minimum detected level of target protein binding to an activatable antibody in the absence of the protease.
  • the dynamic range of an activatable antibody can be calculated as the ratio of the dissociation constant of an activatable antibody cleaving agent (e.g., enzyme) treatment to the dissociation constant of the activatable antibodies cleaving agent treatment. The greater the dynamic range of an activatable antibody, the better the switchable phenotype of the activatable antibody.
  • Activatable antibodies having relatively higher dynamic range values exhibit more desirable switching phenotypes such that target protein binding by the activatable antibodies occurs to a greater extent (e.g., predominantly occurs) in the presence of a cleaving agent (e.g., enzyme) capable of cleaving the CM of the activatable antibodies than in the absence of a cleaving agent.
  • a cleaving agent e.g., enzyme
  • the activatable CD47 antibodies provided herein include a masking moiety (MM).
  • the masking moiety is an amino acid sequence that is coupled or otherwise attached to the CD47 antibody and is positioned within the activatable CD47 antibody construct such that the masking moiety reduces the ability of the CD47 antibody to specifically bind CD47.
  • Suitable masking moieties are identified using any of a variety of known techniques. For example, peptide masking moieties are identified using the methods described in PCT Publication No. WO 2009/025846 by Daugherty et al., the contents of which are hereby incorporated by reference in their entirety.
  • the activatable CD47 antibodies provided herein include a cleavable moiety (CM).
  • the cleavable moiety includes an amino acid sequence that is a substrate for a protease, usually an extracellular protease.
  • Suitable substrates are identified using any of a variety of known techniques. For example, peptide substrates are identified using the methods described in U.S.
  • Exemplary substrates include but are not limited to substrates cleavable by one or more of the following enzymes or proteases listed in Table A. Table A: Exemplary Proteases and/or Enzymes
  • the activatable antibodies in an activated state bind CD47 and include (i) an antibody or an antigen binding fragment thereof (AB) that specifically binds to CD47; (ii) a masking moiety (MM) that, when the activatable antibody is in an uncleaved state, inhibits the binding of the AB to CD47; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
  • the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM- MM.
  • the activatable antibody comprises a linking peptide between the MM and the CM.
  • the activatable antibody comprises a linking peptide between the CM and the AB.
  • the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM- LP2-AB or AB-LP2-CM-LP1-MM.
  • the two linking peptides need not be identical to each other.
  • At least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 116) and (GGGS) n (SEQ ID NO: 117), where n is an integer of at least one.
  • At least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 118), GGSGG (SEQ ID NO: 119), GSGSG (SEQ ID NO: 120), GSGGG (SEQ ID NO: 121), GGGSG (SEQ ID NO: 122), and GSSSG (SEQ ID NO: 123).
  • LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 124), GSSGGSGGSGG (SEQ ID NO: 125), GSSGGSGGSGGS (SEQ ID NO: 126), GSSGGSGGSGGSGGGS (SEQ ID NO: 127), GSSGGSGGSG (SEQ ID NO: 128), GSSGGSGGSGS (SEQ ID NO: 129), or GGGSSGGS (SEQ ID NO: 134).
  • LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 130), GSSGT (SEQ ID NO: 131) or GSSG (SEQ ID NO: 132).
  • the antibody or antigen-binding fragment thereof that binds CD47 is a monoclonal antibody, domain antibody, single chain, 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.
  • such an antibody or antigen-binding fragment thereof that binds CD47 is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.
  • the activatable antibody in an uncleaved state specifically binds to the mammalian CD47 with a dissociation constant less than or equal to 1 nM, less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 15 nM, less than or equal to 20 nM, less than or equal to 25 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 150 nM, less than or equal to 250 nM, less than or equal to 500 nM, less than or equal to 750 nM, less than or equal to 1000 nM, and/or less than or equal to 2000 nM.
  • a dissociation constant less than or equal to 1 nM, less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 15 nM, less than or equal to 20 nM, less than or equal to 25 nM, less than or equal to 50 nM, less
  • the activatable antibody in an uncleaved state specifically binds to the mammalian CD47 with a dissociation constant in the range of 1 nM to 2000 nM, 1 nM to 1000 nM, 1 nM to 750 nM, 1 nM to 500 nM, 1 nM to 250 nM, 1 nM to 150 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 25 nM, 1 nM to 15 nM, 1 nM to 10 nM, 1 nM to 5 nM, 5 nM to 2000 nM, 5 nM to 1000 nM, 5 nM to 750 nM, 5 nM to 500 nM, 5 nM to 250 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 25 nM
  • the activatable antibody in an activated state specifically binds to the mammalian CD47 with a dissociation constant is less than or equal to 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM.
  • the activatable antibody in an activated state specifically binds to the mammalian CD47 with a dissociation constant in the range of 0.01 nM to 100 nM, 0.01 nM to 10 nM, 0.01 nM to 5 nM, 0.01 nM to 1 nM, 0.01 to 0.5 nM, 0.01 nm to 0.1 nM, 0.01 nm to 0.05 nM, 0.05 nM to 100 nM, 0.05 nM to 10 nM, 0.05 nM to 5 nM, 0.05 nM to 1 nM, 0.05 to 0.5 nM, 0.05 nm to 0.1 nM, 0.1 nM to 100 nM, 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM, 0.1 to 0.5 nM, 0.5 nM to 100 nM, 0.1 nM to 10 nM, 0.1 n
  • the Kd of the AB modified with a MM towards the CD47 target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10- 100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000- 100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times greater than the K d of the AB not modified with an MM or of the parental AB towards the CD47 target.
  • the binding affinity of the AB modified with a MM towards the CD47 target is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10- 100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the AB not modified with an MM or of the parental AB towards the CD47 target.
  • the dissociation constant (Kd) of the MM towards the AB is generally greater than the K d of the AB towards the CD47 target.
  • the K d of the MM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times greater than the Kd of the AB towards the CD47 target.
  • the binding affinity of the MM towards the AB is generally lower than the binding affinity of the AB towards the CD47 target.
  • the binding affinity of MM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times lower than the binding affinity of the AB towards the CD47 target.
  • the dissociation constant (K d ) of the MM towards the AB is approximately equal to the Kd of the AB towards the CD47 target. In some embodiments, the dissociation constant (K d ) of the MM towards the AB is no more than the dissociation constant of the AB towards the CD47 target. In some embodiments, the dissociation constant (K d ) of the MM towards the AB is equivalent to the dissociation constant of the AB towards the CD47 target. [000131] In some embodiments, the dissociation constant (K d ) of the MM towards the AB is less than the dissociation constant of the AB towards the CD47 target.
  • the dissociation constant (Kd) of the MM towards the AB is greater than the dissociation constant of the AB towards the CD47 target.
  • the MM has a K d for binding to the AB that is no more than the Kd for binding of the AB to the target.
  • the MM has a Kd for binding to the AB that is no less than the Kd for binding of the AB to the target.
  • the MM has a K d for binding to the AB that is approximately equal to the Kd for binding of the AB to the target.
  • the MM has a Kd for binding to the AB that is less than the K d for binding of the AB to the target. [000137] In some embodiments, the MM has a K d for binding to the AB that is greater than the Kd for binding of the AB to the target. [000138] In some embodiments, the MM has a K d for binding to the AB that is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 fold greater than the K d for binding of the AB to the target.
  • the MM has a Kd for binding to the AB that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 fold greater than the K d for binding of the AB to the target.
  • the MM has an affinity for binding to the AB that is less than the affinity of binding of the AB to the target.
  • the MM has an affinity for binding to the AB that is no more than the affinity of binding of the AB to the target.
  • the MM has an affinity for binding to the AB that is approximately equal of the affinity of binding of the AB to the target.
  • the MM has an affinity for binding to the AB that is no less than the affinity of binding of the AB to the target. [000143] In some embodiments, the MM has an affinity for binding to the AB that is greater than the affinity of binding of the AB to the target. [000144] In some embodiments, the MM has an affinity for binding to the AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 less than the affinity of binding of the AB to the target.
  • the MM has an affinity for binding to the AB that is between 1-5, 2-5, 2- 10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 fold less than the affinity of binding of the AB to the target. In some embodiments, the MM has an affinity for binding to the AB that is 2 to 20-fold less than the affinity of binding of the AB to the target. In some embodiments, a MM not covalently linked to the AB and at equimolar concentration to the AB does not inhibit the binding of the AB to the target.
  • the AB’s ability to bind the target when modified with an MM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more when measured in vivo or in an in vitro assay.
  • the MM inhibits the binding of the AB to the target.
  • the MM binds the antigen binding domain of the AB and inhibits binding of the AB to the target.
  • the MM can sterically inhibit the binding of the AB to the target.
  • the MM can allosterically inhibit the binding of the AB to its target.
  • the AB when the AB is modified or coupled to a MM and in the presence of target there is no binding or substantially no binding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target, as compared to the binding of the AB not modified with an MM, the parental AB, or the AB not coupled to an MM to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay.
  • the MM ‘masks’ or reduces or otherwise inhibits the specific binding of the AB to the target.
  • such coupling or modification can effect a structural change that reduces or inhibits the ability of the AB to specifically bind its target.
  • An AB coupled to or modified with an MM can be represented by the following formulae (in order from an amino (N) terminal region to carboxyl (C) terminal region: (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) where MM is a masking moiety, the AB is an antibody or antibody fragment thereof, and the L is a linker.
  • the MM is not a natural binding partner of the AB.
  • the MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB.
  • the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB.
  • the activatable antibodies include an AB that is modified by an MM and also includes one or more cleavable moieties (CM). Such activatable antibodies exhibit activatable/switchable binding, to the AB’s target. Activatable antibodies generally include an antibody or antibody fragment (AB), modified by or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM).
  • the CM contains an amino acid sequence that serves as a substrate for at least one protease.
  • the elements of the activatable antibodies are arranged so that the MM and CM are positioned such that in a cleaved (or relatively active) state and in the presence of a target, the AB binds a target while the activatable antibody is in an uncleaved (or relatively inactive) state in the presence of the target, specific binding of the AB to its target is reduced or inhibited.
  • the specific binding of the AB to its target can be reduced due to the inhibition or masking of the AB’s ability to specifically bind its target by the MM.
  • the Kd of the AB modified with a MM and a CM towards the CD47 target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10- 10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100- 1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000- 10,000,000 times greater than the Kd of the AB not modified with an MM and a CM or of the parental AB towards the CD47 target.
  • the binding affinity of the AB modified with a MM and a CM towards the CD47 target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10- 10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000- 10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the AB not modified with an MM and a CM or of the parental AB towards the CD47 target.
  • the AB is modified with a MM and a CM and is in the presence of the target but not in the presence of a modifying agent (for example at least one protease)
  • a modifying agent for example at least one protease
  • the AB’s ability to bind the target when modified with an MM and a CM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay.
  • Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulae for activatable antibodies are provided below. It is specifically contemplated that the N- to C-terminal order of the AB, MM and CM can be reversed within an activatable antibody. It is also specifically contemplated that the CM and MM may overlap in amino acid sequence, e.g., such that the CM is contained within the MM.
  • activatable antibodies can be represented by the following formula (in order from an amino (N) terminal region to carboxyl (C) terminal region: (MM)-(CM)-(AB) (AB)-(CM)-(MM) where MM is a masking moiety, CM is a cleavable moiety, and AB is an antibody or fragment thereof.
  • MM and CM are indicated as distinct components in the formulae above, in all exemplary embodiments (including formulae) disclosed herein it is contemplated that the amino acid sequences of the MM and the CM could overlap, e.g., such that the CM is completely or partially contained within the MM.
  • the MM is not a natural binding partner of the AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the AB.
  • the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB.
  • linkers e.g., flexible linkers
  • the AB, MM, and/or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, particularly Gly) to provide the desired flexibility.
  • the switchable phenotype of such activatable antibody constructs may benefit from introduction of one or more amino acids to provide for a flexible linker.
  • an activatable antibody comprises one of the following formulae (where the formula below represents an amino acid sequence in either N- to C-terminal direction or C- to N-terminal direction): (MM)-L1-(CM)-(AB) (MM)-(CM)-L2-(AB) (MM)-L1-(CM)-L2-(AB) wherein MM, CM, and AB are as defined above; wherein L1 and L2 are each independently and optionally present or absent, are the same or different flexible linkers that include at least 1 flexible amino acid (e.g., Gly).
  • the formulae above provide for additional amino acid sequences that can be positioned N-terminal or C-terminal to the activatable antibodies elements.
  • targeting moieties e.g., a ligand for a receptor of a cell present in a target tissue
  • serum half-life extending moieties e.g., polypeptides that bind serum proteins, such as immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HAS)).
  • the CM is specifically cleaved by at least one protease at a rate of about 0.001- 1500 x 10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M -1 S -1 .
  • the CM is specifically cleaved at a rate of about 100,000 M -1 S -1 .
  • the CM is specifically cleaved at a rate from about 1x10E2 to about 1x10E6 M -1 S -1 (i.e., from about 1x10 2 to about 1x10 6 M -1 S -1 ).
  • contact between the enzyme and CM is made.
  • the activatable antibody comprising an AB coupled to a MM and a CM is in the presence of target and sufficient enzyme activity, the CM can be cleaved.
  • Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the CM and effect cleavage.
  • Linkers suitable for use in compositions described herein are generally ones that provide flexibility of the modified AB or the activatable antibodies to facilitate the inhibition of the binding of the AB to the target. Such linkers are generally referred to as flexible linkers.
  • Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 3 or 4, 5 or 6, 7, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
  • 1 amino acid e.g., Gly
  • Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 116) and (GGGS)n (SEQ ID NO: 117), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components.
  • Exemplary flexible linkers include, but are not limited to Gly-Gly-Ser-Gly (SEQ ID NO: 118), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 119), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 120), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 121), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 122), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 123), and the like.
  • an activatable antibodies can include linkers 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 antibodies structure.
  • the disclosure also provides compositions and methods that include an activatable CD47 antibody that includes an antibody or antibody fragment (AB) that specifically binds CD47, where the AB is coupled to a masking moiety (MM) that decreases the ability of the AB to bind its target.
  • the activatable CD47 antibody further includes a cleavable moiety (CM) that is a substrate for a protease.
  • compositions and methods provided herein enable the attachment of one or more agents to one or more cysteine residues in the AB without compromising the activity (e.g., the masking, activating or binding activity) of the activatable CD47 antibody.
  • the compositions and methods provided herein enable the attachment of one or more agents to one or more cysteine residues in the AB without reducing or otherwise disturbing one or more disulfide bonds within the MM.
  • compositions and methods provided herein produce an activatable CD47 antibody that is conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic and/or prophylactic agents, for example, in some embodiments, without any of the agent(s) being conjugated to the MM of the activatable CD47 antibody.
  • the compositions and methods provided herein produce conjugated activatable CD47 antibodies in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in an uncleaved state.
  • the compositions and methods provided herein produce conjugated activatable CD47 antibodies in which the activatable antibody is still activated, i.e., cleaved, in the presence of a protease that can cleave the CM.
  • the activatable antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1. In some embodiments, the activatable antibody comprises a light chain variable region amino acid sequence of SEQ ID NO: 2. [000165] In some embodiments, the activatable antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1. In some embodiments, the activatable antibody comprises a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 2.
  • the activatable antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected of SEQ ID NO: 2.
  • the activatable antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 3) or
  • the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO
  • the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIV
  • the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence that includes a sequence
  • the MM has a dissociation constant for binding to the AB which is greater than the dissociation constant of the AB to CD47. [000172] In some embodiments, the MM has a dissociation constant for binding to the AB which is no more than the dissociation constant of the AB to CD47. [000173] In some embodiments, the MM has a dissociation constant for binding to the AB is equivalent to the dissociation constant of the AB to CD47. [000174] In some embodiments, the MM has a dissociation constant for binding to the AB which is less than the dissociation constant of the AB to CD47.
  • the dissociation constant (Kd) of the MM towards the AB is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or greater, or between 1- 5, 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100- 10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000- 1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000- 1,000,000, or 100,000-10,000,000 times or greater than the dissociation constant of the AB towards the CD47 target.
  • the MM does not interfere or compete with the AB for binding to CD47 when the activatable antibody is in a cleaved state.
  • the MM is a polypeptide of about 2 to 40 amino acids in length. In some embodiments, the MM is a polypeptide of up to about 40 amino acids in length.
  • the MM polypeptide sequence is different from that of CD47. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB.
  • the MM polypeptide sequence is different from that of CD47 and is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of the AB.
  • the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (K d ) of the AB when coupled to the MM towards CD47 is at least two times greater than the Kd of the AB when not coupled to the MM towards CD47.
  • the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least five times greater than the Kd of the AB when not coupled to the MM towards CD47.
  • the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 10 times greater than the Kd of the AB when not coupled to the MM towards CD47.
  • the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 20 times greater than the Kd of the AB when not coupled to the MM towards CD47.
  • the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 40 times greater than the K d of the AB when not coupled to the MM towards CD47.
  • the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 100 times greater than the K d of the AB when not coupled to the MM towards CD47.
  • the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (K d ) of the AB when coupled to the MM towards CD47 is at least 1000 times greater than the K d of the AB when not coupled to the MM towards CD47.
  • the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (K d ) of the AB when coupled to the MM towards CD47 is at least 10,000 times greater than the Kd of the AB when not coupled to the MM towards CD47.
  • the MM reduces the ability of the AB to bind CD47 by at least 90% when the CM is uncleaved, as compared to when the CM is cleaved when assayed in vitro using a target displacement assay such as, for example, the assay described in PCT Publication No.
  • MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16-29. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 23. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 27.
  • the protease that cleaves the CM is active, e.g., up- regulated or otherwise unregulated, in diseased tissue, and the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.
  • the protease is co-localized with CD47 in a tissue, and the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.
  • the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least twofold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds CD47.
  • the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least fivefold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds CD47.
  • the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 10-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds CD47.
  • the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 20-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds CD47.
  • the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 40-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state, the AB binds CD47.
  • the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 50-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state, the AB binds CD47.
  • the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 100-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state, the AB binds CD47.
  • the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 200-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state, the AB binds CD47.
  • the CM is a polypeptide of up to 15 amino acids in length.
  • the CM is a polypeptide that includes a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP).
  • MMP matrix metalloprotease
  • SP serine protease
  • each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1- CM2 substrate is independently a polypeptide of up to 15 amino acids in length.
  • the CM is a substrate for at least one protease that is or is believed to be up-regulated or otherwise unregulated in cancer.
  • the CM is a substrate for at least one protease selected from the group consisting of a matrix metalloprotease (MMP), thrombin, a neutrophil elastase, a cysteine protease, legumain, and a serine protease, such as matriptase (MT-SP1), and urokinase (uPA).
  • MMP matrix metalloprotease
  • thrombin thrombin
  • neutrophil elastase a neutrophil elastase
  • cysteine protease cysteine protease
  • legumain and a serine protease
  • uPA urokinase
  • Exemplary substrates include but are not limited to substrates cleavable by one or more of the following enzymes or proteases listed in Table A.
  • the CM is selected for use with a specific protease, for example a protease that is known to be co-localized with the target of the activatable antibody.
  • the CM is a substrate for at least one MMP. Examples of MMPs include the MMPs listed in the Table A.
  • the CM is a substrate for a protease selected from the group consisting of MMP 9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19.
  • the CM is a substrate for MMP9.
  • the CM is a substrate for MMP14.
  • the CM is a substrate that includes the sequence TGRGPSWV (SEQ ID NO: 356); SARGPSRW (SEQ ID NO: 357); TARGPSFK (SEQ ID NO: 358); LSGRSDNH (SEQ ID NO: 359); GGWHTGRN (SEQ ID NO: 360); HTGRSGAL (SEQ ID NO: 361); PLTGRSGG (SEQ ID NO: 362); AARGPAIH (SEQ ID NO: 363); RGPAFNPM (SEQ ID NO: 364); SSRGPAYL (SEQ ID NO: 365); RGPATPIM (SEQ ID NO: 366); RGPA (SEQ ID NO: 367); GGQPSGMWGW (SEQ ID NO: 368); FPRPLGITGL (SEQ ID NO: 369); VHMPLGFLGP (SEQ ID NO: 370); SPLTGRSG (SEQ ID NO: 371); SAGFSLPA (SEQ ID NO: 372
  • the CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 359). In some embodiments, the CM comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 356). In some embodiments, the CM comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 362). In some embodiments, the CM comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO: 368). In some embodiments, the CM comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 369). In some embodiments, the CM comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 370).
  • the CM comprises the amino acid sequence PLGL (SEQ ID NO: 375). In some embodiments, the CM comprises the amino acid sequence SARGPSRW (SEQ ID NO: 357). In some embodiments, the CM comprises the amino acid sequence TARGPSFK (SEQ ID NO: 358). In some embodiments, the CM comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 360). In some embodiments, the CM comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 361). In some embodiments, the CM comprises the amino acid sequence AARGPAIH (SEQ ID NO: 363). In some embodiments, the CM comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 364).
  • the CM comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 365). In some embodiments, the CM comprises the amino acid sequence RGPATPIM (SEQ ID NO: 366). In some embodiments, the CM comprises the amino acid sequence RGPA (SEQ ID NO: 367). In some embodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ ID NO: 789). In some embodiments, the CM comprises the amino acid sequence SGRSANPRG (SEQ ID NO: 790). In some embodiments, the CM comprises the amino acid sequence LSGRSDDH (SEQ ID NO: 791). In some embodiments, the CM comprises the amino acid sequence LSGRSDIH (SEQ ID NO: 792).
  • the CM comprises the amino acid sequence LSGRSDQH (SEQ ID NO: 793). In some embodiments, the CM comprises the amino acid sequence LSGRSDTH (SEQ ID NO: 794). In some embodiments, the CM comprises the amino acid sequence LSGRSDYH (SEQ ID NO: 795). In some embodiments, the CM comprises the amino acid sequence LSGRSDNP (SEQ ID NO: 796). In some embodiments, the CM comprises the amino acid sequence LSGRSANP (SEQ ID NO: 797). In some embodiments, the CM comprises the amino acid sequence LSGRSANI (SEQ ID NO: 798). In some embodiments, the CM comprises the amino acid sequence LSGRSDNI (SEQ ID NO: 799).
  • the CM comprises the amino acid sequence MIAPVAYR (SEQ ID NO: 800). In some embodiments, the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 801). In some embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ ID NO: 802). In some embodiments, the CM comprises the amino acid sequence FRLLDWQW (SEQ ID NO: 803). In some embodiments, the CM comprises the amino acid sequence ISSGL (SEQ ID NO: 804). In some embodiments, the CM comprises the amino acid sequence ISSGLLS (SEQ ID NO: 805). In some embodiments, the CM comprises the amino acid sequence and/or ISSGLL (SEQ ID NO: 806).
  • the CM is a substrate for an MMP and includes the sequence ISSGLSS (SEQ ID NO: 376); QNQALRMA (SEQ ID NO: 377); AQNLLGMV (SEQ ID NO: 378); STFPFGMF (SEQ ID NO: 379); PVGYTSSL (SEQ ID NO: 380); DWLYWPGI (SEQ ID NO: 381), ISSGLLSS (SEQ ID NO: 382), LKAAPRWA (SEQ ID NO: 383); GPSHLVLT (SEQ ID NO: 384); LPGGLSPW (SEQ ID NO: 385); MGLFSEAG (SEQ ID NO: 386); SPLPLRVP (SEQ ID NO: 387); RMHLRSLG (SEQ ID NO: 388); LAAPLGLL (SEQ ID NO: 389); AVGLLAPP (SEQ ID NO: 390); LLAPSHRA (SEQ ID NO: 391); PAGLWLDP (SEQ ID NO: 392
  • the CM comprises the amino acid sequence ISSGLSS (SEQ ID NO: 376). In some embodiments, the CM comprises the amino acid sequence QNQALRMA (SEQ ID NO: 377). In some embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ ID NO: 378). In some embodiments, the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 379). In some embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 380). In some embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ ID NO: 381). In some embodiments, the CM comprises the amino acid sequence ISSGLLSS (SEQ ID NO: 382).
  • the CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 383). In some embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO: 384). In some embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQ ID NO: 385). In some embodiments, the CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 386). In some embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ ID NO: 387). In some embodiments, the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO: 388). In some embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 389).
  • the CM comprises the amino acid sequence AVGLLAPP (SEQ ID NO: 390). In some embodiments, the CM comprises the amino acid sequence LLAPSHRA (SEQ ID NO: 391). In some embodiments, the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 392). In some embodiments, the CM comprises the amino acid sequence ALAHGLF (SEQ ID NO: 424). In some embodiments, the CM comprises the amino acid sequence DLAHPLL (SEQ ID NO: 425). In some embodiments, the CM comprises the amino acid sequence AFRHLR (SEQ ID NO: 426). In some embodiments, the CM comprises the amino acid sequence PHGFFQ (SEQ ID NO: 427).
  • the CM comprises the amino acid sequence SVHHLI (SEQ ID NO: 428). In some embodiments, the CM comprises the amino acid sequence RGPKLYW (SEQ ID NO: 429). In some embodiments, the CM comprises the amino acid sequence RFPYGVW (SEQ ID NO: 430). In some embodiments, the CM comprises the amino acid sequence HVPRQV (SEQ ID NO: 431). In some embodiments, the CM comprises the amino acid sequence SNPFKY (SEQ ID NO: 432). In some embodiments, the CM comprises the amino acid sequence RFPLKV (SEQ ID NO: 433). In some embodiments, the CM comprises the amino acid sequence PFHLSR (SEQ ID NO: 434).
  • the CM comprises the amino acid sequence STVFHM (SEQ ID NO: 435). In some embodiments, the CM comprises the amino acid sequence MGPWFM (SEQ ID NO: 436). In some embodiments, the CM comprises the amino acid sequence RHLAKL (SEQ ID NO: 437). In some embodiments, the CM comprises the amino acid sequence PLGVRGK (SEQ ID NO: 438). In some embodiments, the CM comprises the amino acid sequence QNQALRIA (SEQ ID NO: 439). [000210] In some embodiments, the CM is a substrate for thrombin.
  • the CM is a substrate for thrombin and includes the sequence GPRSFGL (SEQ ID NO: 393) or GPRSFG (SEQ ID NO: 394). In some embodiments, the CM comprises the amino acid sequence GPRSFGL (SEQ ID NO: 393). In some embodiments, the CM comprises the amino acid sequence GPRSFG (SEQ ID NO: 394).
  • the CM comprises an amino acid sequence selected from the group consisting of NTLSGRSENHSG (SEQ ID NO: 395); NTLSGRSGNHGS (SEQ ID NO: 396); TSTSGRSANPRG (SEQ ID NO: 397); TSGRSANP (SEQ ID NO: 398); VAGRSMRP (SEQ ID NO: 399); VVPEGRRS (SEQ ID NO: 400); ILPRSPAF (SEQ ID NO: 401); MVLGRSLL (SEQ ID NO: 402); QGRAITFI (SEQ ID NO: 403); SPRSIMLA (SEQ ID NO: 404); and SMLRSMPL (SEQ ID NO: 405).
  • NTLSGRSENHSG SEQ ID NO: 395
  • NTLSGRSGNHGS SEQ ID NO: 396
  • TSTSGRSANPRG SEQ ID NO: 397
  • TSGRSANP SEQ ID NO: 398
  • VAGRSMRP SEQ ID NO: 399
  • VVPEGRRS SEQ ID
  • the CM comprises the amino acid sequence NTLSGRSENHSG (SEQ ID NO: 395). In some embodiments, the CM comprises the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 396). In some embodiments, the CM comprises the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 397). In some embodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ ID NO: 398). In some embodiments, the CM comprises the amino acid sequence VAGRSMRP (SEQ ID NO: 399). In some embodiments, the CM comprises the amino acid sequence VVPEGRRS (SEQ ID NO: 400).
  • the CM comprises the amino acid sequence ILPRSPAF (SEQ ID NO: 401). In some embodiments, the CM comprises the amino acid sequence MVLGRSLL (SEQ ID NO: 402). In some embodiments, the CM comprises the amino acid sequence QGRAITFI (SEQ ID NO: 403). In some embodiments, the CM comprises the amino acid sequence SPRSIMLA (SEQ ID NO: 404). In some embodiments, the CM comprises the amino acid sequence SMLRSMPL (SEQ ID NO: 405). [000213] In some embodiments, the CM is a substrate for a neutrophil elastase. In some embodiments, the CM is a substrate for a serine protease.
  • the CM is a substrate for uPA. In some embodiments, the CM is a substrate for legumain. In some embodiments, the CM is a substrate for matriptase. In some embodiments, the CM is a substrate for a cysteine protease. In some embodiments, the CM is a substrate for a cysteine protease, such as a cathepsin.
  • the CM is a CM1-CM2 substrate and includes the sequence ISSGLLSGRSDNH (SEQ ID NO: 406); ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 407); AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 408); TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 409); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 410); TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 411); AVGLLAPPGGLSGRSDNH (SEQ ID NO: 412); LSGRSDNHGGAVGLLAPP (SEQ ID NO: 413); VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 414); LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 415); LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 416); LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 416); L
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNH (SEQ ID NO: 406), which is also referred to herein as substrate 2001.
  • the CM1-CM2 substrate includes the sequence ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 407), which is also referred to herein as substrate 1001/LP'/0001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133).
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 408), which is also referred to herein as substrate 2015 and/or substrate 1004/LP'/0003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 409), which is also referred to herein as substrate 0003/LP'/1004, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 410), which is also referred to herein as substrate 1003/ LP'/0003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 411), which is also referred to herein as substrate 0003/LP'/1003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 412), which is also referred to herein as substrate 3001 and/or substrate 1004/LP'/0001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence LSGRSDNHGGAVGLLAPP (SEQ ID NO: 413), which is also referred to herein as substrate 0001/LP'/1004, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 414), which is also referred to herein as substrate 1003/LP’/0001, wherein LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 415), which is also referred to herein as substrate 0001/ LP'/1003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 416), which is also referred to herein as substrate 0001/LP'/1001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133).
  • the CM1-CM2 substrate includes the sequence LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 417), which is also referred to herein as substrate 0002/LP'/1001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133).
  • the CM1-CM2 substrate includes the sequence ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 418), which is also referred to herein as substrate 1001/LP'/0002, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133).
  • the CM1-CM2 substrate includes the sequence LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 419), which is also referred to herein as substrate 0001/LP'/1002, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133).
  • the CM1-CM2 substrate includes the sequence QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 420), which is also referred to herein as substrate 1002/LP'/0001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133).
  • the CM1-CM2 substrate includes the sequence LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 421), which is also referred to herein as substrate 0002/LP'/1002, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133).
  • the CM1-CM2 substrate includes the sequence QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 422), which is also referred to herein as substrate 1002/LP'/0002, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133).
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSGNH (SEQ ID NO: 423), which is also referred to herein as substrate 2002.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSANPRG (SEQ ID NO: 680), which is also referred to herein as substrate 2003.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPTSGRSANPRG (SEQ ID NO: 681), which is also referred to herein as substrate 2004.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPSGRSANPRG (SEQ ID NO: 682), which is also referred to herein as substrate 2005.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSDDH (SEQ ID NO: 683), which is also referred to herein as substrate 2006.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSDIH (SEQ ID NO: 684), which is also referred to herein as substrate 2007.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSDQH (SEQ ID NO: 685), which is also referred to herein as substrate 2008.
  • the CM1- CM2 substrate includes the sequence ISSGLLSGRSDTH (SEQ ID NO: 686), which is also referred to herein as substrate 2009.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSDYH (SEQ ID NO: 687), which is also referred to herein as substrate 2010.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNP (SEQ ID NO: 688), which is also referred to herein as substrate 2011.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSANP (SEQ ID NO: 689), which is also referred to herein as substrate 2012.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSANI (SEQ ID NO: 690), which is also referred to herein as substrate 2013.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDDH (SEQ ID NO: 691), which is also referred to herein as substrate 3006.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDIH (SEQ ID NO: 692), which is also referred to herein as substrate 3007.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDQH (SEQ ID NO: 693), which is also referred to herein as substrate 3008.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDTH (SEQ ID NO: 694), which is also referred to herein as substrate 3009.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDYH (SEQ ID NO: 695), which is also referred to herein as substrate 3010.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDNP (SEQ ID NO: 696), which is also referred to herein as substrate 3011.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSANP (SEQ ID NO: 697), which is also referred to herein as substrate 3012.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSANI (SEQ ID NO: 698), which is also referred to herein as substrate 3013.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNI (SEQ ID NO: 713), which is also referred to herein as substrate 2014.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 714), which is also referred to herein as substrate 3014.
  • the CM1-CM2 substrate includes the sequence GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 807), which is also referred to herein as substrate 0001/LP'/1004, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM1-CM2 substrate includes the sequence GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 808), which is also referred to herein as substrate 0001/LP'/1003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG.
  • the CM is a CM1-CM2 substrate and includes the sequence LSGRSALAHGLF (SEQ ID NO: 440); ALAHGLFSGRSAN (SEQ ID NO: 441); HVPRQVLSGRS (SEQ ID NO: 442); HVPRQVLSGRSAN (SEQ ID NO: 443); TARGPALAHGLF (SEQ ID NO: 444); TARGPVPRQV (SEQ ID NO: 445); APRSALAHGLF (SEQ ID NO: 446); ALAHGLFAPRSF (SEQ ID NO: 447); HVPRQVAPRSF (SEQ ID NO: 448); ALAHGLPTFVHL (SEQ ID NO: 449); GLPTFVHLPRQV (SEQ ID NO: 450); AANALAHGLF (SEQ ID NO: 451); GPTNALAHGLF (SEQ ID NO: 452); ISSGLLSGRSNI (SEQ ID NO: 453); AVGLLAPPGGLSGRSNI (SEQ ID NO: 453);
  • the CM is a CM1-CM2 substrate and includes the sequence LSGRSALAHGLF (SEQ ID NO: 440), which is also referred to herein as substrate 5001.
  • the CM is a CM1-CM2 substrate and includes the sequence ALAHGLFSGRSAN (SEQ ID NO: 441), which is also referred to herein as substrate 5002.
  • the CM1-CM2 substrate includes the sequence HVPRQVLSGRS (SEQ ID NO: 442), which is also referred to herein as substrate 5003.
  • the CM1- CM2 substrate includes the sequence HVPRQVLSGRSAN (SEQ ID NO: 443), which is also referred to herein as substrate 5004.
  • the CM1-CM2 substrate includes the sequence TARGPALAHGLF (SEQ ID NO: 444), which is also referred to herein as substrate 5005.
  • the CM1-CM2 substrate includes the sequence TARGPVPRQV (SEQ ID NO: 445), which is also referred to herein as substrate 5006.
  • the CM1-CM2 substrate includes the sequence APRSALAHGLF (SEQ ID NO: 446), which is also referred to herein as substrate 5007.
  • the CM1-CM2 substrate includes the sequence ALAHGLFAPRSF (SEQ ID NO: 447), which is also referred to herein as substrate 5008.
  • the CM1-CM2 substrate includes the sequence HVPRQVAPRSF (SEQ ID NO: 448), which is also referred to herein as substrate 5009.
  • the CM1-CM2 substrate includes the sequence ALAHGLPTFVHL (SEQ ID NO: 449), which is also referred to herein as substrate 5010.
  • the CM1-CM2 substrate includes the sequence GLPTFVHLPRQV (SEQ ID NO: 450), which is also referred to herein as substrate 5011.
  • the CM1-CM2 substrate includes the sequence AANALAHGLF (SEQ ID NO: 451), which is also referred to herein as substrate 5012.
  • the CM1-CM2 substrate includes the sequence GPTNALAHGLF (SEQ ID NO: 452), which is also referred to herein as substrate 5013.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSNI (SEQ ID NO: 453), which is also referred to herein as substrate 5014.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSNI (SEQ ID NO: 454), which is also referred to herein as substrate 5015.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSNIGS (SEQ ID NO: 455), which is also referred to herein as substrate 5016.
  • the CM1- CM2 substrate includes the sequence AVGLLAPPGGLSGRSNIGS (SEQ ID NO: 456), which is also referred to herein as substrate 5017.
  • the CM1-CM2 substrate includes the sequence ISSGLLSGRSNIG (SEQ ID NO: 457), which is also referred to herein as substrate 5018.
  • the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSNIG (SEQ ID NO: 458), which is also referred to herein as substrate 5019.
  • the CM is a substrate for at least two proteases.
  • each protease is selected from the group consisting of those shown in Table A.
  • the CM is a substrate for at least two proteases, wherein one of the proteases is selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain and matriptase and the other protease is selected from the group consisting of those shown in Table A.
  • the CM is a substrate for at least two proteases selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain and matriptase.
  • the activatable antibody includes at least a first CM and a second CM.
  • the first CM and the second CM are each polypeptides of no more than 15 amino acids long.
  • the first CM and the second CM in the activatable antibody in the uncleaved state have the structural arrangement from N-terminus to C-terminus as follows: MM-CM1-CM2-AB or AB-CM2-CM1-MM.
  • at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase.
  • the first CM is cleaved by a first cleaving agent selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase in a target tissue and the second CM is cleaved by a second cleaving agent in a target tissue.
  • a first cleaving agent selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase in a target tissue
  • the second CM is cleaved by a second cleaving agent in a target tissue.
  • the other protease is selected from the group consisting of those shown in Table A.
  • the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase, and the first CM and the second CM are different substrates for the enzyme.
  • the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of those shown in Table A.
  • the first cleaving agent and the second cleaving agent are different proteases.
  • the first cleaving agent and the second cleaving agent are co-localized in the target tissue.
  • the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.
  • the activatable antibody is exposed to and cleaved by a protease such that, in the activated or cleaved state, the activated antibody includes a light chain amino acid sequence that includes at least a portion of LP2 and/or CM sequence after the protease has cleaved the CM.
  • Suitable activatable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • Suitable activatable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or
  • Suitable activatable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 to a CD47 antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • Suitable activatable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 to a CD47 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ
  • the activatable CD47 antibody is an activatable antibody that, in an activated state, binds CD47 comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
  • AB antibody or an antigen binding fragment thereof
  • MM masking moiety
  • CM cleavable moiety
  • the MM has a dissociation constant for binding to the AB that is greater than the dissociation constant of the AB to CD47. In some embodiments, the MM does not interfere or compete with the AB for binding to CD47 when the activatable antibody is in a cleaved state. In some embodiments, the MM is a polypeptide of no more than 40 amino acids in length. In some embodiments, the MM polypeptide sequence is different from that of human CD47. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-109.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 107-109.
  • the CM is a substrate for a protease that is active in diseased tissue.
  • the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808.
  • the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
  • the activatable antibody comprises an antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab’)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
  • the AB of the activatable antibody specifically binds human CD47.
  • the AB comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS
  • the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the AB is linked to the CM.
  • the AB is linked directly to the CM.
  • the AB is linked to the CM via a linking peptide.
  • the MM is linked to the CM such that the activatable antibody in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM.
  • the activatable antibody comprises a linking peptide between the MM and the CM.
  • the activatable antibody comprises a linking peptide between the CM and the AB.
  • the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
  • the two linking peptides need not be identical to each other.
  • each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.
  • the activatable antibody comprises the heavy chain sequence selected from the group consisting of SEQ ID NOs: 11-14 and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81-94, and 16-114.
  • the activatable antibody comprises a combination of amino acid sequences, wherein the combination of amino acid sequences is selected from a single row in Table B, wherein for a given combination, (a) the heavy chain of the AB comprises the amino acid sequences of the VH CDR sequences corresponding to the given combination in the single row listed in Table B, (b) the light chain of the AB comprises the amino acid sequences of the VL CDR sequences corresponding to the given combination in the single row listed in Table B, (c) the MM comprises the amino acid sequence of the mask sequence (MM) corresponding to the given combination in the single row listed in Table B, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) corresponding to the given combination in the single row listed in Table B.
  • the activatable antibody comprises a combination of amino acid sequences, wherein for a given combination of amino acid sequences, (a) the heavy chain of the AB comprises the amino acid sequences of the VH sequence or VH CDR sequences selected from the group consisting of: the VH sequence or VH CDR sequences listed in the corresponding column of Table C, (b) the light chain of the AB comprises the amino acid sequences of the VL sequence or VL CDR sequences selected from the group consisting of: the VL sequence or VL CDR sequences listed in the corresponding column of Table C, (c) the MM comprises the amino acid sequence of the mask sequence (MM) selected from the group consisting of: the MM sequences listed in the corresponding column of Table C, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) selected from the group consisting of: the CM sequences listed in the corresponding column of Table C.
  • the heavy chain of the AB comprises the amino acid sequences of the VH sequence or VH CDR sequences selected from
  • the activatable CD47 antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, a MM, and a CM, wherein the activatable antibody comprises: a heavy chain sequence of SEQ ID NOs: 11-14; and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81-94, and 16-114.
  • AB antigen binding fragment thereof
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29
  • the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
  • the AB comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS
  • the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the activatable CD47 antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29, and a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29
  • the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
  • the AB comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS
  • the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the activatable CD47 antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as an isolated antibody of the disclosure; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
  • AB antigen binding fragment thereof
  • the CD47 activatable antibody of the disclosure comprises an isolated antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47.
  • AB antigen binding fragment thereof
  • the antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence GYT
  • the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the activatable CD47 antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically cross-competes with (inhibits the binding of) an isolated antibody of the disclosure for binding to human CD47 and/or cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
  • AB antigen binding fragment thereof
  • the CD47 activatable antibody of the disclosure comprises an isolated antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47.
  • AB antigen binding fragment thereof
  • the antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence GYT
  • the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the activatable antibody also includes an agent conjugated to the AB.
  • the agent conjugated to the AB or the AB of an activatable antibody is a therapeutic agent.
  • the agent is an antineoplastic agent.
  • the agent is a toxin or fragment thereof. As used herein, a fragment of a toxin is a fragment that retains toxic activity.
  • the agent is conjugated to the AB via a cleavable linker.
  • the agent is conjugated to the AB via a linker that includes at least one CM1-CM2 substrate sequence. In some embodiments, the agent is conjugated to the AB via a noncleavable linker. In some embodiments, the agent is conjugated to the AB via a linker that is cleavable in an intracellular or lysosomal environment. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylator, a DNA cleaving agent, a DNA cross-linker, a DNA intercalator, or other DNA damaging agent. In some embodiments, the agent is an agent selected from the group listed in Table E.
  • the agent is a dolastatin. In some embodiments, the agent is an auristatin or derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or derivative thereof. In some embodiments, the agent is a calicheamicin or derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine.
  • the agent is a pyrrolobenzodiazepine dimer.
  • the activatable antibody is conjugated to one or more equivalents of an agent. In some embodiments, the activatable antibody is conjugated to one equivalent of the agent. In some embodiments, the activatable antibody is conjugated to two, three, four, five, six, seven, eight, nine, ten, or greater than ten equivalents of the agent. In some embodiments, the activatable antibody is part of a mixture of activatable antibodies having a homogeneous number of equivalents of conjugated agents. In some embodiments, the activatable antibody is part of a mixture of activatable antibodies having a heterogeneous number of equivalents of conjugated agents.
  • the mixture of activatable antibodies is such that the average number of agents conjugated to each activatable antibody is between zero to one, between one to two, between two and three, between three and four, between four and five, between five and six, between six and seven, between seven and eight, between eight and nine, between nine and ten, and ten and greater. In some embodiments, the mixture of activatable antibodies is such that the average number of agents conjugated to each activatable antibody is one, two, three, four, five, six, seven, eight, nine, ten, or greater.
  • the activatable antibody comprises one or more site-specific amino acid sequence modifications such that the number of lysine and/or cysteine residues is increased or decreased with respect to the original amino acid sequence of the activatable antibody, thus in some embodiments correspondingly increasing or decreasing the number of agents that can be conjugated to the activatable antibody, or in some embodiments limiting the conjugation of the agents to the activatable antibody in a site-specific manner.
  • the modified activatable antibody is modified with one or more non-natural amino acids in a site-specific manner, thus in some embodiments limiting the conjugation of the agents to only the sites of the non-natural amino acids.
  • the agent is an anti-inflammatory agent.
  • the activatable antibody also includes a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. [000244] In some embodiments, the activatable antibody also includes a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is joined directly to the MM of the activatable antibody. In some embodiments, the spacer is joined directly to the MM of the activatable antibody in the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.
  • QGQSGQ SEQ ID NO: 31
  • Other examples of a spacer joined directly to the N-terminus of MM of the activatable antibody include QGQSGQG (SEQ ID NO: 30), QGQSG (SEQ ID NO: 32), QGQS (SEQ ID NO: 33), QGQ, QG, and Q.
  • Other examples of a spacer joined directly to the N-terminus of MM of the activatable antibody include GQSGQG (SEQ ID NO: 34), QSGQG (SEQ ID NO: 35), SGQG (SEQ ID NO: 36), GQG, and G.
  • the spacer includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 31). In some embodiments, the spacer includes at least the amino acid sequence QGQSGQG (SEQ ID NO: 30). In some embodiments, the spacer includes at least the amino acid sequence QGQSG (SEQ ID NO: 32). In some embodiments, the spacer includes at least the amino acid sequence QGQS (SEQ ID NO: 33). In some embodiments, the spacer includes at least the amino acid sequence QGQ. In some embodiments, the spacer includes at least the amino acid sequence QG. In some embodiments, the spacer includes at least the amino acid residue Q.
  • the spacer includes at least the amino acid sequence GQSGQG (SEQ ID NO: 34). In some embodiments, the spacer includes at least the amino acid sequence QSGQG (SEQ ID NO: 35). In some embodiments, the spacer includes at least the amino acid sequence SGQG (SEQ ID NO: 36). In some embodiments, the spacer includes at least the amino acid sequence GQG. In some embodiments, the spacer includes at least the amino acid sequence G. In some embodiments, the spacer is absent. [000245] In some embodiments, the AB of the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to include one or more disulfide bonds.
  • activatable antibody or antigen binding fragment thereof is conjugated to an agent.
  • the activatable antibody comprises an antibody or antigen binding fragment thereof cross-competes with (inhibits the binding of) an isolated antibody that comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO:
  • the activatable antibody comprises an antibody or antigen binding fragment thereof cross-competes with (inhibits the binding of) an isolated antibody that comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the agent is a toxin or fragment thereof.
  • the agent is a microtubule inhibitor.
  • the agent is a nucleic acid damaging agent.
  • the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof.
  • the agent is auristatin E or a derivative thereof.
  • the agent is monomethyl auristatin E (MMAE).
  • the agent is monomethyl auristatin D (MMAD).
  • the agent is a maytansinoid selected from the group consisting of DM1 and DM4.
  • the agent is maytansinoid DM4. In some embodiments, the agent is duocarmycin. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker with which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety. In some embodiments, the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, vc-duocarmycin, or a PEG2-vc-MMAD moiety. In some embodiments, the linker is a cleavable linker.
  • the linker is a non-cleavable linker.
  • the agent is a detectable moiety.
  • the detectable moiety is a diagnostic agent.
  • the conjugated activatable antibody comprises a conjugated activatable antibody that, in an activated state, binds CD47 comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease; and an agent conjugated to the AB.
  • AB antibody or an antigen binding fragment thereof
  • MM masking moiety
  • CM cleavable moiety
  • the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof.
  • the agent is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a duocarmycin, a pyrrolobenzodiazepine, and a pyrrolobenzodiazepine dimer.
  • the agent is conjugated to the AB via a linker.
  • the linker with which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety.
  • the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, vc-duocarmycin, or a PEG2-vc- MMAD moiety.
  • the AB of the conjugated activatable antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL
  • the AB of the conjugated activatable antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16- 29.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23, 24, 25, 27, and 28.
  • the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808.
  • the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
  • the activatable antibody comprises a combination of amino acid sequences, wherein the combination of amino acid sequences is selected from a single row in Table B, wherein for a given combination, (a) the heavy chain of the AB comprises the amino acid sequences of the VH CDR sequences corresponding to the given combination in the single row listed in Table B, (b) the light chain of the AB comprises the amino acid sequences of the VL CDR sequences corresponding to the given combination in the single row listed in Table B, (c) the MM comprises the amino acid sequence of the mask sequence (MM) corresponding to the given combination in the single row listed in Table B, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) corresponding to the given combination in the single row listed in Table B.
  • the activatable antibody comprises a combination of amino acid sequences, wherein for a given combination of amino acid sequences, (a) the heavy chain of the AB comprises the amino acid sequences of the VH sequence or VH CDR sequences selected from the group consisting of: the VH sequence or VH CDR sequences listed in the corresponding column of Table C, (b) the light chain of the AB comprises the amino acid sequences of the VL sequence or VL CDR sequences selected from the group consisting of: the VL sequence or VL CDR sequences listed in the corresponding column of Table C, (c) the MM comprises the amino acid sequence of the mask sequence (MM) selected from the group consisting of: the MM sequences listed in the corresponding column of Table C, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) selected from the group consisting of: the CM sequences listed in the corresponding column of Table C.
  • the heavy chain of the AB comprises the amino acid sequences of the VH sequence or VH CDR sequences selected from
  • the activatable antibody comprises: a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4; and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81-94, and 16-114.
  • the conjugated activatable antibody comprises a conjugated activatable antibody that, in an activated state, binds to CD47, comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease; and an agent conjugated to the AB, wherein the AB comprises: (i) the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (AB) that specifically binds to ma
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23, 24, 25, 27, and 28. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
  • the agent is conjugated to the AB via a linker, and wherein the linker to which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety.
  • the linker and toxin conjugated to the AB comprises an SPDB- DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, vc-duocarmycin, or a PEG2-vc-MMAD moiety.
  • the conjugated activatable antibody comprises a conjugated activatable antibody comprising: an antibody or antigen binding fragment thereof (AB) that, in an activated state, binds CD47; and a toxin conjugated to the AB via a linker, wherein the conjugated activatable antibody comprises amino acid sequences, a linker, and a toxin selected from a single row in Table D, wherein for the given combination: (a) the AB comprises a heavy chain comprising the amino acid sequence of the heavy chain sequence or heavy chain variable domain sequence corresponding to the given combination in the single row listed in Table D, (b) the AB comprises a light chain comprising the amino acid sequence of the light chain sequence or light chain variable domain sequence corresponding to the given combination in the single row listed in Table D, and (c) the linker and the toxin comprise the linker and the toxin corresponding to the given combination in the single row listed in Table D.
  • AB antibody or antigen binding fragment thereof
  • the conjugated activatable antibody comprises amino acid sequences,
  • the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain variable region amino acid sequence of SEQ ID NO: 1. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a heavy chain variable region amino acid sequence of SEQ ID NO: 1.
  • the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a light variable region chain amino acid of SEQ ID NO: 2.
  • the disclosure also provides methods for producing an activatable antibody of the disclosure by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises a nucleic acid molecule of the disclosure or a vector of the disclosure.
  • the disclosure also provides methods of manufacturing an activatable antibody that, in an activated state, binds CD47, the method comprising: (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises an activatable antibody of the disclosure; and (b) recovering the activatable antibody.
  • the activatable antibody includes one or more polypeptides that include the combination of sequences in a given row of Table B or any combination of a mask sequence (MM), a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequences, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequences of Table C.
  • Table B CD47 Activatable Antibody Combinations
  • Table C CD47 Activatable Antibody Components
  • an activatable antibody of the present disclosure includes one or more polypeptides that include the combination of sequences selected from Table B or Table C, where the polypeptide includes a combination of a masking sequence selected from the column titled “Mask Sequence (MM)” of Table B or Table C, a substrate sequence from the column titled “Substrate Sequence (CM)” of Table B or Table C, a light chain variable domain or light chain CDRs from the column titled “VL or VL CDRs” or “VL CDRs SEQ ID NOs” of Table B or Table C, and a heavy chain variable domain or heavy chain CDRs from the column titled “VH or VH CDRs” or “VH CDRs SEQ ID Nos” of Table B or Table C.
  • MM Mesk Sequence
  • CM Substrate Sequence
  • an activatable antibody of the present disclosure may include the amino acid sequences of combination no.147, which includes the masking sequence of SEQ ID NO: 17, the substrate sequence of SEQ ID NO: 412, a light chain variable domain that includes the VL CDR sequences of SEQ ID NOs: 15, 16, and 18, and a heavy chain variable domain that includes the VH CDR sequences of 11, 12, and 13. Therefore, an activatable antibody that includes at least the combination of sequences in any given row of Table B is described herein. Similarly, any combination of a mask sequence (MM), a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequences, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequences of Table C is described herein.
  • An activatable antibody that includes at least any combination of a masking sequence, a substrate sequence, a variable heavy chain or variable heavy chain CDRs, and a variable light chain or variable light chain CDRs selected from the corresponding columns Table B or Table C is also described herein.
  • an activatable antibody that includes at least the combination of sequences in any given row of Table B or any combination of a mask sequence (MM), a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequences, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequences of Table C can be combined with one or more toxins, including a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, or a pyrrolobenzodiazepine or a derivative thereof.
  • toxins including a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, or a pyrrolobenzodiazepine or a derivative thereof.
  • an activatable antibody that includes at least the combination of sequences in any given row of Table B or any combination of a mask sequence (MM), a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequences, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequences of Table C can be combined with one or more toxins, including auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, and/or a duocarmycin.
  • auristatin E monomethyl auristatin F
  • MMAE monomethyl auristatin E
  • MMAD monomethyl auristatin D
  • maytansinoid DM4 maytansinoid DM1, a pyrrolobenzodia
  • any of the combinations in Table B or Table C as described above can be combined with human immunoglobulin constant regions to result in fully human IgGs including IgG1, IgG2, IgG4 or mutated constant regions to result in human IgGs with altered functions such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P.
  • any mask sequence disclosed herein can be used in a combination.
  • any CM disclosed herein can be used in a combination.
  • any light chain variable region sequence or light chain CDR sequences disclosed herein can be used in a combination.
  • any heavy chain variable region sequence or heavy chain CDR sequences disclosed herein can be used in a combination.
  • the serum half-life of the activatable antibody is longer than that of the corresponding antibody, e.g., the pK of the activatable antibody is longer than that of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to that of the corresponding antibody.
  • the serum half- life of the activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 7 days when administered to an organism.
  • the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism.
  • the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism.
  • the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism. [000258]
  • the disclosure also provides methods of producing an activatable CD47 antibody polypeptide by culturing a cell under conditions that lead to expression of the polypeptide, wherein the cell comprises an isolated nucleic acid molecule encoding an antibody and/or an activatable antibody described herein, and/or vectors that include these isolated nucleic acid sequences.
  • the disclosure provides methods of producing an antibody and/or activatable antibody by culturing a cell under conditions that lead to expression of the antibody and/or activatable antibody, wherein the cell comprises an isolated nucleic acid molecule encoding an antibody and/or an activatable antibody described herein, and/or vectors that include these isolated nucleic acid sequences.
  • the invention also provides a method of manufacturing activatable antibodies that in an activated state binds CD47 by (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or an antigen binding fragment thereof (AB) that specifically binds CD47, (i) wherein the CM is a polypeptide that functions as a substrate for a protease; and (ii) wherein the CM is positioned in the activatable antibody such that, when the activatable antibody is in an uncleaved state, the MM interferes with specific binding of the AB to CD47 and in a cleaved state the MM does not interfere or compete with specific binding of the AB to CD47; and (b) recovering the activatable antibody.
  • MM masking moiety
  • CM cleavable moiety
  • Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.
  • the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM- MM.
  • the activatable antibody comprises a linking peptide between the MM and the CM.
  • the activatable antibody comprises a linking peptide between the CM and the AB.
  • the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
  • the two linking peptides need not be identical to each other.
  • the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer.
  • At least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GGS) n , (GSGGS) n (SEQ ID NO: 116) and (GGGS) n (SEQ ID NO: 117), where n is an integer of at least one.
  • At least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 118), GGSGG (SEQ ID NO: 119), GSGSG (SEQ ID NO: 120), GSGGG (SEQ ID NO: 121), GGGSG (SEQ ID NO: 122), and GSSSG (SEQ ID NO: 123).
  • LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 124), GSSGGSGGSGG (SEQ ID NO: 125), GSSGGSGGSGGS (SEQ ID NO: 126), GSSGGSGGSGGSGGGS (SEQ ID NO: 127), GSSGGSGGSG (SEQ ID NO: 128), GSSGGSGGSGS (SEQ ID NO: 129), or GGGSSGGS (SEQ ID NO: 134).
  • LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 130), GSSGT (SEQ ID NO: 131) or GSSG (SEQ ID NO: 132).
  • the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer, where LP1 comprises the amino acid sequence GGGSSGGS (SEQ ID NO: 134) and the LP2 comprises the amino acid sequence GGS.
  • the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer, where LP1 comprises the amino acid sequence GGGSSGGS (SEQ ID NO: 134) and the LP2 comprises the amino acid sequence GGGS (SEQ ID NO: 130).
  • Conjugated CD47 Antibodies and Activatable Antibodies [000267]
  • the CD47 antibodies and activatable antibodies described herein also include an agent conjugated to the antibody/activatable antibody.
  • the conjugated agent is a therapeutic agent, such as an anti-inflammatory and/or an antineoplastic agent.
  • the agent is conjugated to a carbohydrate moiety of the antibody/activatable antibody, for example, in some embodiments, where the carbohydrate moiety is located outside the antigen-binding region of the antibody or antigen-binding fragment in the activatable antibody.
  • the agent is conjugated to a sulfhydryl group of the antibody or antigen-binding fragment in the antibody/activatable antibody.
  • the agent is a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • 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 disclosure also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • cytotoxic agents include, for example, dolastatins and derivatives thereof (e.g. auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE).
  • the agent is monomethyl auristatin E (MMAE) or monomethyl auristatin D (MMAD).
  • the agent is an agent selected from the group listed in Table E. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or derivative thereof. In some embodiments, the agent is a calicheamicin or derivative thereof.
  • the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • the agent is a toxin or fragment thereof.
  • the agent is a microtubule inhibitor.
  • the agent is a nucleic acid damaging agent.
  • the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof.
  • the agent is auristatin E or a derivative thereof.
  • the agent is monomethyl auristatin E (MMAE).
  • the agent is monomethyl auristatin D (MMAD).
  • the agent is a maytansinoid selected from the group consisting of DM1 and DM4.
  • the agent is maytansinoid DM4. In some embodiments, the agent is duocarmycin. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker with which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety. In some embodiments, the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, vc-duocarmycin, or a PEG2-vc-MMAD moiety. In some embodiments, the linker is a cleavable linker.
  • the linker is a non-cleavable linker.
  • the agent is a detectable moiety.
  • the detectable moiety is a diagnostic agent.
  • the conjugated activatable antibody comprises an antibody comprising: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB comprises: (i) the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7);
  • the CD47 antibodies and activatable antibodies of the disclosure have at least one point of conjugation for an agent, but in the methods and compositions provided herein less than all possible points of conjugation are available for conjugation to an agent.
  • the one or more points of conjugation are sulfur atoms involved in disulfide bonds.
  • the one or more points of conjugation are sulfur atoms involved in interchain disulfide bonds.
  • the one or more points of conjugation are sulfur atoms involved in interchain sulfide bonds, but not sulfur atoms involved in intrachain disulfide bonds.
  • the one or more points of conjugation are sulfur atoms of cysteine or other amino acid residues containing a sulfur atom.
  • Such residues may occur naturally in the antibody structure or can be incorporated into the antibody by site-directed mutagenesis, chemical conversion, or mis-incorporation of non-natural amino acids.
  • Also provided are methods of preparing a conjugate of a CD47 antibody/CD47 activatable antibody having one or more interchain disulfide bonds in the AB and one or more intrachain disulfide bonds in the MM, and a drug reactive with free thiols is provided.
  • the method generally includes partially reducing interchain disulfide bonds in the activatable antibody with a reducing agent, such as, for example, TCEP; and conjugating the drug reactive with free thiols to the partially reduced antibody/activatable antibody.
  • partial reduction refers to situations where an act antibody/activatable antibody is contacted with a reducing agent and less than all disulfide bonds, e.g., less than all possible sites of conjugation are reduced. In some embodiments, less than 99%, 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.
  • a method of reducing and conjugating an agent, e.g., a drug, to a CD47 antibody/CD47 activatable antibody resulting in selectivity in the placement of the agent is provided.
  • the method generally includes partially reducing the CD47 antibody/CD47 activatable antibody with a reducing agent such that any conjugation sites in the masking moiety or other non-AB portion of the CD47 antibody/CD47 activatable antibody are not reduced, and conjugating the agent to interchain thiols in the AB.
  • the conjugation site(s) are selected so as to allow desired placement of an agent to allow conjugation to occur at a desired site.
  • the reducing agent is, for example, TCEP.
  • the reduction reaction conditions such as, for example, the ratio of reducing agent to antibody/activatable antibody, the length of incubation, the temperature during the incubation, the pH of the reducing reaction solution, etc., are determined by identifying the conditions that produce a conjugated antibody/activatable antibody e.g. under conditions that produce a conjugated activatable antibody in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in an uncleaved state.
  • the ratio of reduction agent to antibody/activatable antibody will vary depending on the antibody/activatable antibody.
  • the ratio of reducing agent to antibody/activatable antibody 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 about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5.
  • the ratio is in a range of from about 5:1 to 1:1. In some embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In some embodiments, the ratio is in a range of from about 4:1 to 1:1. In some embodiments, the ratio is in a range from about 4:1 to 1.5:1. In some embodiments, the ratio is in a range from about 8:1 to about 1:1. In some embodiments, the ratio is in a range of from about 2.5:1 to 1:1. [000277] In some embodiments, the CD47 antibody undergoes conjugation first and is then further modified to include a CM and MM (resulting in an activatable antibody). In some embodiments, the CD47 activatable antibody is conjugated.
  • a method of reducing interchain disulfide bonds in the AB of an activatable CD47 antibody and conjugating an agent, e.g., a thiol-containing agent such as a drug, to the resulting interchain thiols to selectively locate agent(s) on the AB is provided.
  • the method generally includes partially reducing the AB with a reducing agent to form at least two interchain thiols without forming all possible interchain thiols in the activatable antibody; and conjugating the agent to the interchain thiols of the partially reduced AB.
  • the AB of the activatable antibody is partially reduced for about 1 hour at about 37°C at a desired ratio of reducing agent:activatable antibody.
  • the ratio of reducing agent to activatable antibody 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 about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5.
  • the ratio is in a range of from about 5:1 to 1:1. In some embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In some embodiments, the ratio is in a range of from about 4:1 to 1:1. In some embodiments, the ratio is in a range from about 4:1 to 1.5:1. In some embodiments, the ratio is in a range from about 8:1 to about 1:1. In some embodiments, the ratio is in a range of from about 2.5:1 to 1:1. [000279]
  • the thiol-containing reagent can be, for example, cysteine or N-acetyl cysteine.
  • the reducing agent can be, for example, TCEP.
  • the reduced activatable antibody can be purified prior to conjugation, using for example, column chromatography, dialysis, or diafiltration. Alternatively, the reduced antibody is not purified after partial reduction and prior to conjugation.
  • the invention also provides partially reduced antibodies/activatable antibodies in which at least one interchain disulfide bond in the antibody/activatable antibody has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the antibody/activatable antibody, wherein the activatable antibody includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to CD47, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the CD47 target, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
  • AB antigen binding fragment thereof
  • MM masking moiety
  • CM cleavable moiety
  • the MM is coupled to the AB via the CM.
  • one or more intrachain disulfide bond(s) of the antibody/activatable antibody is not disturbed by the reducing agent.
  • one or more intrachain disulfide bond(s) of the MM within the antibody/activatable antibody is not disturbed by the reducing agent.
  • the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM.
  • reducing agent is TCEP.
  • a method of reducing and conjugating an agent, e.g., a drug, to a CD47 antibody/CD47 activatable antibody resulting in selectivity in the placement of the agent by providing an activatable CD47 antibody with a defined number and positions of lysine and/or cysteine residues.
  • the defined number of lysine and/or cysteine residues is higher or lower than the number of corresponding residues in the amino acid sequence of the parent antibody or activatable antibody.
  • the defined number of lysine and/or cysteine residues may result in a defined number of agent equivalents that can be conjugated to the CD47 antibody or activatable CD47 antibody.
  • the defined number of lysine and/or cysteine residues may result in a defined number of agent equivalents that can be conjugated to the CD47 antibody or activatable CD47 antibody in a site-specific manner.
  • the modified activatable antibody is modified with one or more non-natural amino acids in a site-specific manner, thus in some embodiments limiting the conjugation of the agents to only the sites of the non-natural amino acids.
  • the CD47 antibody or activatable CD47 antibody with a defined number and positions of lysine and/or cysteine residues can be partially reduced with a reducing agent as discussed herein such that any conjugation sites in the masking moiety or other non-AB portion of the activatable antibody are not reduced, and conjugating the agent to interchain thiols in the AB.
  • the disclosure also provides partially reduced activatable antibodies in which at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the activatable antibody includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to the target, e.g., CD47, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the target, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for at least one protease.
  • AB antigen binding fragment thereof
  • MM masking moiety
  • CM cleavable moiety
  • the MM is coupled to the AB via the CM.
  • one or more intrachain disulfide bond(s) of the activatable antibody is not disturbed by the reducing agent.
  • one or more intrachain disulfide bond(s) of the MM within the activatable antibody is not disturbed by the reducing agent.
  • the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM.
  • reducing agent is TCEP.
  • the agent is linked to the AB using a maleimide caproyl- valine-citrulline linker or a maleimide PEG-valine-citrulline linker. In some embodiments, the agent is linked to the AB using a maleimide caproyl-valine-citrulline linker.
  • the agent is linked to the AB using a maleimide PEG-valine-citrulline linker
  • the agent is monomethyl auristatin D (MMAD) linked to the AB using a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as “vc-MMAD.”
  • the agent is monomethyl auristatin E (MMAE) linked to the AB using a maleimide PEG-valine-citrulline-para- aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as “vc- MMAE.”
  • the agent is linked to the AB using a maleimide PEG-valine- citrulline linker
  • the agent is monomethyl auristatin D (MMAD) linked to the AB using a maleimide bis-PEG-valine-
  • PEG2-vc-MMAD [000284]
  • the disclosure also provides conjugated activatable antibodies that include an activatable antibody linked to monomethyl auristatin D (MMAD) payload, wherein the activatable antibody includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the target, and cleavable moiety (CM) coupled to the AB, and the CM is a polypeptide that functions as a substrate for at least one MMP protease.
  • MMAD monomethyl auristatin D
  • the MMAD-conjugated activatable antibody can be conjugated using any of several methods for attaching agents to ABs: (a) attachment to the carbohydrate moieties of the AB, or (b) attachment to sulfhydryl groups of the AB, or (c) attachment to amino groups of the AB, or (d) attachment to carboxylate groups of the AB. [000286]
  • the MMAD payload is conjugated to the AB via a linker.
  • the MMAD payload is conjugated to a cysteine in the AB via a linker.
  • the MMAD payload is conjugated to a lysine in the AB via a linker. In some embodiments, the MMAD payload is conjugated to another residue of the AB via a linker, such as those residues disclosed herein.
  • the linker is a thiol-containing linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non- cleavable linker. In some embodiments, the linker is selected from the group consisting of the linkers shown in Tables 7 and 8. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide caproyl-valine-citrulline linker.
  • the activatable antibody and the MMAD payload are linked via a maleimide PEG-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide caproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide PEG-valine- citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation technology disclosed herein.
  • the polyethylene glycol (PEG) component of a linker of the present disclosure is formed from 2 ethylene glycol monomers, 3 ethylene glycol monomers, 4 ethylene glycol monomers, 5 ethylene glycol monomers, 6 ethylene glycol monomers, 7 ethylene glycol monomers 8 ethylene glycol monomers, 9 ethylene glycol monomers, or at least 10 ethylene glycol monomers.
  • the PEG component is a branched polymer. In some embodiments of the present disclosure, the PEG component is an unbranched polymer.
  • the PEG polymer component is functionalized with an amino group or derivative thereof, a carboxyl group or derivative thereof, or both an amino group or derivative thereof and a carboxyl group or derivative thereof.
  • the PEG component of a linker of the present disclosure is an amino-tetra-ethylene glycol-carboxyl group or derivative thereof.
  • the PEG component of a linker of the present disclosure is an amino-tri-ethylene glycol-carboxyl group or derivative thereof.
  • the PEG component of a linker of the present disclosure is an amino-di-ethylene glycol-carboxyl group or derivative thereof.
  • an amino derivative is the formation of an amide bond between the amino group and a carboxyl group to which it is conjugated.
  • a carboxyl derivative is the formation of an amide bond between the carboxyl group and an amino group to which it is conjugated.
  • a carboxyl derivative is the formation of an ester bond between the carboxyl group and an hydroxyl group to which it is conjugated.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4- dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyr
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94/11026).
  • Table E lists some of the exemplary pharmaceutical agents that can be employed in the herein described disclosure but in no way is meant to be an exhaustive list.
  • Table E Exemplary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTS Auristatins Turbostatin Auristatin E Phenstatins Monomethyl auristatin D (MMAD) Hydroxyphenstatin Monomethyl auristatin E (MMAE) Spongistatin 5 Desmethyl auristatin E (DMAE) Spongistatin 7 Auristatin F Halistatin 1 Monomethyl auristatin F (MMAF) Halistatin 2 Desmethyl auristatin F (DMAF) Halistatin 3 Auristatin derivatives, e.g., amides thereof Modified Bryostatins Auristatin tyramine Halocomstatins Auristatin quinoline Pyrrolobenzimidazoles (PBI) Dolastatins Cibrostatin6 Dolastatin derivatives Doxaliform Dolastatin 16 DmJ Anthracyclins analogues Dolastatin 16 Dpv Maytansinoids, e.g.
  • Coupling can be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. In some embodiments, the binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.
  • bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present disclosure, to other molecules.
  • representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
  • This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun.133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).
  • the activatable antibody in addition to the compositions and methods provided herein, can also be modified for site-specific conjugation through modified amino acid sequences inserted or otherwise included in the activatable antibody sequence. These modified amino acid sequences are designed to allow for controlled placement and/or dosage of the conjugated agent within a conjugated activatable antibody.
  • the activatable antibody can be engineered to include cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not negatively impact protein folding and assembly, nor alter antigen binding.
  • the activatable antibody can be engineered to include or otherwise introduce one or more non-natural amino acid residues within the activatable antibody to provide suitable sites for conjugation.
  • the activatable antibody can be engineered to include or otherwise introduce enzymatically activatable peptide sequences within the activatable antibody sequence.
  • Suitable linkers 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 antibody by way of an oligopeptide linker.
  • 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.
  • Additional linkers include, but are not limited to, SMCC ((succinimidyl 4- (N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC (sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate), SPDB (N-succinimidyl-4-(2-pyridyldithio) butanoate), or sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfo butanoate).
  • SMCC succinimidyl 4- (N-maleimidomethyl)cyclohexane-1-carboxylate)
  • sulfo-SMCC sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate
  • SPDB N-succinimidy
  • linkers 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.
  • the linkers are cleavable.
  • the linkers are non-cleavable.
  • two or more linkers are present. The two or more linkers are all the same, i.e., cleavable or non-cleavable, or the two or more linkers are different, i.e., at least one cleavable and at least one non-cleavable.
  • ABs can be covalently attached to an agent through an intermediate linker having at least two reactive groups, one to react with AB and one to react with the agent.
  • the linker which may include any compatible organic compound, can be chosen such that the reaction with AB (or agent) does not adversely affect AB reactivity and selectivity. Furthermore, the attachment of linker to agent might not destroy the activity of the agent.
  • Suitable linkers for reaction with oxidized antibodies or oxidized antibody fragments include those containing an amine selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups. Such reactive functional groups may exist as part of the structure of the linker, or can be introduced by suitable chemical modification of linkers not containing such groups. [000298] According to the present disclosure, suitable linkers for attachment to reduced ABs include those having certain reactive groups capable of reaction with a sulfhydryl group of a reduced antibody or fragment.
  • Such reactive groups include, but are not limited to: reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercuribenzoate groups and groups capable of Michael-type addition reactions (including, for example, maleimides and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc.16: 3097-3110).
  • suitable linkers for attachment to neither oxidized nor reduced Abs include those having certain functional groups capable of reaction with the primary amino groups present in unmodified lysine residues in the Ab.
  • Such reactive groups include, but are not limited to, NHS carboxylic or carbonic esters, sulfo-NHS carboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonic esters, pentafluorophenyl carboxylic or carbonic esters, acyl imidazoles, isocyanates, and isothiocyanates.
  • suitable linkers for attachment to neither oxidized nor reduced Abs include those having certain functional groups capable of reaction with the carboxylic acid groups present in aspartate or glutamate residues in the Ab, which have been activated with suitable reagents.
  • Suitable activating reagents include EDC, with or without added NHS or sulfo-NHS, and other dehydrating agents utilized for carboxamide formation.
  • the functional groups present in the suitable linkers would include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.
  • the agent can be attached to the linker before or after the linker is attached to the AB. In certain applications it may be desirable to first produce an AB-linker intermediate in which the linker is free of an associated agent. Depending upon the particular application, a specific agent may then be covalently attached to the linker.
  • the AB is first attached to the MM, CM and associated linkers and then attached to the linker for conjugation purposes.
  • Branched Linkers In specific embodiments, branched linkers that have multiple sites for attachment of agents are utilized. For multiple site linkers, a single covalent attachment to an AB would result in an AB-linker intermediate capable of binding an agent at a number of sites. The sites can be aldehyde or sulfhydryl groups or any chemical site to which agents can be attached.
  • higher specific activity or higher ratio of agents to AB
  • This plurality of sites can be introduced into the AB by either of two methods. First, one may generate multiple aldehyde groups and/or sulfhydryl groups in the same AB. Second, one may attach to an aldehyde or sulfhydryl of the AB a "branched linker" having multiple functional sites for subsequent attachment to linkers.
  • the functional sites of the branched linker or multiple site linker can be aldehyde or sulfhydryl groups, or can be any chemical site to which linkers can be attached. Still higher specific activities can be obtained by combining these two approaches, that is, attaching multiple site linkers at several sites on the AB.
  • Cleavable Linkers Peptide linkers that are susceptible to cleavage by enzymes of the complement system, such as but not limited to u-plasminogen activator, tissue plasminogen activator, trypsin, plasmin, or another enzyme having proteolytic activity can be used in one embodiment of the present disclosure.
  • an agent is attached via a linker susceptible to cleavage by complement.
  • the antibody is selected from a class that can activate complement. The antibody-agent conjugate, thus, activates the complement cascade and releases the agent at the target site.
  • an agent is attached via a linker susceptible to cleavage by enzymes having a proteolytic activity such as a u-plasminogen activator, a tissue plasminogen activator, plasmin, or trypsin.
  • cleavable linkers are useful in conjugated activatable antibodies that include an extracellular toxin, e.g., by way of non-limiting example, any of the extracellular toxins shown in Table E.
  • Non-limiting examples of cleavable linker sequences are provided in Table F.
  • Table F Exemplary Linker Sequences for Conjugation Types of Cleavable Sequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinase PRFKIIGG (SEQ ID NO: 615) PRFRIIGG (SEQ ID NO: 616) TGFb SSRHRRALD (SEQ ID NO: 617) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 618) Staphylokinase SSSFDKGKYKKGDDA (SEQ ID NO: 619) SSSFDKGKYKRGDDA (SEQ ID NO: 620) Factor Xa cleavable sequences IEGR (SEQ ID NO: 621) IDGR (SEQ ID NO: 622) GGSIDGR (SEQ ID NO: 623) MMP cleavable sequences Gelatinase A PLGLWA (SEQ ID NO: 624) Collagenase cleavable sequences Calf skin collagen
  • the reducing agent that would modify a CM would also modify the linker of the conjugated activatable antibody.
  • Spacers and Cleavable Elements In some embodiments, it may be necessary to construct the linker in such a way as to optimize the spacing between the agent and the AB of the activatable antibody.
  • the linker may comprise a spacer element and a cleavable element.
  • the spacer element serves to position the cleavable element away from the core of the AB such that the cleavable element is more accessible to the enzyme responsible for cleavage.
  • Certain of the branched linkers described above may serve as spacer elements.
  • linker to agent or of spacer element to cleavable element, or cleavable element to agent
  • Any reaction providing a product of suitable stability and biological compatibility is acceptable.
  • Serum Complement and Selection of Linkers according to one method of the present disclosure, when release of an agent is desired, an AB that is an antibody of a class that can activate complement is used. The resulting conjugate retains both the ability to bind antigen and activate the complement cascade.
  • an agent is joined to one end of the cleavable linker or cleavable element and the other end of the linker group is attached to a specific site on the AB.
  • the agent has an hydroxy group or an amino group, it can be attached to the carboxy terminus of a peptide, amino acid or other suitably chosen linker via an ester or amide bond, respectively.
  • such agents can be attached to the linker peptide via a carbodimide reaction. If the agent contains functional groups that would interfere with attachment to the linker, these interfering functional groups can be blocked before attachment and deblocked once the product conjugate or intermediate is made.
  • Linkers can be of any desired length, one end of which can be covalently attached to specific sites on the AB of the activatable antibody. The other end of the linker or spacer element can be attached to an amino acid or peptide linker.
  • conjugates when administered to a subject, will accomplish delivery and release of the agent at the target site, and are particularly effective for the in vivo delivery of pharmaceutical agents, antibiotics, antimetabolites, antiproliferative agents and the like as presented in but not limited to those in Table E.
  • Linkers for Release without Complement Activation In yet another application of targeted delivery, release of the agent without complement activation is desired since activation of the complement cascade will ultimately lyse the target cell. Hence, this approach is useful when delivery and release of the agent should be accomplished without killing the target cell. Such is the goal when delivery of cell mediators such as hormones, enzymes, corticosteroids, neurotransmitters, genes or enzymes to target cells is desired.
  • conjugates can be prepared by attaching the agent to an AB that is not capable of activating complement via a linker that is mildly susceptible to cleavage by serum proteases.
  • this conjugate is administered to an individual, antigen-antibody complexes will form quickly whereas cleavage of the agent will occur slowly, thus resulting in release of the compound at the target site.
  • Biochemical Cross Linkers In some embodiments, the activatable antibody can be conjugated to one or more therapeutic agents using certain biochemical cross-linkers. Cross- linking reagents form molecular bridges that tie together functional groups of two different molecules. To link two different proteins in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.
  • Peptidyl linkers cleavable by lysosomal proteases are also useful, for example, Val-Cit, Val-Ala or other dipeptides.
  • acid-labile linkers cleavable in the low-pH environment of the lysosome can be used, for example: bis-sialyl ether.
  • Other suitable linkers include cathepsin-labile substrates, particularly those that show optimal function at an acidic pH.
  • Exemplary hetero-bifunctional cross-linkers are referenced in Table G.
  • Table G Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length after cross-linking Linker Reactive Toward Advantages and Applications (Angstroms)
  • SMPT Primary amines Greater stability 11.2 ⁇ Sulfhydryls SPDP Primary amines Thiolation 6.8 ⁇ Sulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extended spacer arm 15.6 ⁇ Sulfhydryls Sulfo-LC-SPDP Primary amines Extender spacer arm 15.6 ⁇ Sulfhydryls Water-soluble SMCC Primary amines Stable maleimide reactive 11.6 ⁇ group Sulfhydryls Enzyme-antibody conjugation Hapten-carrier protein conjugation Sulfo-SMCC Primary amines Stable maleimide reactive 11.6 ⁇ group Sulfhydryls Water-soluble Enzyme-antibody conjugation MBS Primary amines Enzy
  • Non-cleavable linkers may include amino acids, peptides, D-amino acids or other organic compounds that can be modified to include functional groups that can subsequently be utilized in attachment to ABs by the methods described herein.
  • A-general formula for such an organic linker could be W – (CH 2 )n – Q wherein W is either --NH--CH 2 -- or --CH 2 --; Q is an amino acid, peptide; and n is an integer from 0 to 20.
  • Non-Cleavable Conjugates In some embodiments, a compound can be attached to ABs that do not activate complement.
  • the antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab’ fragments of the antibody of the present disclosure can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
  • the antibody drug conjugates (ADCs) and activatable antibody drug conjugates (AADCs) can include one or more polypeptides that include the combination of a light chain sequence or a light chain variable domain sequence, and a heavy chain sequence or a heavy chain variable domain sequence, a linker, and a toxin in a given row of Table D or any combination of a light chain sequence or a light chain variable domain sequence, and a heavy chain sequence or a heavy chain variable domain sequence, a linker, and a toxin of Table D.
  • Combination No.1 comprises the heavy chain of SEQ ID No.1, the light chain of SEQ ID No.160, a vc linker, conjugated to MMAD toxin.
  • Table D CD47 Activatable ADC Combinations
  • any of the combinations in Table D that list a heavy chain and light chain variable region can be combined with human immunoglobulin constant regions to result in fully human IgGs including IgG1, IgG2, IgG4 or mutated constant regions to result in human IgGs with altered functions such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P.
  • the combinations described in Table D are not limited by the particular combinations shown in any given row, and thus can include any heavy chain sequence or heavy chain variable region sequence from column 2 of Table D combined with any light chain sequence or light chain variable region sequence from column 3 of Table D combined with any linker from column 4 combined with any toxin from column 5.
  • any heavy chain sequence or heavy chain variable region sequence disclosed herein can be used in a combination.
  • any light chain sequence or light chain variable region sequence disclosed herein can be used in a combination.
  • any linker disclosed herein can be used in a combination.
  • any toxin disclosed herein can be used in a combination.
  • Multispecific Antibodies and Activatable Antibodies [000324] In some embodiments, the activatable CD47 antibody and/or conjugated activatable CD47 antibody is monospecific.
  • the activatable CD47 antibody and/or conjugated activatable CD47 antibody is multispecific, e.g., by way of non- limiting example, bispecific or trifunctional.
  • the activatable CD47 antibody and/or conjugated activatable CD47 antibody is formulated as part of a pro-Bispecific T Cell Engager (BITE) molecule.
  • the activatable CD47 antibody and/or conjugated activatable CD47 antibody is formulated as part of a pro-Chimeric Antigen Receptor (CAR) modified T cell or other engineered receptor.
  • BITE pro-Bispecific T Cell Engager
  • CAR pro-Chimeric Antigen Receptor
  • the multispecific antibodies provided herein are multispecific antibodies that recognize CD47 and at least one or more different antigens or epitopes.
  • the disclosure also provides multispecific CD47 activatable antibodies.
  • the multispecific activatable antibodies provided herein are multispecific antibodies that recognize CD47 and at least one or more different antigens or epitopes and that include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody such that coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target.
  • MM masking moiety
  • the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM) that functions as a substrate for at least one protease.
  • CM cleavable moiety
  • the activatable multispecific antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to a target that is at least comparable to the corresponding, unmodified multispecific antibody.
  • the activatable antibody or antigen-binding fragment thereof is incorporated in a multispecific activatable antibody or antigen-binding fragment thereof, where at least one arm of the multispecific activatable antibody specifically binds CD47.
  • the activatable antibody or antigen-binding fragment thereof is incorporated in a bispecific antibody or antigen-binding fragment thereof, where at least one arm of the bispecific activatable antibody specifically binds CD47.
  • the antibody or antigen-binding fragment thereof is incorporated in a multispecific antibody or antigen-binding fragment thereof, where at least one arm of the multispecific antibody or antigen-binding fragment thereof specifically binds CD47.
  • the antibody or antigen-binding fragment thereof is incorporated in a bispecific antibody or antigen-binding fragment thereof, where at least one arm of the bispecific antibody or antigen-binding fragment thereof specifically binds CD47.
  • At least one arm of the multispecific antibody or antigen- binding fragment thereof comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1.
  • at least one arm of the multispecific antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence of SEQ ID NO: 2.
  • At least one arm of the multispecific antibody or antigen- binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4.
  • At least one arm of the multispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1.
  • At least one arm of the multispecific antibody or antigen-binding fragment thereof comprises a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 2.
  • At least one arm of the multispecific antibody or antigen- binding fragment thereof comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one complementarity determining region (CDR) sequence is selected from VH CDR1, also referred to herein as CDRH1
  • At least one arm of the multispecific antibody or antigen- binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GFTFSNYWMN (SEQ ID NO: 37) or GFTFSNYWMD (SEQ ID NO: 38); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9
  • At least one arm of the multispecific antibody or antigen- binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NY
  • At least one arm of the multispecific antibody or antigen- binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GFTFSNYWMN (SEQ ID NO: 37) or GFTFSNYWMD (SEQ ID NO: 38); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR
  • the multispecific antibodies/activatable antibodies are designed to engage immune effector cells, also referred to herein as immune-effector cell engaging multispecific activatable antibodies.
  • the multispecific antibodies/activatable antibodies are designed to engage leukocytes, also referred to herein as leukocyte engaging multispecific activatable antibodies.
  • the multispecific antibodies/activatable antibodies are designed to engage T cells, also referred to herein as T-cell engaging multispecific antibodies/activatable antibodies.
  • the multispecific antibodies/activatable antibodies engage a surface antigen on a leukocyte, such as on a T cell, on a natural killer (NK) cell, on a myeloid mononuclear cell, on a macrophage, and/or on another immune effector cell.
  • the immune effector cell is a leukocyte.
  • the immune effector cell is a T cell.
  • the immune effector cell is a NK cell.
  • the immune effector cell is a mononuclear cell, such as a myeloid mononuclear cell.
  • the multispecific activatable antibodies are designed to bind or otherwise interact with more than one target and/or more than one epitope, also referred to herein as multi-antigen targeting activatable antibodies.
  • target and “antigen” are used interchangeably.
  • immune effector cell engaging multispecific activatable antibodies of the disclosure include a targeting antibody or antigen-binding fragment thereof that binds CD47 and an immune effector cell engaging antibody or antigen-binding portion thereof, where at least one of the targeting antibody or antigen-binding fragment thereof and/or the immune effector cell engaging antibody or antigen-binding portion thereof is masked.
  • the immune effector cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, immune effector cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target.
  • the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47.
  • the immune effector cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, immune effector cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47.
  • AB1 antibody or antigen-binding fragment thereof
  • MM1 masking moiety
  • the non-immune effector cell engaging antibody is a cancer targeting antibody. In some embodiments the non-immune cell effector antibody is an IgG. In some embodiments the immune effector cell engaging antibody is a scFv. In some embodiments the CD47-targeting antibody (e.g., non-immune cell effector antibody) is an IgG and the immune effector cell engaging antibody is a scFv. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a myeloid mononuclear cell.
  • T-cell engaging multispecific activatable antibodies of the disclosure include a CD47-targeting antibody or antigen-binding fragment thereof and a T-cell engaging antibody or antigen-binding portion thereof, where at least one of the CD47-targeting antibody or antigen-binding fragment thereof and/or the T-cell engaging antibody or antigen- binding portion thereof is masked.
  • the T-cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, T-cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target.
  • the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47.
  • AB2 second antibody or antigen-binding fragment thereof
  • MM2 masking moiety
  • the T- cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen- binding fragment thereof (AB1) that binds a first, T-cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen- binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47.
  • AB1 antibody or antigen- binding fragment thereof
  • MM1 masking moiety
  • the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen- binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling
  • one antigen is CD47, and another antigen is typically a stimulatory or inhibitory receptor present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.
  • NK natural killer
  • the antigen is a stimulatory receptor present on the surface of a T cell or NK cell; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40.
  • the antigen is an inhibitory receptor present on the surface of a T-cell; examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIRs.
  • the antibody domain conferring specificity to the T-cell surface antigen may also be substituted by a ligand or ligand domain that binds to a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PDL1, PDL2, or TNFSF9.
  • a ligand or ligand domain that binds to a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PDL1, PDL2, or TNFSF9.
  • the T-cell engaging multispecific activatable antibody includes an anti-CD3 epsilon (CD3e, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, where at least one of the anti-CD3e scFv and/or the targeting antibody or antigen-binding portion thereof is masked.
  • the CD3e scFv includes a first antibody or antigen-binding fragment thereof (AB1) that binds CD3e, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind CD3e.
  • the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47.
  • AB2 second antibody or antigen-binding fragment thereof
  • MM2 masking moiety
  • the CD3e scFv includes a first antibody or antigen-binding fragment thereof (AB1) that binds CD3e, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind CD3e, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47.
  • AB1 first antibody or antigen-binding fragment thereof
  • MM1 masking moiety
  • the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47
  • AB2 is attached to a masking moiety
  • the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies include at least a first antibody or antigen-binding fragment thereof that binds a first target and/or first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and/or a second epitope.
  • the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind two or more different targets.
  • the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind two or more different epitopes on the same target.
  • the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind a combination of two or more different targets and two or more different epitopes on the same target.
  • a multispecific activatable antibody comprising an IgG has the IgG variable domains masked.
  • a multispecific activatable antibody comprising a scFv has the scFv domains masked.
  • a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the IgG variable domains is coupled to a masking moiety.
  • a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the scFv domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where each of the IgG variable domains and the scFv domains is coupled to its own masking moiety.
  • one antibody domain of a multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for another target antigen. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for an epitope of a target antigen and another antibody domain has specificity for another epitope of the target antigen.
  • a scFv in a multispecific activatable antibody, can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, to the carboxyl terminus of the light chain of an IgG activatable antibody, or to the carboxyl termini of both the heavy and light chains of an IgG activatable antibody.
  • a scFv in a multispecific activatable antibody, can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, to the amino terminus of the light chain of an IgG activatable antibody, or to the amino termini of both the heavy and light chains of an IgG activatable antibody.
  • a scFv can be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody.
  • a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of the IgG.
  • a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of at least one scFv.
  • a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of an IgG and a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of at least one scFv.
  • the disclosure provides examples of multispecific activatable antibody structures which include, but are not limited to, the following: (VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*- L3-VL*-L2-CM-L1-MM)2; (VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1- MM) 2 ; (MM-L1-CM-L2-VL-CL) 2 :(VH-CH1-CH2-CH3-L4-VH*-L3-VL*) 2 ; (MM-L1-CM-L2- VL-CL) 2 :(VH-CH1-CH2-CH3-L4-VL*-L3-VH*) 2 ; (VL-CL) 2 :(MM-L1-CM-L2-VL*-L3-VH*- L4-VH-CH1-CH
  • the first and second specificities can be toward any antigen or epitope.
  • one antigen is CD47, and another antigen is typically a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.
  • the antibody domain conferring specificity to the T-cell surface antigen may also be substituted by a ligand or ligand domain that binds to a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/or other immune effector cell receptor.
  • the targeting antibody is a CD47 antibody disclosed herein.
  • the targeting antibody can be in the form an activatable antibody.
  • the scFv(s) can be in the form of a Pro-scFv (see, e.g., WO 2009/025846, WO 2010/081173).
  • the scFv is specific for binding CD3e, and comprises or is derived from an antibody or fragment thereof that binds CD3e, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9.
  • the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4).
  • the anti-CTLA-4 scFv includes the amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKV EIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQ APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYW YFDLWGRGTLVTVSSAS (SEQ ID NO: 643) [000349] In some embodiments, the anti-CTLA-4 scFv includes the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
  • the anti-CD3 ⁇ scFv includes the amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRP GQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC LDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGS GTKLEINR (SEQ ID NO: 644) [000351] In some embodiments, the anti-CD3 ⁇ scFv includes the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
  • the scFv is specific for binding one or more T-cells, one or more NK-cells and/or one or more macrophages.
  • the scFv is specific for binding a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.
  • the multispecific antibody/activatable antibody also includes an agent conjugated to the AB.
  • the agent is a therapeutic agent. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is conjugated to the multispecific antibody/activatable antibody via a linker. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylator or DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker.
  • the agent is an agent selected from the group listed in Table E. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or derivative thereof. In some embodiments, the agent is a calicheamicin or derivative thereof.
  • the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer.
  • the multispecific antibody/activatable antibody also includes a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. [000355] In some embodiments, the multispecific antibody/activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the multispecific activatable antibody can be engineered to include one or more disulfide bonds.
  • the disclosure also provides an isolated nucleic acid molecule encoding a multispecific antibody/activatable antibody described herein, as well as vectors that include these isolated nucleic acid sequences.
  • the disclosure provides methods of producing a multispecific antibody/activatable antibody by culturing a cell under conditions that lead to expression of the antibody/activatable antibody, wherein the cell comprises such a nucleic acid molecule. In some embodiments, the cell comprises such a vector.
  • the disclosure also provides a method of manufacturing multispecific CD47 antibodies of the disclosure by (a) culturing a cell comprising a nucleic acid construct that encodes the multispecific antibody under conditions that lead to expression of the multispecific antibody.
  • the disclosure also provides a method of manufacturing multispecific activatable CD47 antibodies of the disclosure by (a) culturing a cell comprising a nucleic acid construct that encodes the multispecific activatable antibody under conditions that lead to expression of the multispecific activatable antibody, and (b) recovering the multispecific activatable antibody.
  • Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.
  • the disclosure also provides multispecific activatable antibodies and/or multispecific activatable antibody compositions that include at least a first antibody or antigen- binding fragment thereof (AB1) that specifically binds a first target or first epitope and a second antibody or antigen-biding fragment thereof (AB2) that binds a second target or a second epitope, where at least AB1 is coupled or otherwise attached to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind its target.
  • AB1 antibody or antigen- binding fragment thereof
  • AB2 second antibody or antigen-biding fragment thereof
  • the MM1 is coupled to AB1 via a first cleavable moiety (CM1) sequence that includes a substrate for a protease, for example, a protease that is co-localized with the target of AB1 at a treatment site or a diagnostic site in a subject.
  • CM1 first cleavable moiety
  • the multispecific activatable antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to the target of AB1 that is at least comparable to the corresponding, unmodified multispecific antibody.
  • Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.
  • compositions and methods that include a multispecific activatable antibody that includes at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), where at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that decreases the ability of AB1 to bind its target.
  • a multispecific activatable antibody that includes at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), where at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that decreases the ability of AB1 to bind its target.
  • MM1 masking moiety
  • each AB is coupled to a MM that decreases the ability of its corresponding AB to each target.
  • AB1 is coupled to a first masking moiety (MM1) that decreases the ability of AB1 to bind its target
  • AB2 is coupled to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target.
  • the multispecific activatable antibody comprises more than two AB regions; in such embodiments, AB1 is coupled to a first masking moiety (MM1) that decreases the ability of AB1 to bind its target, AB2 is coupled to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target, AB3 is coupled to a third masking moiety (MM3) that decreases the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody.
  • Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.
  • the multispecific activatable antibody further includes at least one cleavable moiety (CM) that is a substrate for a protease, where the CM links a MM to an AB.
  • the multispecific activatable antibody includes at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), where at least the first AB in the multispecific activatable antibody is coupled via a first cleavable moiety (CM1) to a masking moiety (MM1) that decreases the ability of AB1 to bind its target.
  • AB1 is coupled via CM1 to MM1, and AB2 is coupled via a second cleavable moiety (CM2) to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target.
  • the multispecific activatable antibody comprises more than two AB regions; in some of these embodiments, AB1 is coupled via CM1 to MM1, AB2 is coupled via CM2 to MM2, and AB3 is coupled via a third cleavable moiety (CM3) to a third masking moiety (MM3) that decreases the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody.
  • Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.
  • Activatable Antibodies Having Non-Binding Steric Moieties or Binding Partners for Non-Binding Steric Moieties [000362]
  • the disclosure also provides activatable antibodies that include non-binding steric moieties (NB) or binding partners (BP) for non-binding steric moieties, where the BP recruits or otherwise attracts the NB to the activatable antibody.
  • NB non-binding steric moieties
  • BP binding partners
  • the activatable antibodies provided herein include, for example, an activatable antibody that includes a non-binding steric moiety (NB), a cleavable linker (CL) and antibody or antibody fragment (AB) that binds a target; an activatable antibody that includes a binding partner for a non-binding steric moiety (BP), a CL and an AB; and an activatable antibody that includes a BP to which an NB has been recruited, a CL and an AB that binds the target.
  • NB non-binding steric moiety
  • CL cleavable linker
  • AB antibody or antibody fragment
  • NB-containing activatable antibodies Activatable antibodies in which the NB is covalently linked to the CL and AB of the activatable antibody or is associated by interaction with a BP that is covalently linked to the CL and AB of the activatable antibody are referred to herein as “NB-containing activatable antibodies.”
  • activatable or switchable is meant that the activatable antibody exhibits a first level of binding to a target when the activatable antibody is in an inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target when the activatable antibody is in an uninhibited, unmasked and/or cleaved state (i.e., a second conformation, i.e., activated antibody), where the second level of target binding is greater than the first level of target binding.
  • activatable antibody compositions can exhibit increased bioavailability and more favorable biodistribution compared to conventional antibody therapeutics.
  • activatable antibodies provide for reduced toxicity and/or adverse side effects that could otherwise result from binding of the at non-treatment sites and/or non-diagnostic sites if the AB were not masked or otherwise inhibited from binding to such a site.
  • CD47 activatable antibodies that include a non-binding steric moiety (NB) can be made using the methods set forth in PCT Publication No. WO 2013/192546, the contents of which are hereby incorporated by reference in their entirety.
  • the invention provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating a CD47-mediated disease in a subject by administering a therapeutically effective amount of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody described herein to a subject in need thereof.
  • a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody for use in preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating a CD47-mediated disease.
  • the invention also provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating cancer in a subject by administering a therapeutically effective amount of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody described herein to a subject in need thereof.
  • a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody for use in preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating cancer.
  • the invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with aberrant expression and/or activity of CD47 in a subject using antibodies/activatable antibodies that bind CD47, particularly activatable antibodies that bind and neutralize or otherwise inhibit at least one biological activity of CD47 and/or CD47-mediated signaling.
  • antibodies/activatable antibodies that bind CD47 for use in treating, preventing and/or delaying the onset or progression of, ameliorating, or alleviating a symptom associated with aberrant expression and/or activity of CD47.
  • the invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with the presence, growth, proliferation, metastasis, and/or activity of cells which are expressing CD47 or aberrantly expressing CD47 in a subject using antibodies/activatable antibodies that bind CD47, particularly activatable antibodies that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are expressing or aberrantly expressing CD47.
  • the invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with the presence, growth, proliferation, metastasis, and/or activity of cells which are expressing CD47 in a subject using antibodies/activatable antibodies that bind CD47, particularly antibodies/activatable antibodies that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are expressing CD47.
  • the invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with the presence, growth, proliferation, metastasis, and/or activity of cells which are aberrantly expressing CD47 in a subject using antibodies/activatable antibodies that bind CD47, particularly antibodies/activatable antibodies that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are aberrantly expressing CD47.
  • CD47 is known to be expressed in a variety of cancers, such as, by way of non- limiting example, adenocarcinoma, bile duct (biliary) cancer, bladder cancer, breast cancer, e.g., triple-negative breast cancer and Her2-negative breast cancer; carcinoid cancer; cervical cancer; cholangiocarcinoma; colorectal; endometrial; esophageal cancer; glioma; head and neck cancer, e.g., head and neck squamous cell cancer; leukemia; liver cancer; lung cancer, e.g., NSCLC, SCLC; lymphoma; melanoma; osopharyngeal cancer; ovarian cancer; pancreatic cancer; prostate cancer, e.g., metastatic castration-resistant prostate carcinoma; renal cancer; skin cancer; squamous cell cancer, stomach cancer; testis cancer; thyroid cancer; and urothelial cancer.
  • adenocarcinoma bile
  • the cancer is associated with a CD47-expressing tumor. In some embodiments, the cancer is due to a CD47-expressing tumor.
  • a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody used in any of the embodiments of these methods and uses can be administered at any stage of the disease.
  • such a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody can be administered to a patient suffering cancer of any stage, from early to metastatic.
  • the terms subject and patient are used interchangeably herein.
  • the subject is a mammal, such as a human, non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal.
  • the subject is a human.
  • the subject is a companion animal.
  • the subject is an animal in the care of a veterinarian.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder associated with aberrant CD47 expression and/or activity.
  • a subject suffering from or susceptible to a disease or disorder associated with aberrant CD47 expression and/or activity is identified using any of a variety of methods known in the art.
  • subjects suffering from cancer or other neoplastic condition are identified using any of a variety of clinical and/or laboratory tests such as, physical examination and blood, urine and/or stool analysis to evaluate health status.
  • subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests such as physical examination and/or bodily fluid analysis, e.g., blood, urine and/or stool analysis, to evaluate health status.
  • a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with aberrant CD47 expression and/or activity is considered successful if any of a variety of laboratory or clinical objectives is achieved.
  • administration of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with aberrant CD47 expression and/or activity is considered successful if one or more of the symptoms associated with the disease or disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state.
  • CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worse, state.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder, such as subjects suffering from cancer or other neoplastic condition, wherein the subject’s diseased cells are expressing CD47.
  • the diseased cells are associated with aberrant CD47 expression and/or activity. In some embodiments, the diseased cells are associated with normal CD47 expression and/or activity.
  • a subject suffering from or susceptible to a disease or disorder wherein the subject’s diseased cells express CD47 is identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic condition are identified using any of a variety of clinical and/or laboratory tests such as, physical examination and blood, urine and/or stool analysis to evaluate health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests such as physical examination and/or bodily fluid analysis, e.g., blood, urine and/or stool analysis, to evaluate health status.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder associated with cells expressing CD47 or the presence, growth, proliferation, metastasis, and/or activity of such cells, such as subjects suffering from cancer or other neoplastic conditions.
  • the cells are associated with aberrant CD47 expression and/or activity.
  • the cells are associated with normal CD47 expression and/or activity.
  • a subject suffering from or susceptible to a disease or disorder associated with cells that express CD47 is identified using any of a variety of methods known in the art.
  • subjects suffering from cancer or other neoplastic condition are identified using any of a variety of clinical and/or laboratory tests such as, physical examination and blood, urine and/or stool analysis to evaluate health status.
  • subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests such as physical examination and/or bodily fluid analysis, e.g., blood, urine and/or stool analysis, to evaluate health status.
  • Administration of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with cells expressing CD47 is considered successful if any of a variety of laboratory or clinical objectives is achieved.
  • a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with cells expressing CD47 is considered successful if one or more of the symptoms associated with the disease or disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state.
  • Administration of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with cells expressing CD47 is considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worse, state.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is administered during and/or after treatment in combination with one or more additional agents such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent.
  • additional agents such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) are administered simultaneously.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) are administered sequentially.
  • the disclosure also provides methods of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in which diseased cells express CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof.
  • the disorder or disease is cancer.
  • the disclosure also provides methods of treating, alleviating a symptom of, or delaying the progression of a disorder or disease associated with cells expressing CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof.
  • the disorder or disease associated with cells expressing CD47 is cancer.
  • the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2-negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non-small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell
  • the expression and/or activity of the mammalian CD47 is aberrant.
  • the method comprises administering an additional agent.
  • the additional agent is a therapeutic agent.
  • the disclosure also provides methods of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing mammalian CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof.
  • the expression and/or activity of the mammalian CD47 is aberrant.
  • the method comprises administering an additional agent.
  • the additional agent is a therapeutic agent.
  • the disclosure also provides methods of inhibiting, blocking, or preventing the binding of a natural ligand to mammalian CD47, comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof.
  • the expression and/or activity of the mammalian CD47 is aberrant.
  • the method comprises administering an additional agent.
  • the additional agent is a therapeutic agent.
  • the disclosure also provides methods of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in which diseased cells express CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof.
  • the disorder or disease is cancer.
  • the disclosure also provides methods of treating, alleviating a symptom of, or delaying the progression of a disorder or disease associated with cells expressing CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof.
  • the disorder or disease associated with cells expressing CD47 is cancer.
  • the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2-negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non-small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell
  • the expression and/or activity of the mammalian CD47 is aberrant.
  • the method comprises administering an additional agent.
  • the additional agent is a therapeutic agent.
  • the disclosure also provides methods of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing mammalian CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof.
  • the expression and/or activity of the mammalian CD47 is aberrant.
  • the method comprises administering an additional agent.
  • the additional agent is a therapeutic agent.
  • the disclosure also provides methods of inhibiting, blocking, or preventing the binding of a natural ligand to mammalian CD47, comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof.
  • the expression and/or activity of the mammalian CD47 is aberrant.
  • the method comprises administering an additional agent.
  • the additional agent is a therapeutic agent.
  • suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • a multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LipofectinTM), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration.
  • Therapeutic formulations of the disclosure which include a CD47 antibody and/or activatable CD47 antibody, such as by way of non-limiting example, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody, are used to prevent, treat or otherwise ameliorate a disease or disorder associated with aberrant target expression and/or activity.
  • therapeutic formulations of the disclosure which include an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody, are used to treat or otherwise ameliorate a cancer or other neoplastic condition, inflammation, an inflammatory disorder, and/or an autoimmune disease.
  • the cancer is a solid tumor or a hematologic malignancy where the target is expressed.
  • the cancer is a solid tumor where the target is expressed. In some embodiments, the cancer is a hematologic malignancy where the target is expressed. In some embodiments, the target is expressed on parenchyma (e.g., in cancer, the portion of an organ or tissue that often carries out function(s) of the organ or tissue). In some embodiments, the target is expressed on a cell, tissue, or organ. In some embodiments, the target is expressed on stroma (i.e., the connective supportive framework of a cell, tissue, or organ). In some embodiments, the target is expressed on an osteoblast. In some embodiments, the target is expressed on the endothelium (vasculature). In some embodiments, the target is expressed on a cancer stem cell.
  • parenchyma e.g., in cancer, the portion of an organ or tissue that often carries out function(s) of the organ or tissue.
  • the target is expressed on a cell, tissue, or organ.
  • the target is expressed on stroma (i.
  • the agent to which the antibody and/or the activatable antibody is conjugated is a microtubule inhibitor. In some embodiments, the agent to which the antibody and/or the activatable antibody is conjugated is a nucleic acid damaging agent. [000394] Efficaciousness of prevention, amelioration or treatment is determined in association with any known method for diagnosing or treating the disease or disorder associated with target expression and/or activity, such as, for example, aberrant target expression and/or activity.
  • an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody can be administered in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub.
  • the smallest fragment that specifically binds to the binding domain of the target protein is selected.
  • peptide molecules can be designed that retain the ability to bind the target protein sequence.
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology.
  • the formulation can also contain more than one active compounds as necessary for the particular indication being treated, for example, in some embodiments, those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth- inhibitory agent.
  • cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth- inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients can also 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
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained-release preparations can be prepared.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, 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 (U.S. Pat.
  • copolymers of L-glutamic acid and g ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • 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.
  • the CD47 antibodies, conjugated CD47 antibodies, activatable CD47 antibodies and/or conjugated activatable CD47 antibodies described herein are used in conjunction with one or more additional agents or a combination of additional agents.
  • additional agents include current pharmaceutical and/or surgical therapies for an intended application, such as, for example, cancer.
  • the CD47 antibodies, conjugated CD47 antibodies, activatable CD47 antibodies and/or conjugated activatable CD47 antibodies can be used in conjunction with an additional chemotherapeutic agent, anti-neoplastic agent, anti-inflammatory agent, an immunosuppressive agent, an alkylating agent, an anti- metabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and/or any other nucleic acid damaging agent.
  • the additional agent(s) is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.
  • the additional agent(s) is a checkpoint inhibitor, a kinase inhibitor, an agent targeting inhibitors in the tumor microenvironment, and/or a T cell or NK agonist.
  • the additional agent(s) is radiation therapy, alone or in combination with another additional agent(s) such as a chemotherapeutic or anti-neoplastic agent.
  • the additional agent(s) is a vaccine, an oncovirus, and/or a DC- activating agent such as, by way of non-limiting example, a toll-like receptor (TLR) agonist and/or a-CD40.
  • the additional agent(s) is a tumor-targeted antibody designed to kill the tumor via ADCC or via direct conjugation to a toxin (e.g., an antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of CTLA-4, LAG-3, PD-1, CD47, TIGIT, TIM-3, B7H4, and Vista.
  • the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib.
  • the kinase inhibitor is crizotinib.
  • the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an a-CSF1R inhibitor, an a-CCR4 inhibitor, a TGF-beta, a myeloid-derived suppressor cell, or a T-regulatory cell.
  • the agonist is selected from the group consisting of Ox40, GITR, CD137, ICOS, CD27, and HVEM.
  • the inhibitor is a CTLA-4 inhibitor.
  • the inhibitor is a LAG-3 inhibitor.
  • the inhibitor is a PD-1 inhibitor.
  • the inhibitor is a CD47 inhibitor.
  • the inhibitor is a TIGIT inhibitor.
  • the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an a-CSF1R inhibitor. In some embodiments, the inhibitor is an a-CCR4 inhibitor. In some embodiments, the inhibitor is a TGF-beta.
  • the inhibitor is a myeloid-derived suppressor cell. In some embodiments, the inhibitor is a T-regulatory cell. [000405] In some embodiments, the agonist is Ox40. In some embodiments, the agonist is GITR. In some embodiments, the agonist is CD137. In some embodiments, the agonist is ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM.
  • the CD47 antibody, conjugated antibody, activatable antibody and/or conjugated activatable antibody is administered during and/or after treatment in combination with one or more additional agents such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent.
  • additional agents such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are formulated into a single therapeutic composition, and the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and additional agent are administered simultaneously.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are administered simultaneously, or the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are administered at different times during a treatment regimen.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is administered prior to the administration of the additional agent
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is administered subsequent to the administration of the additional agent
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are administered in an alternating fashion.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and additional agent are administered in single doses or in multiple doses.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) are administered simultaneously.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) are administered sequentially, or the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are administered at different times during a treatment regimen.
  • the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is administered during and/or after treatment in combination with one or more additional agents such as, by way of non- limiting example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent, such as an alkylating agent, an anti-metabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and/or any other nucleic acid damaging agent.
  • the additional agent is a taxane, such as paclitaxel (e.g., Abraxane®).
  • the additional agent is an anti-metabolite, such as gemcitabine.
  • the additional agent is an alkylating agent, such as platinum- based chemotherapy, such as carboplatin or cisplatin.
  • the additional agent is a targeted agent, such as a kinase inhibitor, e.g., sorafenib or erlotinib.
  • the additional agent is a targeted agent, such as another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody.
  • the additional agent is a proteosome inhibitor, such as bortezomib or carfilzomib.
  • the additional agent is an immune modulating agent, such as lenolidominde or IL- 2.
  • the additional agent is radiation.
  • the additional agent is an agent considered standard of care by those skilled in the art.
  • the additional agent is a chemotherapeutic agent well known to those skilled in the art. [000409]
  • the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activatable antibody or antigen-binding fragment thereof and/or another conjugated activatable antibody or antigen-binding fragment thereof.
  • the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen- binding fragment thereof, another activatable antibody or antigen-binding fragment thereof and/or another conjugated activatable antibody or antigen-binding fragment thereof against the same target as the first antibody or antigen-binding fragment thereof, the first conjugated antibody or antigen-binding fragment thereof, activatable antibody or antigen-binding fragment thereof and/or a conjugated activatable antibody or antigen-binding fragment thereof, e.g., against CD47.
  • the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activatable antibody or antigen-binding fragment thereof and/or another conjugated activatable antibody or antigen-binding fragment thereof against a target different than the target of the first antibody or antigen-binding fragment thereof, the first conjugated antibody or antigen-binding fragment thereof, activatable antibody or antigen-binding fragment thereof and/or a conjugated activatable antibody or antigen-binding fragment thereof.
  • the antibody, antigen-binding fragment and/or the AB of an activatable antibody (of the additional agent) is a binding partner for any target listed in Table H. Table H: Exemplary Targets
  • the antibody, antigen-binding fragment and/or the AB of an activatable antibody is or is derived from an antibody listed in Table J.
  • Table J Exemplary sources for Abs
  • the additional antibody or antigen binding fragment thereof, conjugated antibody or antigen binding fragment thereof, activatable antibody or antigen binding fragment thereof, and/or conjugated activatable antibody or antigen binding fragment thereof is a monoclonal antibody, domain antibody, single chain, 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.
  • the additional antibody or antigen binding fragment thereof, conjugated antibody or antigen binding fragment thereof, activatable antibody or antigen binding fragment thereof, and/or conjugated activatable antibody or antigen binding fragment thereof is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody. Detection, Diagnostics, Imaging, Patient Selection [000413]
  • the invention also provides methods and kits for using the antibodies/conjugated antibodies/activatable antibodies/conjugated activatable antibodies provided herein in a variety of diagnostic and/or prophylactic indications.
  • the invention provides methods and kits for detecting the presence or absence of a cleaving agent and a target of interest in a subject or a sample by (i) contacting a subject or sample with a CD47 activatable antibody, wherein the CD47 activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the CD47 activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to CD47, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein,
  • the CD47 activatable antibody can bind both human and cynomolgus CD47.
  • the activatable CD47 antibody is an activatable CD47 antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent.
  • the activatable CD47 antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.
  • the activatable CD47 antibody includes a detectable label.
  • the detectable label includes an imaging agent, a contrasting agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label.
  • the imaging agent comprises a radioisotope.
  • the radioisotope is indium or technetium.
  • the contrasting agent comprises iodine, gadolinium or iron oxide.
  • the enzyme comprises horseradish peroxidase, alkaline phosphatase, or b- galactosidase.
  • the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative.
  • the luminescent label comprises an N-methylacrydium derivative.
  • the label comprises an Alexa Fluor ® label, such as Alex Fluor ® 680 or Alexa Fluor ® 750.
  • the ligand-based label comprises biotin, avidin, streptavidin or one or more haptens.
  • the subject is a mammal. In some embodiments of these methods, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal. In some embodiments, the subject is a rodent.
  • the method is an in vivo method. In some embodiments of these methods, the method is an in situ method.
  • the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method. [000418] In some embodiments of the methods and kits, the method is used to identify or otherwise refine a patient population suitable for treatment with a CD47 activatable antibody of the disclosure, followed by treatment by administering that activatable CD47 antibody and/or conjugated activatable CD47 antibody to a subject in need thereof.
  • patients that test positive for both the target (e.g., CD47) and a protease that cleaves the substrate in the cleavable moiety (CM) of CD47 activatable antibody being tested in these methods are identified as suitable candidates for treatment with such a CD47 activatable antibody comprising such a CM, and the patient is then administered a therapeutically effective amount of the activatable CD47 antibody and/or conjugated activatable CD47 antibody that was tested.
  • patients that test negative for either or both of the target (e.g., CD47) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy.
  • such patients can be tested with other CD47 activatable antibodies until a suitable CD47 activatable antibody for treatment is identified (e.g., a CD47 activatable antibody comprising a CM that is cleaved by the patient at the site of disease).
  • a suitable CD47 activatable antibody for treatment e.g., a CD47 activatable antibody comprising a CM that is cleaved by the patient at the site of disease.
  • the patient is then administered a therapeutically effective amount of the activatable CD47 antibody and/or conjugated for which the patient tested positive.
  • Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.
  • the antibody, the conjugated antibody, activatable antibody and/or conjugated activatable antibody contains a detectable label.
  • an intact antibody, or a fragment thereof is used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the disclosure can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, immunochemical staining, and immunofluorescence.
  • In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol.42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P.
  • analyte protein in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti- analyte protein antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies of the disclosure are also useful in a variety of diagnostic and prophylactic formulations.
  • an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to patients that are at risk of developing one or more of the aforementioned disorders.
  • a patient’s or organ’s predisposition to one or more of the aforementioned disorders can be determined using genotypic, serological or biochemical markers.
  • an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders.
  • an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to mitigate or reverse the effects of the clinical indication.
  • an antibody, a conjugated antibody, an activatable antibody, and/or a conjugated activatable antibody of the disclosure is also useful in the detection of a target in patient samples and accordingly are useful as diagnostics.
  • the antibodies and/or activatable antibodies, and conjugated versions thereof, of the disclosure are used in in vitro assays, e.g., ELISA, to detect target levels in a patient sample.
  • an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody of the disclosure is immobilized on a solid support (e.g., the well(s) of a microtiter plate).
  • the immobilized antibody, conjugated antibody, activatable antibody and/or conjugated activatable antibody serves as a capture antibody for any target that may be present in a test sample.
  • the solid support Prior to contacting the immobilized antibody and/or activatable antibody, and/or conjugated versions thereof, with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.
  • a test sample suspected of containing the antigen or with a solution containing a standard amount of the antigen.
  • a sample is, e.g., a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology.
  • the solid support After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled.
  • the labeled second antibody serves as a detecting antibody.
  • the level of detectable label is measured, and the concentration of target antigen in the test sample is determined by comparison with a standard curve developed from the standard samples.
  • An antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody can also be used in diagnostic and/or imaging methods.
  • such methods are in vitro methods.
  • such methods are in vivo methods.
  • such methods are in situ methods.
  • such methods are ex vivo methods.
  • activatable antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM.
  • Such activatable antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of an activatable antibody) in a given cell or tissue of a given host organism.
  • activated antibodies i.e., antibodies resulting from cleavage of an activatable antibody
  • Such accumulation of activated antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses target to which the activated antibody binds.
  • the CM can be selected to be substrate for at least one protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like.
  • the AB can be one that binds a target antigen.
  • a detectable label e.g., a fluorescent label or radioactive label or radiotracer
  • Suitable detectable labels are discussed in the context of the above screening methods and additional specific examples are provided below.
  • activatable antibodies will exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated antibodies in the disease tissue is enhanced relative to non-disease tissues.
  • activatable antibodies can be used to detect the presence or absence of a cleaving agent in a sample.
  • the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample.
  • the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample.
  • the activatable antibodies can be detectably labeled, and can be bound to a support (e.g., a solid support, such as a slide or bead).
  • the detectable label can be positioned on a portion of the activatable antibody that is not released following cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage has occurred.
  • the assay can be conducted by, for example, contacting the immobilized, detectably labeled activatable antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants.
  • the presence or absence of the cleaving agent (e.g., enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the activatable antibodies prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the activatable antibody by the cleaving agent in the sample.
  • the detection methods can be adapted to also provide for detection of the presence or absence of a target that is capable of binding the AB of the activatable antibodies when cleaved.
  • the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest.
  • Activatable antibodies are also useful in in situ imaging for the validation of activatable antibody activation, e.g., by protease cleavage, and binding to a particular target. In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections.
  • a detectable label e.g., a fluorescent label.
  • a detectable label e.g., a fluorescent label.
  • an activatable antibody is labeled with a detectable label.
  • the detectable label can be a fluorescent dye, (e.g.
  • a fluorophore Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), an Alexa Fluor® label), a near infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin and an amplification reagent such as streptavidin, or an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).
  • Detection of the label in a sample that has been incubated with the labeled, activatable antibody indicates that the sample contains the target and contains a protease that is specific for the CM of the activatable antibody.
  • the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and/or by using an agent that is specific for the protease, for example, an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase; see e.g., International Publication Number WO 2010/129609, published 11 November 2010.
  • the same approach of using broad spectrum protease inhibitors such as those described herein, and/or by using a more selective inhibitory agent can be used to identify a protease that is specific for the CM of the activatable antibody.
  • the presence of the target can be confirmed using an agent that is specific for the target, e.g., another antibody, or the detectable label can be competed with unlabeled target.
  • unlabeled activatable antibody could be used, with detection by a labeled secondary antibody or more complex detection system.
  • Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the activatable antibody.
  • These techniques are also useful in kits and/or as reagents for the detection, identification or characterization of protease activity in a variety of cells, tissues, and organisms based on the protease-specific CM in the activatable antibody.
  • the disclosure provides methods of using the antibodies and/or activatable antibodies in a variety of diagnostic and/or prophylactic indications.
  • the disclosure provides methods of detecting presence or absence of a cleaving agent and a target of interest in a subject or a sample by (i) contacting a subject or sample with an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C- terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b)
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.
  • the disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or sample with an activatable antibody in the presence of a target of interest, e.g., the target, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form
  • MM
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.
  • kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample where the kits include at least an activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C- terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, in an uncleaved
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.
  • the disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or sample with an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, an antigen binding domain (AB) that specifically binds the target, and a detectable label, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C- terminus as follows: MM-CM-AB or AB-CM-MM; wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; wherein, in an uncleaved
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.
  • kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample where the kits include at least an activatable antibody and/or conjugated activatable antibody (e.g., an activatable antibody to which a therapeutic agent is conjugated) described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample.
  • the disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody in the presence of the target, and (ii) measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample.
  • Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent.
  • the detectable label is attached to the masking moiety. In some embodiments, the detectable label is attached to the cleavable moiety N-terminal to the protease cleavage site. In some embodiments, a single antigen binding site of the AB is masked. In some embodiments wherein an antibody of the disclosure has at least two antigen binding sites, at least one antigen binding site is masked and at least one antigen binding site is not masked. In some embodiments all antigen binding sites are masked.
  • the measuring step includes use of a secondary reagent comprising a detectable label.
  • kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample with an activatable antibody in the presence of the target, and measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample.
  • Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM- MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent.
  • the detectable label is attached to the masking moiety. In some embodiments, the detectable label is attached to the cleavable moiety N-terminal to the protease cleavage site. In some embodiments, a single antigen binding site of the AB is masked. In some embodiments wherein an antibody of the disclosure has at least two antigen binding sites, at least one antigen binding site is masked and at least one antigen binding site is not masked. In some embodiments all antigen binding sites are masked.
  • the measuring step includes use of a secondary reagent comprising a detectable label.
  • kits for use in methods of detecting presence or absence of a cleaving agent in a subject or a sample where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein no detectable level of activated activatable antibody in
  • the disclosure provides methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM and (ii) measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample.
  • a reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.
  • Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target.
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.
  • kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample.
  • a reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.
  • the disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM; and (ii) measuring a level of detectable label in the subject or biological sample, wherein a detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample.
  • a reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.
  • Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target.
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.
  • kits for use in methods of detecting presence or absence of a cleaving agent of interest in a subject or a sample where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM, wherein a detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent, the target, or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of the detectable label in the subject or biological sample indicates that the
  • a reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.
  • the activatable antibody includes a detectable label.
  • the detectable label includes an imaging agent, a contrasting agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label.
  • the imaging agent comprises a radioisotope.
  • the radioisotope is indium or technetium.
  • the contrasting agent comprises iodine, gadolinium or iron oxide.
  • the enzyme comprises horseradish peroxidase, alkaline phosphatase, or b- galactosidase.
  • the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative.
  • the luminescent label comprises an N-methylacrydium derivative.
  • the label comprises an Alexa Fluor ® label, such as Alex Fluor ® 680 or Alexa Fluor ® 750.
  • the ligand-based label comprises biotin, avidin, streptavidin or one or more haptens.
  • the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method.
  • in situ imaging and/or in vivo imaging are useful in methods to identify which patients to treat. For example, in in situ imaging, the activatable antibodies are used to screen patient samples to identify those patients having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site.
  • in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure.
  • patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM.
  • patients that test negative for either or both of the target (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy.
  • such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease).
  • a suitable activatable antibody for treatment e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease.
  • the patient is then administered a therapeutically effective amount of the activatable antibody for which the patient tested positive.
  • in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure.
  • patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM.
  • patients that test negative might be identified as suitable candidates for another form of therapy.
  • such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease).
  • the patient is then administered a therapeutically effective amount of the activatable antibody for which the patient tested positive.
  • the method or kit is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested in these methods are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM.
  • the target e.g., the target
  • CM cleavable moiety
  • patients that test negative for both of the targets (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy.
  • such patients can be tested with other activatable antibodies until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease).
  • patients that test negative for either of the target (e.g., the target) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM.
  • patients that test negative for either of the target are identified as not being suitable candidates for treatment with such an activatable antibody comprising such a CM.
  • such patients can be tested with other activatable antibodies until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease).
  • the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated.
  • the activatable antibody is not conjugated to an agent.
  • the activatable antibody comprises a detectable label.
  • the detectable label is positioned on the AB.
  • measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label.
  • the secondary reagent is an antibody comprising a detectable label.
  • a method or kit is used to identify or otherwise refine a patient population suitable for treatment with an anti-the target activatable antibody and/or conjugated activatable antibody (e.g., activatable antibody to which a therapeutic agent is conjugated) of the disclosure, followed by treatment by administering that activatable antibody and/or conjugated activatable antibody to a subject in need thereof.
  • patients that test positive for both the targets (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody and/or conjugated activatable antibody being tested in these methods are identified as suitable candidates for treatment with such antibody and/or such a conjugated activatable antibody comprising such a CM, and the patient is then administered a therapeutically effective amount of the activatable antibody and/or conjugated activatable antibody that was tested.
  • patients that test negative for either or both of the target (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy.
  • such patients can be tested with other antibody and/or conjugated activatable antibody until a suitable antibody and/or conjugated activatable antibody for treatment is identified (e.g., an activatable antibody and/or conjugated activatable antibody comprising a CM that is cleaved by the patient at the site of disease).
  • a suitable antibody and/or conjugated activatable antibody for treatment e.g., an activatable antibody and/or conjugated activatable antibody comprising a CM that is cleaved by the patient at the site of disease.
  • the patient is then administered a therapeutically effective amount of the activatable antibody and/or conjugated activatable antibody for which the patient tested positive.
  • the MM is a peptide having a length from about 4 to 40 amino acids.
  • the activatable antibody comprises a linker peptide, wherein the linker peptide is positioned between the MM and the CM. In some embodiments of these methods and kits, the activatable antibody comprises a linker peptide, where the linker peptide is positioned between the AB and the CM. In some embodiments of these methods and kits, the activatable antibody comprises a first linker peptide (L1) and a second linker peptide (L2), wherein the first linker peptide is positioned between the MM and the CM and the second linker peptide is positioned between the AB and the CM.
  • L1 linker peptide
  • L2 second linker peptide
  • each of L1 and L2 is a peptide of about 1 to 20 amino acids in length, and wherein each of L1 and L2 need not be the same linker.
  • one or both of L1 and L2 comprises a glycine-serine polymer.
  • at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GSGGS)n (SEQ ID NO: 116) and (GGGS)n (SEQ ID NO: 117), where n is an integer of at least one.
  • At least one of L1 and L2 comprises an amino acid sequence having the formula (GGS)n, where n is an integer of at least one.
  • at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO: 118), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 119), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 120), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 121), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 122), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 123).
  • the AB comprises an antibody or antibody fragment sequence selected from the cross-reactive antibody sequences presented herein.
  • the AB comprises a Fab fragment, a scFv or a single chain antibody (scAb).
  • the cleaving agent is a protease that is co-localized in the subject or sample with the target and the CM is a polypeptide that functions as a substrate for the protease, wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.
  • the CM is a polypeptide of up to 15 amino acids in length. In some embodiments of these methods and kits, the CM is coupled to the N-terminus of the AB. In some embodiments of these methods and kits, the CM is coupled to the C-terminus of the AB. In some embodiments of these methods and kits, the CM is coupled to the N-terminus of a VL chain of the AB. [000458]
  • the antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies of the disclosure are used in diagnostic and prophylactic formulations. In one embodiment, an activatable antibody is administered to patients that are at risk of developing one or more of the aforementioned inflammation, inflammatory disorders, cancer or other disorders.
  • a patient’s or organ’s predisposition to one or more of the aforementioned disorders can be determined using genotypic, serological or biochemical markers.
  • an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to mitigate or reverse the effects of the clinical indication.
  • Antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies of the disclosure are also useful in the detection of the target in patient samples and accordingly are useful as diagnostics.
  • the antibodies, conjugated antibodies, the activatable antibodies and/or conjugated activatable antibodies of the disclosure are used in in vitro assays, e.g., ELISA, to detect target levels in a patient sample.
  • an antibody and/or activatable antibody of the disclosure is immobilized on a solid support (e.g., the well(s) of a microtiter plate).
  • the immobilized antibody and/or activatable antibody serves as a capture antibody for any target that may be present in a test sample.
  • the solid support Prior to contacting the immobilized antibody and/or activatable antibody with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.
  • a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.
  • the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen.
  • a sample is, e.g., a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology.
  • the solid support After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled.
  • the labeled second antibody serves as a detecting antibody.
  • the level of detectable label is measured, and the concentration of target antigen in the test sample is determined by comparison with a standard curve developed from the standard samples.
  • Antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies can also be used in diagnostic and/or imaging methods.
  • such methods are in vitro methods.
  • such methods are in vivo methods.
  • such methods are in situ methods.
  • such methods are ex vivo methods.
  • activatable antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM.
  • Such activatable antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of an activatable antibody) in a given cell or tissue of a given host organism.
  • activated antibodies i.e., antibodies resulting from cleavage of an activatable antibody
  • Such accumulation of activated antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses target to which the activated antibody binds.
  • the CM can be selected to be a protease substrate for a protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like.
  • the AB can be one that binds a target antigen.
  • a detectable label e.g., a fluorescent label or radioactive label or radiotracer
  • Suitable detectable labels are discussed in the context of the above screening methods and additional specific examples are provided below.
  • activatable antibodies will exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated antibodies in the disease tissue is enhanced relative to non-disease tissues.
  • activatable antibodies can be used to detect the presence or absence of a cleaving agent in a sample.
  • the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample.
  • the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample.
  • the activatable antibodies can be detectably labeled, and can be bound to a support (e.g., a solid support, such as a slide or bead).
  • the detectable label can be positioned on a portion of the activatable antibody that is not released following cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage has occurred.
  • the assay can be conducted by, for example, contacting the immobilized, detectably labeled activatable antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants.
  • the presence or absence of the cleaving agent (e.g., enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the activatable antibodies prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the activatable antibody by the cleaving agent in the sample.
  • the detection methods can be adapted to also provide for detection of the presence or absence of a target that is capable of binding the AB of the activatable antibodies when cleaved.
  • the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest.
  • Activatable antibodies are also useful in in situ imaging for the validation of activatable antibody activation, e.g., by protease cleavage, and binding to a particular target. In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections.
  • a detectable label e.g., a fluorescent label.
  • a detectable label e.g., a fluorescent label.
  • an activatable antibody is labeled with a detectable label.
  • the detectable label can be a fluorescent dye, (e.g.
  • Fluorescein Isothiocyanate FITC
  • Rhodamine Isothiocyanate TRITC
  • NIR near infrared
  • colloidal metal e.g., a hapten
  • a radioactive marker e.g., biotin and an amplification reagent such as streptavidin
  • an enzyme e.g. horseradish peroxidase or alkaline phosphatase.
  • the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and/or by using an agent that is specific for the protease, for example, an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase; see e.g., International Publication Number WO 2010/129609, published 11 November 2010.
  • an agent that is specific for the protease for example, an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase; see e.g., International Publication Number WO 2010/129609, published 11 November 2010.
  • A11 an antibody
  • the same approach of using broad spectrum protease inhibitors such as those described herein, and/or by using a more selective inhibitory agent can be used to identify a protease or class of proteases specific for the CM of the activatable antibody.
  • the presence of the target can be confirmed using an agent that is specific for the target, e.g., another antibody, or the detectable label can be competed with unlabeled target.
  • unlabeled activatable antibody could be used, with detection by a labeled secondary antibody or more complex detection system.
  • Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the activatable antibody.
  • in situ imaging and/or in vivo imaging are useful in methods to identify which patients to treat.
  • the activatable antibodies are used to screen patient samples to identify those patients having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site.
  • in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure.
  • patients that test positive for both the target and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM.
  • patients that test negative for either or both of the target and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the activatable antibody being tested).
  • such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease).
  • a suitable activatable antibody for treatment e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease.
  • in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure.
  • CM cleavable moiety
  • patients that test positive for both the target and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM.
  • patients that test negative are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the activatable antibody being tested).
  • such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease).
  • a suitable activatable antibody for treatment e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease.
  • Pharmaceutical Compositions [000478]
  • the antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies of the disclosure also referred to herein as “active compounds”
  • derivatives, fragments, analogs and homologs thereof can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the antibody, the conjugated antibody, activatable antibody and/or conjugated activatable antibody and a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 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.
  • compositions according to the invention can include an antibody/activatable antibody of the invention and a carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.
  • the pharmaceutical composition comprises an antibody of the disclosure, an activatable antibody of the disclosure, a conjugated antibody or the disclosure, and/or a conjugated activatable antibody of the disclosure, and a carrier.
  • the pharmaceutical composition comprises an additional agent. In some embodiments, the additional agent is a therapeutic agent.
  • a pharmaceutical composition of the disclosure is 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 (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include 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 can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • 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 ⁇ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists.
  • the carrier can 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 such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can 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 filtered sterilization.
  • dispersions are 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.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • compositions for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. [000489] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. [000491] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES EXAMPLE 1.
  • the studies provided herein were designed to evaluate binding of anti-human CD47 antibodies of the disclosure.
  • An anti-human CD47 monoclonal antibody of the present disclosure was obtained using mouse hybridoma technology in accordance with techniques known in the art. Mice were immunized with human CD47 and subsequent hybridomas were screened for binding to human CD47 by ELISA and subsequently confirmed to be cytotoxic in a piggyback assay and confirmed to bind cell surfaces by FACS.
  • the mouse anti-human CD47 H4L2 monoclonal antibody of the present disclosure includes a heavy chain variable region (VH) of SEQ ID NO: 1, and a light chain variable region (VL) of SEQ ID NO: 2.
  • an exemplary antibody of the present disclosure includes a heavy chain having the H4L2 heavy chain variable region of SEQ ID NO: 1 and a light chain having the H4L2 light chain variable region of SEQ ID NO: 2.
  • the mouse anti-human CD47 monoclonal antibody H4L2 was humanized to form humanized anti-human CD47 heavy and light chains.
  • exemplary embodiments of these humanized antibody heavy chains of the present disclosure include H4L2- hIgG1 heavy chain with an human IgG1 Fc domain (SEQ ID NO: 11), H4L2-hIgG1-enhanced heavy chain with a modified human IgG1 Fc domain (SEQ ID NO: 12), H4L2-hIgG4 heavy chain with a human IgG4 Fc domain (SEQ ID NO: 13), and H4L2-hIgG2s heavy chain with a modified human IgG2 Fc domain (SEQ ID NO: 14).
  • Exemplary embodiments of these humanized antibody light chains of the present disclosure include H4L2-h-kappa light chain with a human light chain kappa domain (SEQ ID NO: 15).
  • the amino acids sequences of these exemplary heavy and light chains of the present disclosure are shown below in Table 1.
  • an exemplary antibody of the present disclosure includes the heavy chain H4L2-hIgG1 of SEQ ID NO: 11 with the light chain H4L2-h-kappa of SEQ ID NO: 15.
  • an exemplary antibody of the present disclosure includes the heavy chain H4L2-hIgG1-enhanced of SEQ ID NO: 12 with the light chain H4L2-h-kappa of SEQ ID NO: 15. In some embodiments of the present disclosure, an exemplary antibody of the present disclosure includes the heavy chain H4L2-hIgG4 of SEQ ID NO: 13 with the light chain H4L2-h-kappa of SEQ ID NO: 15. In some embodiments of the present disclosure, an exemplary antibody of the present disclosure includes the heavy chain H4L2-hIgG2s of SEQ ID NO: 14 with the light chain H4L2-h-kappa of SEQ ID NO: 15. Table 1.
  • Anti-Human CD47 Mouse and Humanized Antibody Sequences 1. Anti-huCD47 H4L2-hIgG1 H4 Heavy Chain (amino acid sequence) QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDKSTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRE
  • Anti-huCD47 H4L2-hkappa L2 Light Chain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC (SEQ ID NO: 15) 6.
  • Anti-huCD47 H4L2 L2 VL domain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 2) 8.
  • Anti-huCD47 parental hybridoma M1-vh1 VH domain (amino acid sequence) QVQLQQPGAELVRPGASVKLSCRASGYTFTNYWMTWVKQRPEQGLEWIGRIDPYDVETHYNHKFQDKAIL TVDKSSSTAYMQLSSLTSEDSAVYYCARGGVGGMDYWGQGTSVTVSS (SEQ ID NO: 57) 9.
  • Anti-huCD47 H1 VH domain (amino acid sequence) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMTWVRQAPGQGLEWMGRIDPYDVETHYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 58) 10.
  • Anti-huCD47 H2 VH domain (amino acid sequence) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRVTI TVDESTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 59) 11.
  • Anti-huCD47 H3 VH domain (amino acid sequence) QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDESTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 60) 12.
  • Anti-huCD47 parental hybridoma M1-vk1 VL domain (amino acid sequence) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGHSPNLLIYRVSKRFSGVPDRFSGS GSGTDFTLKITRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK (SEQ ID NO: 75) 13.
  • Anti-huCD47 L1 VL domain (amino acid sequence) DIVMTQSPDSLAVSLGERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSLQAEDVAVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 76) 14.
  • Anti-huCD47 L3 VH domain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSSGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 77) 15.
  • Anti-huCD47 L4 VH domain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSSGQTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 78) 16.
  • Anti-huCD47 L5 VH domain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSSGSTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 79)
  • Exemplary anti-CD47 antibodies of the present disclosure were assayed for their affinity to human and cynomolgus CD47 antigens using standard ELISA methods. These binding affinities were compared to anti-CD47 antibodies B6H12-IgG1 and 5F9-IgG4.
  • Table 3 Blocking Assay of human CD47-SIRPa interaction EXAMPLE 2.
  • Mask Discovery [000498] The studies provided herein were designed to identify and characterize masking moieties for use in activatable anti-human CD47 antibodies of the disclosure.
  • Humanized anti-human CD47 hu H4L2 monoclonal antibody of the present disclosure (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2), was used to screen a random X 15 peptide library with a total diversity of 4 x 16 0 , where X is any amino acid, using a method similar to that described in PCT International Publication Number WO 2010/081173, published 15 July 2010.
  • the screening included magnetic-activated cell sorting (MACS) and fluorescence- activated cell sorting (FACS).
  • Amino acid sequences of exemplary individual clones of masking peptides (MM) that were isolated and sequenced are shown in Table 4.
  • these anti-human CD47 activatable antibodies include cleavable moiety 2001 (ISSGLLSGRSDNH; SEQ ID NO: 406), cleavable moiety 3001 (AVGLLAPPGGLSGRSDNH; SEQ ID NO: 412), cleavable moiety 2007 (ISSGLLSGRSDIH; SEQ ID NO: 684), cleavable moiety 2008 (ISSGLLSGRSDQH; SEQ ID NO: 685), cleavable moiety 2011 (ISSGLLSGRSDNP; SEQ ID NO: 688), cleavable moiety 2012 (ISSGLLSGRSANP; SEQ ID NO: 689), cleavable moiety 2013 (ISSGLLSGRSANI; SEQ ID NO: 690), cleavable moiety 3007 (AVGLLAPPGGLSGRSDIH; SEQ ID NO: 692), cleavable moiety 3008 (AVGLLAPP
  • the activatable anti-human CD47 antibodies of the disclosure can include any suitable spacer sequence, such as, for example, a spacer sequence selected from the group consisting of QGQSGQG (SEQ ID NO: 30), QGQSGQ (SEQ ID NO: 31), QGQSG (SEQ ID NO: 32), QGQS (SEQ ID NO: 33), QGQ, QG, GQSGQG (SEQ ID NO: 34), QSGQG (SEQ ID NO: 35), SGQG (SEQ ID NO: 36), GQG, G, or Q.
  • a spacer sequence selected from the group consisting of QGQSGQG (SEQ ID NO: 30), QGQSGQ (SEQ ID NO: 31), QGQSG (SEQ ID NO: 32), QGQS (SEQ ID NO: 33), QGQ, QG, GQSGQG (SEQ ID NO: 34), QSGQG (SEQ ID NO: 35), SGQG (SEQ ID NO: 36), GQ
  • the activatable anti-human CD47 antibodies of the disclosure can have no spacer sequence joined to its N-terminus.
  • Anti-Human CD47 Activatable Antibody Sequences 8 [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.1-5007 VL domain (SEQ ID NO: 41)] [QGQSGQG][LYDCYALSKSYCMGLGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 61) 9.
  • the activatable antibodies of the present disclosure that were assayed in this exemplary study were (a) hu H4L2-IgG4 HC / H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 41), hu H4L2-IgG4 HC/ H4L2-47.2-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 42), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 43), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 44), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 45), hu H4L2-Ig
  • Humanized anti-human CD47 antibodies of the present disclosure hu H4L2-IgG4 HC/ H4L2 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2) were used as a control.
  • Masking efficiencies i.e., the ratio of the binding affinity of the activatable antibody to the binding affinity of the parental antibody
  • Table 6 Masking efficiencies (i.e., the ratio of the binding affinity of the activatable antibody to the binding affinity of the parental antibody) of these masking peptides of the present disclosure were determined and shown in Table 6.
  • Table 6 Masking Efficiencies of Anti-CD47 Activatable Antibodies [000503] As shown in Figure 1B, certain of the exemplary anti-CD47 activatable antibodies were treated with the protease matriptase (overnight at 37o C) and then assayed with ELISA to determine their binding affinity to human CD47 antigen. As shown in these exemplary results, cleavage of the activatable antibodies with a protease that can specifically cleave the cleavable moiety restored the binding affinity of the activatable antibodies such that they were essentially the same as the parental anti-CD47 antibody.
  • exemplary anti-CD47 activatable antibodies of present disclosure when activated by matriptase, can specifically bind to their CD47 target with an affinity comparable to that of the parental antibody.
  • EXAMPLE 3 Antibody-Dependent Cellular Phagocytosis (ADCP) Assays of Masking Peptides [000504] The studies provided herein were designed to assay the activatable anti-human CD47 antibodies of the present disclosure to induce antibody-dependent cellular phagocytosis (ADCP) via effecting the FcgRIIa receptor pathway.
  • ADCP antibody-dependent cellular phagocytosis
  • the assayed test articles were hu anti-CD47 H4L2-hIgG1 (“H4L2-IgG1”; HC of SEQ ID NO: 11, LC of SEQ ID NO: 15), hu anti-CD47 H4L2-hIgG1 enhanced (“H4L2-IgG1-en”; HC of SEQ ID NO: 12, LC of SEQ ID NO: 15), hu anti-CD47 H4L2-hIgG4 (“H4L2-IgG4”; HC of SEQ ID NO: 13, LC of SEQ ID NO: 15), hu anti- CD47 H4L2-hIgG1-47.3-5007 (“H4L2-IgG1”; HC of SEQ ID NO: 11, VL of SEQ ID NO: 83), hu anti-CD47 H4L2-hIgG1-47.10-5007 (“H4L2-IgG1”; HC of SEQ ID NO: 11, VL of SEQ ID NO: 90), and hu anti
  • EXAMPLE 5 Antibody-Dependent Cellular Phagocytosis (ADCP) Assays of Fc Effector Domains
  • ADCP antibody-dependent cellular phagocytosis
  • the studies provided herein were designed to assay the activatable anti-human CD47 antibodies of the present disclosure to measure their ability to induce antibody-dependent cellular phagocytosis (ADCP). These exemplary results also show that antibodies and activatable antibodies of the present disclosure with IgG1 Fc effector domains showed higher levels of ADCP that those with IgG2s Fc effector domains.
  • PBMCs peripheral blood mononuclear cells
  • the PBMCs were sorted and purified using anti- CD14 micromagnetic beads and validated with anti-CD14 APC antibody and FACS analysis.
  • the purified cells were cultured for 14 days and differentiated macrophages were validated using CD11b PE antibody followed with FACS analysis.
  • Approximately 25,000 macrophage cells per well were plated into a 96 well plate.
  • Each test article was prepared in PBS at the indicated concentration an added to the plate.
  • Approximately 1x10 6 target cells were washed and labeled with the fluorescent dye and added to the 96 well plate containing the macrophages.
  • the plates were scanned at 10x imaging for every 30 min using Incucyte S3 and the intensity was measured as shown.
  • the assayed test articles were hu anti-CD47 H4L2- hIgG1 (“H4L2-IgG1”; HC of SEQ ID NO: 11, LC of SEQ ID NO: 15), hu anti-CD47 H4L2- hIgG1 enhanced (“H4L2-IgG1-en”; HC of SEQ ID NO: 12, LC of SEQ ID NO: 15), and hu anti- CD47 H4L2-hIgG2s (“H4L2-IgG2s”; HC of SEQ ID NO: 14, LC of SEQ ID NO: 15).
  • the target cells tested in these assays were Daudi cells, derived from human B lymphoblast cells.
  • FIGS.5A and 5B shows the efficacy of anti-human CD47 antibodies of the present disclosure hu anti-CD47 H4L2-hIgG1 (“H4L2-IgG1”; HC of SEQ ID NO: 11, LC of SEQ ID NO: 15), hu anti-CD47 H4L2-hIgG1 enhanced (“H4L2-IgG1 en”; HC of SEQ ID NO: 12, LC of SEQ ID NO: 15), hu anti-CD47 H4L2-hIgG4 (“H4L2-IgG4”; HC of SEQ ID NO: 13, LC of SEQ ID NO: 15), and hu anti-CD47 H4L2-hIgG2s (“H4L2-IgG2s”; HC of SEQ ID NO: 14, LC of SEQ ID NO: 15).
  • the indicated anti-CD47 antibodies of the present disclosure were tested for in vivo efficacy in a NOD/SCID mice with CD20-expressing Raji cell xenografts (derived from human B-cell lymphoma). These antibodies of the present disclosure were tested against a PBS buffer vehicle control and the anti-CD20 antibody rituximab.
  • the test articles were dosed at 10 mg/kg every other day for 4 weeks and the tumor size was measured at the indicated days.
  • the test articles were also compared to rituximab, either alone or in combination with the H4L2-IgG4 antibody of the present disclosure.

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Abstract

The invention relates generally to activatable antibodies that bind to CD47 and methods of making and using these activatable antibodies in a variety of therapeutic, prophylactic, and diagnostic contexts. In some embodiments, the CD47 activatable antibodies bind human and cynomolgus monkey CD47.

Description

ANTI-CD47 ANTIBODIES, ACTIVATABLE ANTI-CD47 ANTIBODIES, AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The invention claims the benefit of U.S. Provisional Application No.62/904,534, filed on September 23, 2019, the contents of which are incorporated herein by reference in their entirety. FIELD OF THE INVENTION [0002] The invention relates generally to antibodies that specifically bind to CD47, activatable antibodies that specifically bind to CD47, and methods of making and using these antibodies and activatable antibodies in a variety of therapeutic, prophylactic, and diagnostic contexts. REFERENCE TO SEQUENCE LISTING [0003] The “Sequence Listing” submitted electronically concurrently herewith pursuant to 37 C.F.R. § 1.821 in computer readable form (CFR) via EFS-Web as file name “CYTX-066- PCT_ST25” is incorporated herein by reference. The electronic copy of the Sequence Listing was created on September 23, 2020, and the disk size is 197 kilobytes. BACKGROUND OF THE INVENTION [0004] Antibody-based therapies have proven effective treatments for several diseases 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. [0005] In the realm of small molecule therapeutics, strategies have been developed to provide prodrugs of an active chemical entity. Such prodrugs are administered in a relatively inactive (or significantly less active) form. Once administered, the prodrug is metabolized in vivo into the active compound. Such prodrug strategies can provide for increased selectivity of the drug for its intended target and for a reduction of adverse effects. [0006] Accordingly, there is a continued need in the field of antibody-based therapeutics for antibodies that mimic the desirable characteristics of the small molecule prodrug. BRIEF SUMMARY OF THE INVENTION [0007] Provided herein are activatable antibodies that bind to CD47 and methods of making and using these activatable antibodies in a variety of therapeutic, prophylactic, and diagnostic contexts. In some embodiments, the activatable antibodies bind human and cynomolgus monkey CD47. [0008] In one aspect of the invention, provided herein is an activatable antibody that, in an activated state, binds CD47 comprising: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to human CD47 and cynomolgus monkey CD47; (b) a masking moiety (MM) coupled to the AB, wherein the MM inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved (unactivated state) state, and wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16- 29; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. [0009] In another aspect provided herein, any one of the activatable antibodies provided herein are conjugated to an agent, generating a conjugated activatable antibody. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent. In some embodiments, the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. In a related aspect, provided herein is a conjugated activatable antibody comprising: an antibody or antigen binding fragment thereof (AB) that, in an activated state, binds CD47; and a toxin conjugated to the AB via a linker, wherein the conjugated activatable antibody comprises amino acid sequences, a linker, and a toxin selected from a single row in Table D, wherein for the given combination: (a) the AB comprises a heavy chain comprising the amino acid sequence of the heavy chain sequence or heavy chain variable domain sequence corresponding to the given combination in the single row listed in Table D; (b) the AB comprises a light chain comprising the amino acid sequence of the light chain sequence or light chain variable domain sequence corresponding to the given combination in the single row listed in Table D; and (c) the linker and the toxin comprise the linker and the toxin corresponding to the given combination in the single row listed in Table D. [00010] In another aspect, provided herein is a pharmaceutical composition comprising any of the activatable antibodies or conjugated activatable antibodies provided herein; and a carrier. In some embodiments, the pharmaceutical composition of comprises an additional agent. In some embodiments, the additional agent is a therapeutic agent. [00011] In another aspect, provided herein is an isolated nucleic acid molecule encoding any one of the activatable antibodies described herein. In a related aspect, provided herein is a vector comprising the isolated nucleic acid molecule. In another related aspect provided herein is a method of producing an activatable antibody by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises the nucleic acid molecules or the vectors provided herein. [00012] In another aspect, provided herein is a method of manufacturing any one of the activatable antibodies provided herein that, in an activated state, binds CD47, the method comprising: (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to the expression of any one of the activatable antibodies described herein, and (b) recovering the activatable antibody. [00013] In another aspect, provided herein is a method of preventing, treating, alleviating a symptom of, delaying the progression of, or otherwise ameliorating a disorder or disease in which diseased cells express CD47 or a CD47-medidated disease or disorder comprising administering a therapeutically effective amount of any one of the activatable antibodies or conjugated activatable antibodies provided herein, or pharmaceutical compositions of the same to a subject in need thereof. In another aspect, provided herein is a method of preventing, treating, alleviating a symptom of, delaying the progression of, or otherwise ameliorating a disorder or disease associated with cells expressing CD47 or a CD47-medidated disease or disorder comprising administering a therapeutically effective amount of any one of the activatable antibodies or conjugated activatable antibodies provided herein, or pharmaceutical compositions of the same to a subject in need thereof. In some embodiments the disorder or disease is cancer. In some embodiments, the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2- negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non- small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer. [00014] In a related aspect, provided herein is a method of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing CD47 comprising administering a therapeutically effective amount of any one of activatable antibodies or conjugated activatable antibodies provided herein, or pharmaceutical compositions of the same to a subject in need thereof. In some embodiments, the expression and/or activity of the CD47 is aberrant. [00015] In another aspect, provided herein is a method of inhibiting, blocking, or preventing the binding of a natural ligand to CD47, comprising administering a therapeutically effective amount of any one of the activatable antibodies or conjugated activatable antibodies provided herein, or pharmaceutical compositions of the same to a subject in need thereof. In some embodiments, the expression and/or activity of the CD47 is aberrant. [00016] In another aspect, provided herein are activatable antibodies, conjugated activatable antibody, pharmaceutical composition thereof, and methods of use thereof wherein the activatable antibodies or conjugated activatable when bound to CD47, blocks the interaction of CD47 with SIRPa. BRIEF DESCRIPTION OF THE DRAWINGS [00017] FIGS.1A and 1B are graphs depicting exemplary in vitro studies of the ability of anti-human CD47 activatable antibodies of the disclosure to bind human CD47 antigen. [00018] FIG.2 is a graph depicting exemplary in vitro studies of the ability of anti-human CD47 activatable antibodies of the disclosure to activate antibody-dependent cell phagocytosis (ADCP). [00019] FIGS.3A-3D are graphs depicting exemplary in vitro studies of the ability of anti- human CD47 antibodies and anti-CD47 activatable antibodies of the disclosure to activate ADCP. [00020] FIGS.4A-4C are graphs depicting exemplary in vitro studies of the ability of anti- human CD47 antibodies of the disclosure to activate ADCP. [00021] FIGS.5A and 5B are graphs depicting an exemplary study of the in vivo efficacy of anti-human CD47 antibodies in mouse xenograft models. DETAILED DESCRIPTION OF THE INVENTION [00022] CD47, also known as integrin associated protein (IAP), is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD47 has been shown to interact with membrane integrins on other cells. CD47 has also been shown to bind the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPa). CD47 can protect cancer cells from the immune system by preventing consumption of the cells by myeloid cells, such as a macrophages. CD47 has been shown to be expressed in human cells and overexpressed on many tumor cells. [00023] Blocking of the interaction between CD47 on a target cell and the SIRPa on a macrophage or other phagocytic cell can induce phagocytosis of the target cell. In some cases, blocking of the interaction between CD47 on a target cell and the SIRPa on a macrophage or other phagocytic cell can induce phagocytosis of the target cell along with engagement by the Fc receptor on the phagocytic cell. In some cases, the Fc receptor engagement can be performed by a Fc domain on a CD47-binding antibody. [00024] CD47 is desirable target because it is shown to be overexpressed in multiple cancer indications. [00025] Accordingly, the present disclosure provides antibodies, activatable antibodies, conjugated antibodies, and conjugated activatable antibodies that specifically bind mammalian CD47, methods of making and use thereof. [00026] More specifically, the disclosure provides anti-mammalian CD47 antibodies and fragments thereof (interchangeably referred to herein as CD47 antibodies, or ABs), conjugated CD47 antibodies, activatable CD47 antibodies, and conjugated activatable CD47 antibodies that are useful in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a disease or disorder associated with cells expressing CD47. In some embodiments, the cells are associated with normal CD47 expression and/or activity. In some embodiments, the cells are associated with aberrant CD47 expression and/or activity. In some embodiments, the cells are associated with CD47 expression and/or activity in diseased cells. For example, any of the antibodies/activatable antibodies described herein can be used in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a cancer or other neoplastic condition. Any of the antibodies/activatable antibodies described herein can also be used for detection/diagnostic applications. [00027] In some embodiments of the disclosure, the antibodies and activatable antibodies specifically bind human CD47 and cynomolgus monkey CD47. In some embodiments, the antibodies and activatable antibodies bind human CD47. In some embodiments, the antibodies and activatable antibodies bind cynomolgus monkey CD47. In some embodiments, the antibodies and activatable antibodies are internalized by CD47-containing cells. In some embodiments, the antibodies and activatable antibodies bind both the glycosylated and deglycosylated forms of the CD47 antigen. [00028] In some embodiments of the disclosure, the antibodies and activatable antibodies specifically bind human CD47 and cynomolgus monkey CD47 and block interaction with SIRPa. In some embodiments of the disclosure, the antibodies and activatable antibodies specifically bind human CD47 and cynomolgus monkey CD47 and activate antibody-dependent cell phagocytosis. In some embodiments of the disclosure, the antibodies and activatable antibodies specifically bind human CD47 and cynomolgus monkey CD47 and engage Fc receptor pathway. Definitions: [00029] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The term “a” entity or “an” entity refers to one or more of that entity. For example, a compound refers to one or more compounds. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. [00030] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: [00031] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” or “immunospecifically bind” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd > 10-6). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab, and F(ab’)2 fragments, scFvs, and an Fab expression library. The antibodies provided herein can be of any of the IgG, IgM, IgA, IgE and IgD classes (or subclasses thereof). [00032] The term “monoclonal antibody” (mAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it. [00033] The term “antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L ” ains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus, the term “FR” refers to amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three- dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989). [00034] As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin, a scFv, or a T-cell receptor. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N-terminal or C-terminal peptides of a polypeptide. An antibody is said to specifically bind an antigen when the dissociation constant is £ 1 µM; in some embodiments, £ 100 nM and in some embodiments, £ 10 nM. [00035] As used herein, the terms “specific binding,” “immunological binding,” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen- binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the on rate constant (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:185-87 (1993)). The ratio of Koff /Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present disclosure is said to specifically bind to the target, when the binding constant (Kd) is £ 1 µM, in some embodiments £100 nM, in some embodiments ^ 10 nM, and in some embodiments £ 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. [00036] The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides in accordance with the disclosure include the nucleic acid molecules encoding the heavy chain immunoglobulin molecules shown herein, and nucleic acid molecules encoding the light chain immunoglobulin molecules shown herein. [00037] The term “isolated protein” referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of murine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature. [00038] The term “polypeptide” is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the disclosure comprise the heavy chain immunoglobulin molecules shown herein, and the light chain immunoglobulin molecules shown herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof. [00039] The term naturally-occurring as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory or otherwise is naturally occurring. [00040] The term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. [00041] The term “control sequence” as used herein refers to polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term “polynucleotide” as referred to herein means nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. [00042] The term oligonucleotide referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. In some embodiments, oligonucleotides are 10 to 60 bases in length and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes, although oligonucleotides may be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides of the disclosure are either sense or antisense oligonucleotides. [00043] The term “naturally occurring nucleotides” referred to herein includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term “oligonucleotide linkages” referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselerloate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res.14:9081 (1986); Stec et al. J. Am. Chem. Soc.106:6077 (1984), Stein et al. Nucl. Acids Res.16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp.87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Patent No.5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotide can include a label for detection, if desired. [00044] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Green, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a-, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present disclosure. Examples of unconventional amino acids include: 4 hydroxyproline, g-carboxyglutamate, e-N,N,N-trimethyllysine, e -N- acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. [00045] Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5’ end the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction sequence regions on the DNA strand having the same sequence as the RNA and that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”, sequence regions on the DNA strand having the same sequence as the RNA and that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences”. [00046] As applied to polypeptides, the term substantial identity means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, in some embodiments, at least 90 percent sequence identity, in some embodiments, at least 95 percent sequence identity, and in some embodiments, at least 99 percent sequence identity. [00047] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. [00048] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, in some embodiments, at least 80%, 90%, 95%, and in some embodiments, 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Suitable amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three- dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the disclosure. [00049] Suitable amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (for example, conservative amino acid substitutions) can be made in the naturally occurring sequence (for example, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991). [00050] The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion and/or one or more internal deletion(s), but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, in some embodiments, at least 14 amino acids long, in some embodiments, at least 20 amino acids long, usually at least 50 amino acids long, and in some embodiments, at least 70 amino acids long. The term analog as used herein refers to polypeptides that are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and that has specific binding to the target, under suitable binding conditions. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally occurring sequence. Analogs typically are at least 20 amino acids long, in some embodiments, at least 50 amino acids long or longer, and can often be as long as a full-length naturally occurring polypeptide. [00051] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. [00052] As used herein, the terms “label” or “labeled” refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of 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). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term “pharmaceutical agent or drug” as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. [00053] Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw- Hill, San Francisco (1985)). [00054] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and in some embodiments, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. [00055] Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. [00056] The term patient includes human and veterinary subjects. CD47 Antibodies [00057] Provided herein are antibodies and antigen binding fragments thereof (ABs) that specifically bind to mammalian CD47. In some embodiments, the AB specifically binds human CD47 and cynomolgus monkey CD47. [00058] The ABs provided herein that bind CD47 includes a monoclonal antibody, a domain antibody, a single chain antibody, 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. In some embodiments, such an ABs that binds CD47 is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody. [00059] Also provided herein are activatable CD47 antibodies that include an antibody or antigen-binding fragment thereof (AB) that specifically binds CD47 coupled to a masking moiety (MM), such that coupling of the MM reduces the ability of the antibody or antigen- binding fragment thereof to bind CD47. In some embodiments, the MM is coupled via a sequence that includes a substrate for a protease (cleavable moiety, CM), for example, a protease that is co-localized with CD47 at a treatment site in a subject. (The activatable CD47 antibodies of the disclosure are described in greater detail in a below section). [00060] The CD47 antibodies of the disclosure specifically bind a mammalian CD47 target, such as, for example, human CD47. Also included in the disclosure are CD47 antibodies and ABs that bind to the same CD47 epitope as an antibody of the disclosure and/or an activated activatable antibody described herein. Also included in the disclosure are CD47 antibodies compete with a CD47 antibody described herein for binding to a CD47 target, e.g., human CD47. Also included in the disclosure are CD47 antibodies that cross-compete with (inhibit the binding of) a CD47 antibody and/or an activated CD47 activatable antibody described herein for binding to a CD47 target, e.g., human CD47. [00061] Antibodies and/or activatable antibodies of the disclosure specifically bind a mammalian CD47, e.g. human CD47 and cynomologous CD47. Also included in the disclosure are antibodies and/or activatable antibodies that bind to the same epitope as any of the antibodies and/or activatable antibodies described herein. Also included in the disclosure are antibodies and/or antibodies activatable antibodies that compete with a CD47 antibody (inhibit the binding of) and/or a CD47 activatable antibody described herein for binding to CD47, e.g., human CD47. Also included in the disclosure are antibodies and/or antibodies activatable antibodies that cross- compete with a CD47 antibody and/or a CD47 activatable antibody described herein for binding to CD47 (inhibits the binding to CD47), e.g., human CD47. [00062] In some embodiments, the mammalian CD47 is selected from the group consisting of a human CD47, a murine CD47, a rat CD47, and a cynomolgus monkey CD47. In some embodiments, the AB specifically binds to human CD47, murine CD47 or cynomolgus monkey CD47 with a dissociation constant of less than 1 nM. In some embodiments, the mammalian CD47 is a human CD47. [00063] In some embodiments, the AB has one or more of the following characteristics: (a) the AB specifically binds to human CD47; and (b) the AB specifically binds to human CD47 and cynomolgus monkey CD47. [00064] In some embodiments, the AB has one or more of the following characteristics: (a) the AB specifically binds human CD47 and cynomolgus monkey CD47; (b) the AB inhibits binding of one or more of the natural mammalian ligands of CD47 to mammalian CD47; (c) the AB inhibits binding of one or more of the natural human ligands of CD47 to human CD47; and (d) the AB inhibits binding of one or more of the natural cynomolgus monkey ligands of CD47 to cynomolgus monkey CD47. [00065] In some embodiments the AB binds both glycosylated and deglycosylated forms of CD47. [00066] In some embodiments, the AB blocks the ability of a natural ligand to bind to the mammalian CD47 with an EC50 less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 500 nM, and/or less than or equal to 1000 nM. In some embodiments, the AB blocks the ability of a natural ligand to bind to the mammalian CD47 with an EC50 less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 500 nM, and/or less than or equal to 1000 nM. [00067] In some embodiments, the AB blocks the ability of a natural ligand to bind to the mammalian CD47 with an EC50 of 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM 10 nM to 50 nM, 50 nM to 1000 nM, 50 nM to 500 nM, 50 nM to 100 nM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 1000 nM. In some embodiments, the AB blocks the ability of a natural ligand to bind to the mammalian CD47 with an EC50 of 5 nM to 1000 nM, 5 nM to 500 nM, 5 nM to 100 nM 5 nM to 50 nM, 5 nM to 10 nM, 10 nM to 1000 nM, 10 nM to 500 nM, 10 nM to 100 nM 10 nM to 50 nM, 50 nM to 1000 nM, 50 nM to 500 nM, 50 nM to 100 nM, 100 nM to 1000 nM, 100 nM to 500 nM, 500 nM to 1000 nM. [00068] In some embodiments, the AB of the present disclosure inhibits or reduces the growth, proliferation, and/or metastasis of cells expressing mammalian CD47. Without intending to be bound by any theory, the AB of the present disclosure may inhibit or reduce the growth, proliferation, and/or metastasis of cells expressing mammalian CD47 by specifically binding to CD47 and inhibiting, blocking, and/or preventing the binding of a natural ligand to mammalian CD47. [00069] In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to mammalian CD47. In some embodiments, the AB has a dissociation constant of about 10 nM or less for binding to mammalian CD47. In some embodiments, the AB has a dissociation constant of about 5 nM or less for binding to CD47. In some embodiments, the AB has a dissociation constant of about 1 nM or less for binding to CD47. In some embodiments, the AB has a dissociation constant of about 0.5 nM or less for binding to CD47. In some embodiments, the AB has a dissociation constant of about 0.1 nM or less for binding to CD47. In some embodiments, the AB has a dissociation constant of 0.01 nM to 100 nM, 0.01 nM to 10 nM, 0.01 nM to 5 nM, 0.01 nM to 1 nM, 0.01 to 0.5 nM, 0.01 nm to 0.1 nM, 0.01 nm to 0.05 nM, 0.05 nM to 100 nM, 0.05 nM to 10 nM, 0.05 nM to 5 nM, 0.05 nM to 1 nM, 0.05 to 0.5 nM, 0.05 nm to 0.1 nM, 0.1 nM to 100 nM, 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM, 0.1 to 0.5 nM, 0.5 nM to 100 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 1 nM, 1 nM to 100 nM, 1 nM to 10 nM, 1 nM to 5 nM, 5 nM to 100 nM, 5 nM to 10 nM, or 10 nM to 100 nM, for binding to mammalian CD47. [00070] Exemplary CD47 antibodies and activatable CD47 antibodies of the invention may include a heavy chain and a light chain that are, or are derived from, the heavy chain variable and light chain variable sequences shown below (CDR sequences are shown in bold and underline): hu H4L2 H4 VH: QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDKSTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 1) hu H4L2 L2 VL: DVVMTQSPDSLAVSLGERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 2) [00071] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 1 and 57-59. [00072] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a light chain variable region amino acid sequence of any one of SEQ ID NOs: 2 and 76-79. [00073] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1. [00074] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a light chain variable region amino acid sequence of SEQ ID NO: 2. [00075] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. [00076] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1. [00077] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 2. [00078] In some embodiments, the antibody or antigen-binding fragment thereof of the CD47 antibody/activatable antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 2. [00079] Exemplary CD47 antibodies and activatable CD47 antibodies of the invention include a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [00080] In some embodiments, the CD47 antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [00081] In some embodiments, the CD47 antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [00082] In some embodiments, the CD47 antibody or antigen-binding fragment thereof comprises an amino acid sequence comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [00083] In some embodiments, the CD47 antibody or antigen-binding fragment thereof comprises an amino acid sequence comprising the VH CDR1 sequence comprising the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [00084] In some embodiments, the CD47 antibody or antigen-binding fragment thereof comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, comprising a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [00085] Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 1 and 57-59, and a light chain variable region amino acid sequence of any one of SEQ ID NOs: 2 and 76-79. [00086] Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a heavy chain variable region amino acid sequence SEQ ID NO: 1, and a light chain variable region amino acid sequence SEQ ID NO: 2. [00087] Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [00088] Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a heavy chain variable region amino acid sequence of any one of SEQ ID NOs: 1 and 57-59, and a light chain variable region amino acid sequence of any one of SEQ ID NOs: 2 and 76-79. [00089] Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 to a CD47 antibody comprising a heavy chain variable region amino acid sequence SEQ ID NO: 1, and a light chain variable region amino acid sequence SEQ ID NO: 2.. [00090] Suitable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 to a CD47 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [00091] In some embodiments, the CD47 antibody of the disclosure comprises an isolated antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47. In some embodiments, the antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2. [00092] In some embodiments, the isolated antibody or antigen binding fragment thereof binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as an isolated antibody that comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2. [00093] In some embodiments, the isolated antibody or antigen binding fragment thereof cross-competes with (inhibits the binding of) an isolated antibody that comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2. [00094] In some embodiments, the CD47 antibody/activatable CD47 antibody comprises or is derived from an antibody that is manufactured, secreted or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) disclosed in US Patent No.5,330,896 and deposited at ATCC under deposit number HB 8214. [00095] In some embodiments, the CD47 antibody/activatable CD47 antibody comprises or is derived from an antibody that is manufactured, secreted or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) designated BA120 as disclosed in US Patent No.7,736,647 and deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) (Institut Pasteur, Paris, France, 25, Rue du Docteur Roux, F-75724, Paris, Cedex 15) on Jun.14, 2005, under number CNCM I-3449; the hybridoma(s) disclosed in US Patent No. 7,572,895 and deposited at the ATCC under PTA-6055; the hybridoma(s) disclosed in PCT Publication No. WO 2014/020140 and WO 2005/111082 and deposited with CNCM on May 10 2001, under number 1-2665; the hybridoma(s) disclosed in US Patent No.4,434,156 and deposited at the ATCC under HB-8094; the hybridoma(s) disclosed in US Patent No.5,648,469 and deposited at the ATCC under HB-1161 and HB-1160. [00096] In some embodiments, the CD47 antibody/activatable CD47 antibody includes a heavy chain that comprises or is derived from a heavy chain amino acid sequence shown in PCT Publication Nos. WO 2014/144060, WO 2014/189973, WO 2014/020140, in US Patent Nos. 8,663,598; 8,129,503; 7,736,647; 7,572,895; 4,434,156; in US Patent Application Publication Nos. US2014114054, US20140212423, US2013177579, US2013045206, US20130216476, US20120282176, and/or in Chinese Patent No. CN16245107B, the contents of each of which are hereby incorporated by reference in their entirety. [00097] The disclosure also provides methods for producing a CD47 AB of the disclosure by culturing a cell under conditions that lead to expression of the antibody or fragment thereof, wherein the cell comprises a nucleic acid molecule of the disclosure or a vector of the disclosure. [00098] In some embodiments, the CD47 antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain variable region amino acid sequence comprising an amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14. [00099] In some embodiments, the CD47 antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence come rising an amino acid sequence of SEQ ID NO: 15. [000100] In some embodiments, the CD47 antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a heavy chain variable region amino acid sequence comprising an amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14. [000101] In some embodiments, the CD47 antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a light chain variable region amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment thereof is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a light chain amino acid sequence come rising an amino acid sequence of SEQ ID NO: 15. Activatable CD47 Antibodies [000102] As described above, the disclosure also provides activatable antibodies that include an antibody or antigen-binding fragment thereof that specifically binds CD47 coupled to a masking moiety (MM), such that coupling of the MM reduces the ability of the antibody or antigen-binding fragment thereof to bind CD47. In some embodiments, the MM is coupled via a sequence that includes a substrate for a protease (CM, cleavable moiety), for example, a protease that is active in diseased tissue and/or a protease that is co-localized with CD47 at a treatment site in a subject. The activatable CD47 antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, e.g., healthy tissue or other tissue not targeted for treatment and/or diagnosis, and, when activated, exhibit binding to CD47 that is at least comparable to the corresponding, unmodified antibody, also referred to herein as the parental antibody. [000103] The activatable CD47 antibodies described herein overcome a limitation of antibody therapeutics, particularly antibody therapeutics that are known to be toxic to at least some degree in vivo. Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. The activatable CD47 antibodies provided herein are designed to address the toxicity associated with the inhibition of the target in normal tissues by traditional therapeutic antibodies. These activatable CD47 antibodies remain masked until proteolytically activated at the site of disease. Starting with a CD47 antibody as a parental therapeutic antibody, the activatable CD47 antibodies of the invention were engineered by coupling the antibody to an inhibitory mask through a linker that incorporates a protease substrate. [000104] As used herein, the term cleaved state of the activatable antibody refers to the condition of the activatable antibodies following modification of the CM by at least one protease. The term uncleaved state, as used herein, refers to the condition of the activatable antibodies in the absence of cleavage of the CM by a protease. As discussed above, the term “activatable antibodies” is used herein to refer to an activatable antibody in both its uncleaved (native) state, as well as in its cleaved state. It will be apparent to the ordinarily skilled artisan that in some embodiments a cleaved activatable antibody may lack an MM due to cleavage of the CM by protease, resulting in release of at least the MM (e.g., where the MM is not joined to the activatable antibodies by a covalent bond (e.g., a disulfide bond between cysteine residues). [000105] By activatable or switchable is meant that the activatable antibody exhibits a first level of binding to a target when the activatable antibody is in a inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target in the uninhibited, unmasked and/or cleaved state (i.e., a second conformation), where the second level of target binding is greater than the first level of binding. In general, the access of target to the AB of the activatable antibody is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease, than in the absence of such a cleaving agent. Thus, when the activatable antibody is in the uncleaved state, the AB is inhibited from target binding and can be masked from target binding (i.e., the first conformation is such the AB cannot bind the target), and in the cleaved state the AB is not inhibited or is unmasked to target binding. [000106] The CM and AB of the activatable antibodies are selected so that the AB represents a binding moiety for a given target, and the CM represents a substrate for a protease. In some embodiments, the protease is co-localized with the target at a treatment site or diagnostic site in a subject. As used herein, co-localized refers to being at the same site or relatively close nearby. In some embodiments, a protease cleaves a CM yielding an activated antibody that binds to a target located nearby the cleavage site. The activatable antibodies disclosed herein find particular use where, for example, a protease capable of cleaving a site in the CM, i.e., a protease, is present at relatively higher levels in target-containing tissue of a treatment site or diagnostic site than in tissue of non-treatment sites (for example in healthy tissue). In some embodiments, a CM of the disclosure is also cleaved by one or more other proteases. In some embodiments, it is the one or more other proteases that is co-localized with the target and that is responsible for cleavage of the CM in vivo. [000107] In some embodiments activatable antibodies provide for reduced toxicity and/or adverse side effects that could otherwise result from binding of the AB at non-treatment sites if the AB were not masked or otherwise inhibited from binding to the target. [000108] In general, an activatable antibody can be designed by selecting an AB of interest (such as any CD47 antibody or fragment thereof described herein) and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides for masking of the AB or reduction of binding of the AB to its target. Structural design criteria can be to be taken into account to provide for this functional feature. [000109] Activatable antibodies exhibiting a switchable phenotype of a desired dynamic range for target binding in an inhibited versus an uninhibited conformation are provided. Dynamic range generally refers to a ratio of (a) a maximum detected level of a parameter under a first set of conditions to (b) a minimum detected value of that parameter under a second set of conditions. For example, in the context of an activatable antibody, the dynamic range refers to the ratio of (a) a maximum detected level of target protein binding to an activatable antibody in the presence of at least one protease capable of cleaving the CM of the activatable antibodies to (b) a minimum detected level of target protein binding to an activatable antibody in the absence of the protease. The dynamic range of an activatable antibody can be calculated as the ratio of the dissociation constant of an activatable antibody cleaving agent (e.g., enzyme) treatment to the dissociation constant of the activatable antibodies cleaving agent treatment. The greater the dynamic range of an activatable antibody, the better the switchable phenotype of the activatable antibody. Activatable antibodies having relatively higher dynamic range values (e.g., greater than 1) exhibit more desirable switching phenotypes such that target protein binding by the activatable antibodies occurs to a greater extent (e.g., predominantly occurs) in the presence of a cleaving agent (e.g., enzyme) capable of cleaving the CM of the activatable antibodies than in the absence of a cleaving agent. [000110] As described above, the activatable CD47 antibodies provided herein include a masking moiety (MM). In some embodiments, the masking moiety is an amino acid sequence that is coupled or otherwise attached to the CD47 antibody and is positioned within the activatable CD47 antibody construct such that the masking moiety reduces the ability of the CD47 antibody to specifically bind CD47. Suitable masking moieties are identified using any of a variety of known techniques. For example, peptide masking moieties are identified using the methods described in PCT Publication No. WO 2009/025846 by Daugherty et al., the contents of which are hereby incorporated by reference in their entirety. [000111] As described above, the activatable CD47 antibodies provided herein include a cleavable moiety (CM). In some embodiments, the cleavable moiety includes an amino acid sequence that is a substrate for a protease, usually an extracellular protease. Suitable substrates are identified using any of a variety of known techniques. For example, peptide substrates are identified using the methods described in U.S. Patent No.7,666,817 by Daugherty et al.; in U.S. Patent No.8,563,269 by Stagliano et al.; and in PCT Publication No. WO 2014/026136 by La Porte et al., the contents of each of which are hereby incorporated by reference in their entirety. (See also Boulware et al. “Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics.” Biotechnol Bioeng.106.3 (2010): 339-46). [000112] Exemplary substrates include but are not limited to substrates cleavable by one or more of the following enzymes or proteases listed in Table A. Table A: Exemplary Proteases and/or Enzymes
Figure imgf000032_0001
Figure imgf000033_0001
[000113] The activatable antibodies in an activated state bind CD47 and include (i) an antibody or an antigen binding fragment thereof (AB) that specifically binds to CD47; (ii) a masking moiety (MM) that, when the activatable antibody is in an uncleaved state, inhibits the binding of the AB to CD47; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. [000114] In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM- MM. [000115] In some embodiments, the activatable antibody comprises a linking peptide between the MM and the CM. [000116] In some embodiments, the activatable antibody comprises a linking peptide between the CM and the AB. [000117] In some embodiments, the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM- LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need not be identical to each other. [000118] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 116) and (GGGS)n (SEQ ID NO: 117), where n is an integer of at least one. [000119] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 118), GGSGG (SEQ ID NO: 119), GSGSG (SEQ ID NO: 120), GSGGG (SEQ ID NO: 121), GGGSG (SEQ ID NO: 122), and GSSSG (SEQ ID NO: 123). [000120] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 124), GSSGGSGGSGG (SEQ ID NO: 125), GSSGGSGGSGGS (SEQ ID NO: 126), GSSGGSGGSGGSGGGS (SEQ ID NO: 127), GSSGGSGGSG (SEQ ID NO: 128), GSSGGSGGSGS (SEQ ID NO: 129), or GGGSSGGS (SEQ ID NO: 134). [000121] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 130), GSSGT (SEQ ID NO: 131) or GSSG (SEQ ID NO: 132). [000122] In some embodiments, the antibody or antigen-binding fragment thereof that binds CD47 is a monoclonal antibody, domain antibody, single chain, 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. In some embodiments, such an antibody or antigen-binding fragment thereof that binds CD47 is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody. [000123] In some embodiments, the activatable antibody in an uncleaved state specifically binds to the mammalian CD47 with a dissociation constant less than or equal to 1 nM, less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 15 nM, less than or equal to 20 nM, less than or equal to 25 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 150 nM, less than or equal to 250 nM, less than or equal to 500 nM, less than or equal to 750 nM, less than or equal to 1000 nM, and/or less than or equal to 2000 nM. [000124] In some embodiments, the activatable antibody in an uncleaved state specifically binds to the mammalian CD47 with a dissociation constant in the range of 1 nM to 2000 nM, 1 nM to 1000 nM, 1 nM to 750 nM, 1 nM to 500 nM, 1 nM to 250 nM, 1 nM to 150 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 25 nM, 1 nM to 15 nM, 1 nM to 10 nM, 1 nM to 5 nM, 5 nM to 2000 nM, 5 nM to 1000 nM, 5 nM to 750 nM, 5 nM to 500 nM, 5 nM to 250 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 25 nM, 5 nM to 15 nM, 5 nM to 10 nM, 10 nM to 2000 nM, 10 nM to 1000 nM, 10 nM to 750 nM, 10 nM to 500 nM, 10 nM to 250 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 25 nM, 10 nM to 15 nM, 15 nM to 2000 nM, 15 nM to 1000 nM, 15 nM to 750 nM, 15 nM to 500 nM, 15 nM to 250 nM, 15 nM to 150 nM, 15 nM to 100 nM, 15 nM to 50 nM, 15 nM to 25 nM, 25 nM to 2000 nM, 25 nM to 1000 nM, 25 nM to 750 nM, 25 nM to 500 nM, 25 nM to 250 nM, 25 nM to 150 nM, 25 nM to 100 nM, 25 nM to 50 nM, 50 nM to 2000 nM, 50 nM to 1000 nM, 50 nM to 750 nM, 50 nM to 500 nM, 50 nM to 250 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to 2000 nM, 100 nM to 1000 nM, 100 nM to 750 nM, 100 nM to 500 nM, 100 nM to 250 nM, 100 nM to 150 nM, 150 nM to 2000 nM, 150 nM to 1000 nM, 150 nM to 750 nM, 150 nM to 500 nM, 150 nM to 250 nM, 250 nM to 2000 nM, 250 nM to 1000 nM, 250 nM to 750 nM, 250 nM to 500 nM, 500 nM to 2000 nM, 500 nM to 1000 nM, 500 nM to 750 nM, 500 nM to 500 nM, 500 nM to 250 nM, 500 nM to 150 nM, 500 nM to 100 nM, 500 nM to 50 nM, 750 nM to 2000 nM, 750 nM to 1000 nM, or 1000 nM to 2000 nM. [000125] In some embodiments, the activatable antibody in an activated state specifically binds to the mammalian CD47 with a dissociation constant is less than or equal to 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM. [000126] In some embodiments, the activatable antibody in an activated state specifically binds to the mammalian CD47 with a dissociation constant in the range of 0.01 nM to 100 nM, 0.01 nM to 10 nM, 0.01 nM to 5 nM, 0.01 nM to 1 nM, 0.01 to 0.5 nM, 0.01 nm to 0.1 nM, 0.01 nm to 0.05 nM, 0.05 nM to 100 nM, 0.05 nM to 10 nM, 0.05 nM to 5 nM, 0.05 nM to 1 nM, 0.05 to 0.5 nM, 0.05 nm to 0.1 nM, 0.1 nM to 100 nM, 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM, 0.1 to 0.5 nM, 0.5 nM to 100 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 1 nM, 1 nM to 100 nM, 1 nM to 10 nM, 1 nM to 5 nM, 5 nM to 100 nM, 5 nM to 10 nM, or 10 nM to 100 nM. [000127] When the AB is modified with a MM and is in the presence of the target, specific binding of the AB to its target is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM or the specific binding of the parental AB to the target. [000128] The Kd of the AB modified with a MM towards the CD47 target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10- 100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000- 100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times greater than the Kd of the AB not modified with an MM or of the parental AB towards the CD47 target. Conversely, the binding affinity of the AB modified with a MM towards the CD47 target is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10- 100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the AB not modified with an MM or of the parental AB towards the CD47 target. [000129] The dissociation constant (Kd) of the MM towards the AB is generally greater than the Kd of the AB towards the CD47 target. The Kd of the MM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times greater than the Kd of the AB towards the CD47 target. Conversely, the binding affinity of the MM towards the AB is generally lower than the binding affinity of the AB towards the CD47 target. The binding affinity of MM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times lower than the binding affinity of the AB towards the CD47 target. [000130] In some embodiments, the dissociation constant (Kd) of the MM towards the AB is approximately equal to the Kd of the AB towards the CD47 target. In some embodiments, the dissociation constant (Kd) of the MM towards the AB is no more than the dissociation constant of the AB towards the CD47 target. In some embodiments, the dissociation constant (Kd) of the MM towards the AB is equivalent to the dissociation constant of the AB towards the CD47 target. [000131] In some embodiments, the dissociation constant (Kd) of the MM towards the AB is less than the dissociation constant of the AB towards the CD47 target. [000132] In some embodiments, the dissociation constant (Kd) of the MM towards the AB is greater than the dissociation constant of the AB towards the CD47 target. [000133] In some embodiments, the MM has a Kd for binding to the AB that is no more than the Kd for binding of the AB to the target. [000134] In some embodiments, the MM has a Kd for binding to the AB that is no less than the Kd for binding of the AB to the target. [000135] In some embodiments, the MM has a Kd for binding to the AB that is approximately equal to the Kd for binding of the AB to the target. [000136] In some embodiments, the MM has a Kd for binding to the AB that is less than the Kd for binding of the AB to the target. [000137] In some embodiments, the MM has a Kd for binding to the AB that is greater than the Kd for binding of the AB to the target. [000138] In some embodiments, the MM has a Kd for binding to the AB that is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 fold greater than the Kd for binding of the AB to the target. In some embodiments, the MM has a Kd for binding to the AB that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 fold greater than the Kd for binding of the AB to the target. [000139] In some embodiments, the MM has an affinity for binding to the AB that is less than the affinity of binding of the AB to the target. [000140] In some embodiments, the MM has an affinity for binding to the AB that is no more than the affinity of binding of the AB to the target. [000141] In some embodiments, the MM has an affinity for binding to the AB that is approximately equal of the affinity of binding of the AB to the target. [000142] In some embodiments, the MM has an affinity for binding to the AB that is no less than the affinity of binding of the AB to the target. [000143] In some embodiments, the MM has an affinity for binding to the AB that is greater than the affinity of binding of the AB to the target. [000144] In some embodiments, the MM has an affinity for binding to the AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 less than the affinity of binding of the AB to the target. I In some embodiments, the MM has an affinity for binding to the AB that is between 1-5, 2-5, 2- 10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 fold less than the affinity of binding of the AB to the target. In some embodiments, the MM has an affinity for binding to the AB that is 2 to 20-fold less than the affinity of binding of the AB to the target. In some embodiments, a MM not covalently linked to the AB and at equimolar concentration to the AB does not inhibit the binding of the AB to the target. [000145] When the AB is modified with a MM and is in the presence of the target specific binding of the AB to its target is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM or the specific binding of the parental AB to the target. When compared to the binding of the AB not modified with an MM or the binding of the parental AB to the target the AB’s ability to bind the target when modified with an MM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more when measured in vivo or in an in vitro assay. [000146] The MM inhibits the binding of the AB to the target. The MM binds the antigen binding domain of the AB and inhibits binding of the AB to the target. The MM can sterically inhibit the binding of the AB to the target. The MM can allosterically inhibit the binding of the AB to its target. In these embodiments when the AB is modified or coupled to a MM and in the presence of target there is no binding or substantially no binding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target, as compared to the binding of the AB not modified with an MM, the parental AB, or the AB not coupled to an MM to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay. [000147] When an AB is coupled to or modified by a MM, the MM ‘masks’ or reduces or otherwise inhibits the specific binding of the AB to the target. When an AB is coupled to or modified by a MM, such coupling or modification can effect a structural change that reduces or inhibits the ability of the AB to specifically bind its target. [000148] An AB coupled to or modified with an MM can be represented by the following formulae (in order from an amino (N) terminal region to carboxyl (C) terminal region: (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) where MM is a masking moiety, the AB is an antibody or antibody fragment thereof, and the L is a linker. In many embodiments, it can be desirable to insert one or more linkers, e.g., flexible linkers, into the composition so as to provide for flexibility. [000149] In certain embodiments, the MM is not a natural binding partner of the AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB. [000150] In some embodiments, the activatable antibodies include an AB that is modified by an MM and also includes one or more cleavable moieties (CM). Such activatable antibodies exhibit activatable/switchable binding, to the AB’s target. Activatable antibodies generally include an antibody or antibody fragment (AB), modified by or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM). In some embodiments, the CM contains an amino acid sequence that serves as a substrate for at least one protease. [000151] The elements of the activatable antibodies are arranged so that the MM and CM are positioned such that in a cleaved (or relatively active) state and in the presence of a target, the AB binds a target while the activatable antibody is in an uncleaved (or relatively inactive) state in the presence of the target, specific binding of the AB to its target is reduced or inhibited. The specific binding of the AB to its target can be reduced due to the inhibition or masking of the AB’s ability to specifically bind its target by the MM. [000152] The Kd of the AB modified with a MM and a CM towards the CD47 target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10- 10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100- 1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000- 10,000,000 times greater than the Kd of the AB not modified with an MM and a CM or of the parental AB towards the CD47 target. Conversely, the binding affinity of the AB modified with a MM and a CM towards the CD47 target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10- 10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000- 10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the AB not modified with an MM and a CM or of the parental AB towards the CD47 target. [000153] When the AB is modified with a MM and a CM and is in the presence of the target but not in the presence of a modifying agent (for example at least one protease), specific binding of the AB to its target is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM and a CM or of the parental AB to the target. When compared to the binding of the parental AB or the binding of an AB not modified with an MM and a CM to its target, the AB’s ability to bind the target when modified with an MM and a CM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay. [000154] Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulae for activatable antibodies are provided below. It is specifically contemplated that the N- to C-terminal order of the AB, MM and CM can be reversed within an activatable antibody. It is also specifically contemplated that the CM and MM may overlap in amino acid sequence, e.g., such that the CM is contained within the MM. [000155] For example, activatable antibodies can be represented by the following formula (in order from an amino (N) terminal region to carboxyl (C) terminal region: (MM)-(CM)-(AB) (AB)-(CM)-(MM) where MM is a masking moiety, CM is a cleavable moiety, and AB is an antibody or fragment thereof. It should be noted that although MM and CM are indicated as distinct components in the formulae above, in all exemplary embodiments (including formulae) disclosed herein it is contemplated that the amino acid sequences of the MM and the CM could overlap, e.g., such that the CM is completely or partially contained within the MM. In addition, the formulae above provide for additional amino acid sequences that can be positioned N-terminal or C-terminal to the activatable antibodies elements. [000156] In certain embodiments, the MM is not a natural binding partner of the AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB. [000157] In many embodiments it may be desirable to insert one or more linkers, e.g., flexible linkers, into the activatable antibody construct so as to provide for flexibility at one or more of the MM-CM junction, the CM-AB junction, or both. For example, the AB, MM, and/or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, particularly Gly) to provide the desired flexibility. As such, the switchable phenotype of such activatable antibody constructs may benefit from introduction of one or more amino acids to provide for a flexible linker. In addition, as described below, where the activatable antibody is provided as a conformationally constrained construct, a flexible linker can be operably inserted to facilitate formation and maintenance of a cyclic structure in the uncleaved activatable antibody. [000158] For example, in certain embodiments an activatable antibody comprises one of the following formulae (where the formula below represents an amino acid sequence in either N- to C-terminal direction or C- to N-terminal direction): (MM)-L1-(CM)-(AB) (MM)-(CM)-L2-(AB) (MM)-L1-(CM)-L2-(AB) wherein MM, CM, and AB are as defined above; wherein L1 and L2 are each independently and optionally present or absent, are the same or different flexible linkers that include at least 1 flexible amino acid (e.g., Gly). In addition, the formulae above provide for additional amino acid sequences that can be positioned N-terminal or C-terminal to the activatable antibodies elements. Examples include, but are not limited to, targeting moieties (e.g., a ligand for a receptor of a cell present in a target tissue) and serum half-life extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HAS)). [000159] The CM is specifically cleaved by at least one protease at a rate of about 0.001- 1500 x 104 M-1S-1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 104 M-1S-1. In some embodiments, the CM is specifically cleaved at a rate of about 100,000 M-1S-1. In some embodiments, the CM is specifically cleaved at a rate from about 1x10E2 to about 1x10E6 M-1S-1 (i.e., from about 1x102 to about 1x106 M-1S-1). [000160] For specific cleavage by an enzyme, contact between the enzyme and CM is made. When the activatable antibody comprising an AB coupled to a MM and a CM is in the presence of target and sufficient enzyme activity, the CM can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the CM and effect cleavage. It can readily be envisioned that an enzyme may be in the vicinity of the CM but unable to cleave because of other cellular factors or protein modification of the enzyme. [000161] Linkers suitable for use in compositions described herein are generally ones that provide flexibility of the modified AB or the activatable antibodies to facilitate the inhibition of the binding of the AB to the target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 3 or 4, 5 or 6, 7, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. [000162] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 116) and (GGGS)n (SEQ ID NO: 117), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to Gly-Gly-Ser-Gly (SEQ ID NO: 118), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 119), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 120), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 121), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 122), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 123), and the like. The ordinarily skilled artisan will recognize that design of an activatable antibodies can include linkers 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 antibodies structure. [000163] The disclosure also provides compositions and methods that include an activatable CD47 antibody that includes an antibody or antibody fragment (AB) that specifically binds CD47, where the AB is coupled to a masking moiety (MM) that decreases the ability of the AB to bind its target. In some embodiments, the activatable CD47 antibody further includes a cleavable moiety (CM) that is a substrate for a protease. The compositions and methods provided herein enable the attachment of one or more agents to one or more cysteine residues in the AB without compromising the activity (e.g., the masking, activating or binding activity) of the activatable CD47 antibody. In some embodiments, the compositions and methods provided herein enable the attachment of one or more agents to one or more cysteine residues in the AB without reducing or otherwise disturbing one or more disulfide bonds within the MM. The compositions and methods provided herein produce an activatable CD47 antibody that is conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic and/or prophylactic agents, for example, in some embodiments, without any of the agent(s) being conjugated to the MM of the activatable CD47 antibody. The compositions and methods provided herein produce conjugated activatable CD47 antibodies in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in an uncleaved state. The compositions and methods provided herein produce conjugated activatable CD47 antibodies in which the activatable antibody is still activated, i.e., cleaved, in the presence of a protease that can cleave the CM. [000164] In some embodiments, the activatable antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1. In some embodiments, the activatable antibody comprises a light chain variable region amino acid sequence of SEQ ID NO: 2. [000165] In some embodiments, the activatable antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1. In some embodiments, the activatable antibody comprises a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 2. [000166] In some embodiments, the activatable antibody comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected of SEQ ID NO: 2. [000167] In some embodiments, the activatable antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000168] In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000169] In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000170] In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000171] In some embodiments, the MM has a dissociation constant for binding to the AB which is greater than the dissociation constant of the AB to CD47. [000172] In some embodiments, the MM has a dissociation constant for binding to the AB which is no more than the dissociation constant of the AB to CD47. [000173] In some embodiments, the MM has a dissociation constant for binding to the AB is equivalent to the dissociation constant of the AB to CD47. [000174] In some embodiments, the MM has a dissociation constant for binding to the AB which is less than the dissociation constant of the AB to CD47. [000175] In some embodiments, the dissociation constant (Kd) of the MM towards the AB is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or greater, or between 1- 5, 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100- 10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000- 1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000- 1,000,000, or 100,000-10,000,000 times or greater than the dissociation constant of the AB towards the CD47 target. [000176] In some embodiments, the MM does not interfere or compete with the AB for binding to CD47 when the activatable antibody is in a cleaved state. [000177] In some embodiments, the MM is a polypeptide of about 2 to 40 amino acids in length. In some embodiments, the MM is a polypeptide of up to about 40 amino acids in length. [000178] In some embodiments, the MM polypeptide sequence is different from that of CD47. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM polypeptide sequence is different from that of CD47 and is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of the AB. [000179] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least two times greater than the Kd of the AB when not coupled to the MM towards CD47. [000180] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least five times greater than the Kd of the AB when not coupled to the MM towards CD47. [000181] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 10 times greater than the Kd of the AB when not coupled to the MM towards CD47. [000182] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 20 times greater than the Kd of the AB when not coupled to the MM towards CD47. [000183] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 40 times greater than the Kd of the AB when not coupled to the MM towards CD47. [000184] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 100 times greater than the Kd of the AB when not coupled to the MM towards CD47. [000185] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 1000 times greater than the Kd of the AB when not coupled to the MM towards CD47. [000186] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind CD47 such that the dissociation constant (Kd) of the AB when coupled to the MM towards CD47 is at least 10,000 times greater than the Kd of the AB when not coupled to the MM towards CD47. [000187] In some embodiments, in the presence of CD47, the MM reduces the ability of the AB to bind CD47 by at least 90% when the CM is uncleaved, as compared to when the CM is cleaved when assayed in vitro using a target displacement assay such as, for example, the assay described in PCT Publication No. WO 2010/081173, the contents of which are hereby incorporated by reference in their entirety. [000188] In some embodiments, MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16-29. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 23. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the MM comprises an amino acid sequence of SEQ ID NO: 27. [000189] In some embodiments, the protease that cleaves the CM is active, e.g., up- regulated or otherwise unregulated, in diseased tissue, and the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease. [000190] In some embodiments, the protease is co-localized with CD47 in a tissue, and the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease. [000191] In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least twofold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds CD47. [000192] In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least fivefold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds CD47. [000193] In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 10-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds CD47. [000194] In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 20-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds CD47. [000195] In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 40-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state, the AB binds CD47. [000196] In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 50-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state, the AB binds CD47. [000197] In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 100-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state, the AB binds CD47. [000198] In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to CD47 is reduced to occur with a dissociation constant that is at least 200-fold greater than the dissociation constant of an unmodified AB binding to CD47, whereas in the cleaved state, the AB binds CD47. [000199] In some embodiments, the CM is a polypeptide of up to 15 amino acids in length. [000200] In some embodiments, the CM is a polypeptide that includes a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). In some embodiments, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1- CM2 substrate is independently a polypeptide of up to 15 amino acids in length. [000201] In some embodiments, the CM is a substrate for at least one protease that is or is believed to be up-regulated or otherwise unregulated in cancer. [000202] In some embodiments, the CM is a substrate for at least one protease selected from the group consisting of a matrix metalloprotease (MMP), thrombin, a neutrophil elastase, a cysteine protease, legumain, and a serine protease, such as matriptase (MT-SP1), and urokinase (uPA). Without being bound by theory, it is believed that these proteases are up-regulated or otherwise unregulated in at least one of cancer. [000203] Exemplary substrates include but are not limited to substrates cleavable by one or more of the following enzymes or proteases listed in Table A. [000204] In some embodiments, the CM is selected for use with a specific protease, for example a protease that is known to be co-localized with the target of the activatable antibody. [000205] In some embodiments, the CM is a substrate for at least one MMP. Examples of MMPs include the MMPs listed in the Table A. In some embodiments, the CM is a substrate for a protease selected from the group consisting of MMP 9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments the CM is a substrate for MMP9. In some embodiments, the CM is a substrate for MMP14. [000206] In some embodiments, the CM is a substrate that includes the sequence TGRGPSWV (SEQ ID NO: 356); SARGPSRW (SEQ ID NO: 357); TARGPSFK (SEQ ID NO: 358); LSGRSDNH (SEQ ID NO: 359); GGWHTGRN (SEQ ID NO: 360); HTGRSGAL (SEQ ID NO: 361); PLTGRSGG (SEQ ID NO: 362); AARGPAIH (SEQ ID NO: 363); RGPAFNPM (SEQ ID NO: 364); SSRGPAYL (SEQ ID NO: 365); RGPATPIM (SEQ ID NO: 366); RGPA (SEQ ID NO: 367); GGQPSGMWGW (SEQ ID NO: 368); FPRPLGITGL (SEQ ID NO: 369); VHMPLGFLGP (SEQ ID NO: 370); SPLTGRSG (SEQ ID NO: 371); SAGFSLPA (SEQ ID NO: 372); LAPLGLQRR (SEQ ID NO: 373); SGGPLGVR (SEQ ID NO: 374); PLGL (SEQ ID NO: 375); LSGRSGNH (SEQ ID NO: 789); SGRSANPRG (SEQ ID NO: 790); LSGRSDDH (SEQ ID NO: 791); LSGRSDIH (SEQ ID NO: 792); LSGRSDQH (SEQ ID NO: 793); LSGRSDTH (SEQ ID NO: 794); LSGRSDYH (SEQ ID NO: 795); LSGRSDNP (SEQ ID NO: 796); LSGRSANP (SEQ ID NO: 797); LSGRSANI (SEQ ID NO: 798); LSGRSDNI (SEQ ID NO: 799); MIAPVAYR (SEQ ID NO: 800); RPSPMWAY (SEQ ID NO: 801); WATPRPMR (SEQ ID NO: 802); FRLLDWQW (SEQ ID NO: 803); ISSGL (SEQ ID NO: 804); ISSGLLS (SEQ ID NO: 805); and/or ISSGLL (SEQ ID NO: 806). [000207] In some embodiments, the CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 359). In some embodiments, the CM comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 356). In some embodiments, the CM comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 362). In some embodiments, the CM comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO: 368). In some embodiments, the CM comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 369). In some embodiments, the CM comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 370). In some embodiments, the CM comprises the amino acid sequence PLGL (SEQ ID NO: 375). In some embodiments, the CM comprises the amino acid sequence SARGPSRW (SEQ ID NO: 357). In some embodiments, the CM comprises the amino acid sequence TARGPSFK (SEQ ID NO: 358). In some embodiments, the CM comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 360). In some embodiments, the CM comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 361). In some embodiments, the CM comprises the amino acid sequence AARGPAIH (SEQ ID NO: 363). In some embodiments, the CM comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 364). In some embodiments, the CM comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 365). In some embodiments, the CM comprises the amino acid sequence RGPATPIM (SEQ ID NO: 366). In some embodiments, the CM comprises the amino acid sequence RGPA (SEQ ID NO: 367). In some embodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ ID NO: 789). In some embodiments, the CM comprises the amino acid sequence SGRSANPRG (SEQ ID NO: 790). In some embodiments, the CM comprises the amino acid sequence LSGRSDDH (SEQ ID NO: 791). In some embodiments, the CM comprises the amino acid sequence LSGRSDIH (SEQ ID NO: 792). In some embodiments, the CM comprises the amino acid sequence LSGRSDQH (SEQ ID NO: 793). In some embodiments, the CM comprises the amino acid sequence LSGRSDTH (SEQ ID NO: 794). In some embodiments, the CM comprises the amino acid sequence LSGRSDYH (SEQ ID NO: 795). In some embodiments, the CM comprises the amino acid sequence LSGRSDNP (SEQ ID NO: 796). In some embodiments, the CM comprises the amino acid sequence LSGRSANP (SEQ ID NO: 797). In some embodiments, the CM comprises the amino acid sequence LSGRSANI (SEQ ID NO: 798). In some embodiments, the CM comprises the amino acid sequence LSGRSDNI (SEQ ID NO: 799). In some embodiments, the CM comprises the amino acid sequence MIAPVAYR (SEQ ID NO: 800). In some embodiments, the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 801). In some embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ ID NO: 802). In some embodiments, the CM comprises the amino acid sequence FRLLDWQW (SEQ ID NO: 803). In some embodiments, the CM comprises the amino acid sequence ISSGL (SEQ ID NO: 804). In some embodiments, the CM comprises the amino acid sequence ISSGLLS (SEQ ID NO: 805). In some embodiments, the CM comprises the amino acid sequence and/or ISSGLL (SEQ ID NO: 806). [000208] In some embodiments, the CM is a substrate for an MMP and includes the sequence ISSGLSS (SEQ ID NO: 376); QNQALRMA (SEQ ID NO: 377); AQNLLGMV (SEQ ID NO: 378); STFPFGMF (SEQ ID NO: 379); PVGYTSSL (SEQ ID NO: 380); DWLYWPGI (SEQ ID NO: 381), ISSGLLSS (SEQ ID NO: 382), LKAAPRWA (SEQ ID NO: 383); GPSHLVLT (SEQ ID NO: 384); LPGGLSPW (SEQ ID NO: 385); MGLFSEAG (SEQ ID NO: 386); SPLPLRVP (SEQ ID NO: 387); RMHLRSLG (SEQ ID NO: 388); LAAPLGLL (SEQ ID NO: 389); AVGLLAPP (SEQ ID NO: 390); LLAPSHRA (SEQ ID NO: 391); PAGLWLDP (SEQ ID NO: 392); ALAHGLF (SEQ ID NO: 424); DLAHPLL (SEQ ID NO: 425); AFRHLR (SEQ ID NO: 426); PHGFFQ (SEQ ID NO: 427); SVHHLI (SEQ ID NO: 428); RGPKLYW (SEQ ID NO: 429); RFPYGVW (SEQ ID NO: 430); HVPRQV (SEQ ID NO: 431); SNPFKY (SEQ ID NO: 432); RFPLKV (SEQ ID NO: 433); PFHLSR (SEQ ID NO: 434); STVFHM (SEQ ID NO: 435); MGPWFM (SEQ ID NO: 436); RHLAKL (SEQ ID NO: 437); PLGVRGK (SEQ ID NO: 438); and/or QNQALRIA (SEQ ID NO: 439). [000209] In some embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ ID NO: 376). In some embodiments, the CM comprises the amino acid sequence QNQALRMA (SEQ ID NO: 377). In some embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ ID NO: 378). In some embodiments, the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 379). In some embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 380). In some embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ ID NO: 381). In some embodiments, the CM comprises the amino acid sequence ISSGLLSS (SEQ ID NO: 382). In some embodiments, the CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 383). In some embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO: 384). In some embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQ ID NO: 385). In some embodiments, the CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 386). In some embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ ID NO: 387). In some embodiments, the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO: 388). In some embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 389). In some embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ ID NO: 390). In some embodiments, the CM comprises the amino acid sequence LLAPSHRA (SEQ ID NO: 391). In some embodiments, the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 392). In some embodiments, the CM comprises the amino acid sequence ALAHGLF (SEQ ID NO: 424). In some embodiments, the CM comprises the amino acid sequence DLAHPLL (SEQ ID NO: 425). In some embodiments, the CM comprises the amino acid sequence AFRHLR (SEQ ID NO: 426). In some embodiments, the CM comprises the amino acid sequence PHGFFQ (SEQ ID NO: 427). In some embodiments, the CM comprises the amino acid sequence SVHHLI (SEQ ID NO: 428). In some embodiments, the CM comprises the amino acid sequence RGPKLYW (SEQ ID NO: 429). In some embodiments, the CM comprises the amino acid sequence RFPYGVW (SEQ ID NO: 430). In some embodiments, the CM comprises the amino acid sequence HVPRQV (SEQ ID NO: 431). In some embodiments, the CM comprises the amino acid sequence SNPFKY (SEQ ID NO: 432). In some embodiments, the CM comprises the amino acid sequence RFPLKV (SEQ ID NO: 433). In some embodiments, the CM comprises the amino acid sequence PFHLSR (SEQ ID NO: 434). In some embodiments, the CM comprises the amino acid sequence STVFHM (SEQ ID NO: 435). In some embodiments, the CM comprises the amino acid sequence MGPWFM (SEQ ID NO: 436). In some embodiments, the CM comprises the amino acid sequence RHLAKL (SEQ ID NO: 437). In some embodiments, the CM comprises the amino acid sequence PLGVRGK (SEQ ID NO: 438). In some embodiments, the CM comprises the amino acid sequence QNQALRIA (SEQ ID NO: 439). [000210] In some embodiments, the CM is a substrate for thrombin. In some embodiments, the CM is a substrate for thrombin and includes the sequence GPRSFGL (SEQ ID NO: 393) or GPRSFG (SEQ ID NO: 394). In some embodiments, the CM comprises the amino acid sequence GPRSFGL (SEQ ID NO: 393). In some embodiments, the CM comprises the amino acid sequence GPRSFG (SEQ ID NO: 394). [000211] In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of NTLSGRSENHSG (SEQ ID NO: 395); NTLSGRSGNHGS (SEQ ID NO: 396); TSTSGRSANPRG (SEQ ID NO: 397); TSGRSANP (SEQ ID NO: 398); VAGRSMRP (SEQ ID NO: 399); VVPEGRRS (SEQ ID NO: 400); ILPRSPAF (SEQ ID NO: 401); MVLGRSLL (SEQ ID NO: 402); QGRAITFI (SEQ ID NO: 403); SPRSIMLA (SEQ ID NO: 404); and SMLRSMPL (SEQ ID NO: 405). [000212] In some embodiments, the CM comprises the amino acid sequence NTLSGRSENHSG (SEQ ID NO: 395). In some embodiments, the CM comprises the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 396). In some embodiments, the CM comprises the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 397). In some embodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ ID NO: 398). In some embodiments, the CM comprises the amino acid sequence VAGRSMRP (SEQ ID NO: 399). In some embodiments, the CM comprises the amino acid sequence VVPEGRRS (SEQ ID NO: 400). In some embodiments, the CM comprises the amino acid sequence ILPRSPAF (SEQ ID NO: 401). In some embodiments, the CM comprises the amino acid sequence MVLGRSLL (SEQ ID NO: 402). In some embodiments, the CM comprises the amino acid sequence QGRAITFI (SEQ ID NO: 403). In some embodiments, the CM comprises the amino acid sequence SPRSIMLA (SEQ ID NO: 404). In some embodiments, the CM comprises the amino acid sequence SMLRSMPL (SEQ ID NO: 405). [000213] In some embodiments, the CM is a substrate for a neutrophil elastase. In some embodiments, the CM is a substrate for a serine protease. In some embodiments, the CM is a substrate for uPA. In some embodiments, the CM is a substrate for legumain. In some embodiments, the CM is a substrate for matriptase. In some embodiments, the CM is a substrate for a cysteine protease. In some embodiments, the CM is a substrate for a cysteine protease, such as a cathepsin. [000214] In some embodiments, the CM is a CM1-CM2 substrate and includes the sequence ISSGLLSGRSDNH (SEQ ID NO: 406); ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 407); AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 408); TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 409); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 410); TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 411); AVGLLAPPGGLSGRSDNH (SEQ ID NO: 412); LSGRSDNHGGAVGLLAPP (SEQ ID NO: 413); VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 414); LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 415); LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 416); LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 417); ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 418); LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 419); QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 420); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 421); QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 422); ISSGLLSGRSGNH (SEQ ID NO: 423); ISSGLLSGRSANPRG (SEQ ID NO: 680); AVGLLAPPTSGRSANPRG (SEQ ID NO: 681); AVGLLAPPSGRSANPRG (SEQ ID NO: 682); ISSGLLSGRSDDH (SEQ ID NO: 683); ISSGLLSGRSDIH (SEQ ID NO: 684); ISSGLLSGRSDQH (SEQ ID NO: 685); ISSGLLSGRSDTH (SEQ ID NO: 686); ISSGLLSGRSDYH (SEQ ID NO: 687); ISSGLLSGRSDNP (SEQ ID NO: 688); ISSGLLSGRSANP (SEQ ID NO: 689); ISSGLLSGRSANI (SEQ ID NO: 690); AVGLLAPPGGLSGRSDDH (SEQ ID NO: 691); AVGLLAPPGGLSGRSDIH (SEQ ID NO: 692); AVGLLAPPGGLSGRSDQH (SEQ ID NO: 693); AVGLLAPPGGLSGRSDTH (SEQ ID NO: 694); AVGLLAPPGGLSGRSDYH (SEQ ID NO: 695); AVGLLAPPGGLSGRSDNP (SEQ ID NO: 696); AVGLLAPPGGLSGRSANP (SEQ ID NO: 697); AVGLLAPPGGLSGRSANI (SEQ ID NO: 698), ISSGLLSGRSDNI (SEQ ID NO: 713); AVGLLAPPGGLSGRSDNI (SEQ ID NO: 714); GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 807); and/or GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 808). [000215] In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNH (SEQ ID NO: 406), which is also referred to herein as substrate 2001. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 407), which is also referred to herein as substrate 1001/LP'/0001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133). In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 408), which is also referred to herein as substrate 2015 and/or substrate 1004/LP'/0003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 409), which is also referred to herein as substrate 0003/LP'/1004, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 410), which is also referred to herein as substrate 1003/ LP'/0003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 411), which is also referred to herein as substrate 0003/LP'/1003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 412), which is also referred to herein as substrate 3001 and/or substrate 1004/LP'/0001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSDNHGGAVGLLAPP (SEQ ID NO: 413), which is also referred to herein as substrate 0001/LP'/1004, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 414), which is also referred to herein as substrate 1003/LP’/0001, wherein LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 415), which is also referred to herein as substrate 0001/ LP'/1003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 416), which is also referred to herein as substrate 0001/LP'/1001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133). In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 417), which is also referred to herein as substrate 0002/LP'/1001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133). In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 418), which is also referred to herein as substrate 1001/LP'/0002, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133). In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 419), which is also referred to herein as substrate 0001/LP'/1002, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133). In some embodiments, the CM1-CM2 substrate includes the sequence QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 420), which is also referred to herein as substrate 1002/LP'/0001, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133). In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 421), which is also referred to herein as substrate 0002/LP'/1002, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133). In some embodiments, the CM1-CM2 substrate includes the sequence QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 422), which is also referred to herein as substrate 1002/LP'/0002, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 133). In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSGNH (SEQ ID NO: 423), which is also referred to herein as substrate 2002. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSANPRG (SEQ ID NO: 680), which is also referred to herein as substrate 2003. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPTSGRSANPRG (SEQ ID NO: 681), which is also referred to herein as substrate 2004. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPSGRSANPRG (SEQ ID NO: 682), which is also referred to herein as substrate 2005. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDDH (SEQ ID NO: 683), which is also referred to herein as substrate 2006. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDIH (SEQ ID NO: 684), which is also referred to herein as substrate 2007. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDQH (SEQ ID NO: 685), which is also referred to herein as substrate 2008. In some embodiments, the CM1- CM2 substrate includes the sequence ISSGLLSGRSDTH (SEQ ID NO: 686), which is also referred to herein as substrate 2009. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDYH (SEQ ID NO: 687), which is also referred to herein as substrate 2010. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNP (SEQ ID NO: 688), which is also referred to herein as substrate 2011. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSANP (SEQ ID NO: 689), which is also referred to herein as substrate 2012. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSANI (SEQ ID NO: 690), which is also referred to herein as substrate 2013. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDDH (SEQ ID NO: 691), which is also referred to herein as substrate 3006. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDIH (SEQ ID NO: 692), which is also referred to herein as substrate 3007. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDQH (SEQ ID NO: 693), which is also referred to herein as substrate 3008. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDTH (SEQ ID NO: 694), which is also referred to herein as substrate 3009. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDYH (SEQ ID NO: 695), which is also referred to herein as substrate 3010. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDNP (SEQ ID NO: 696), which is also referred to herein as substrate 3011. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSANP (SEQ ID NO: 697), which is also referred to herein as substrate 3012. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSANI (SEQ ID NO: 698), which is also referred to herein as substrate 3013. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNI (SEQ ID NO: 713), which is also referred to herein as substrate 2014. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 714), which is also referred to herein as substrate 3014. In some embodiments, the CM1-CM2 substrate includes the sequence GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 807), which is also referred to herein as substrate 0001/LP'/1004, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 808), which is also referred to herein as substrate 0001/LP'/1003, where LP' as used in this CM1-CM2 substrate is the amino acid sequence GG. [000216] In some embodiments, the CM is a CM1-CM2 substrate and includes the sequence LSGRSALAHGLF (SEQ ID NO: 440); ALAHGLFSGRSAN (SEQ ID NO: 441); HVPRQVLSGRS (SEQ ID NO: 442); HVPRQVLSGRSAN (SEQ ID NO: 443); TARGPALAHGLF (SEQ ID NO: 444); TARGPVPRQV (SEQ ID NO: 445); APRSALAHGLF (SEQ ID NO: 446); ALAHGLFAPRSF (SEQ ID NO: 447); HVPRQVAPRSF (SEQ ID NO: 448); ALAHGLPTFVHL (SEQ ID NO: 449); GLPTFVHLPRQV (SEQ ID NO: 450); AANALAHGLF (SEQ ID NO: 451); GPTNALAHGLF (SEQ ID NO: 452); ISSGLLSGRSNI (SEQ ID NO: 453); AVGLLAPPGGLSGRSNI (SEQ ID NO: 454); ISSGLLSGRSNIGS (SEQ ID NO: 455); AVGLLAPPGGLSGRSNIGS (SEQ ID NO: 456); ISSGLLSGRSNIG (SEQ ID NO: 457); AVGLLAPPGGLSGRSNIG (SEQ ID NO: 458). [000217] In some embodiments, the CM is a CM1-CM2 substrate and includes the sequence LSGRSALAHGLF (SEQ ID NO: 440), which is also referred to herein as substrate 5001. In some embodiments, the CM is a CM1-CM2 substrate and includes the sequence ALAHGLFSGRSAN (SEQ ID NO: 441), which is also referred to herein as substrate 5002. In some embodiments, the CM1-CM2 substrate includes the sequence HVPRQVLSGRS (SEQ ID NO: 442), which is also referred to herein as substrate 5003. In some embodiments, the CM1- CM2 substrate includes the sequence HVPRQVLSGRSAN (SEQ ID NO: 443), which is also referred to herein as substrate 5004. In some embodiments, the CM1-CM2 substrate includes the sequence TARGPALAHGLF (SEQ ID NO: 444), which is also referred to herein as substrate 5005. In some embodiments, the CM1-CM2 substrate includes the sequence TARGPVPRQV (SEQ ID NO: 445), which is also referred to herein as substrate 5006. In some embodiments, the CM1-CM2 substrate includes the sequence APRSALAHGLF (SEQ ID NO: 446), which is also referred to herein as substrate 5007. In some embodiments, the CM1-CM2 substrate includes the sequence ALAHGLFAPRSF (SEQ ID NO: 447), which is also referred to herein as substrate 5008. In some embodiments, the CM1-CM2 substrate includes the sequence HVPRQVAPRSF (SEQ ID NO: 448), which is also referred to herein as substrate 5009. In some embodiments, the CM1-CM2 substrate includes the sequence ALAHGLPTFVHL (SEQ ID NO: 449), which is also referred to herein as substrate 5010. In some embodiments, the CM1-CM2 substrate includes the sequence GLPTFVHLPRQV (SEQ ID NO: 450), which is also referred to herein as substrate 5011. In some embodiments, the CM1-CM2 substrate includes the sequence AANALAHGLF (SEQ ID NO: 451), which is also referred to herein as substrate 5012. In some embodiments, the CM1-CM2 substrate includes the sequence GPTNALAHGLF (SEQ ID NO: 452), which is also referred to herein as substrate 5013. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSNI (SEQ ID NO: 453), which is also referred to herein as substrate 5014. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSNI (SEQ ID NO: 454), which is also referred to herein as substrate 5015. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSNIGS (SEQ ID NO: 455), which is also referred to herein as substrate 5016. In some embodiments, the CM1- CM2 substrate includes the sequence AVGLLAPPGGLSGRSNIGS (SEQ ID NO: 456), which is also referred to herein as substrate 5017. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSNIG (SEQ ID NO: 457), which is also referred to herein as substrate 5018. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSNIG (SEQ ID NO: 458), which is also referred to herein as substrate 5019. [000218] In some embodiments, the CM is a substrate for at least two proteases. In some embodiments, each protease is selected from the group consisting of those shown in Table A. In some embodiments, the CM is a substrate for at least two proteases, wherein one of the proteases is selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain and matriptase and the other protease is selected from the group consisting of those shown in Table A. In some embodiments, the CM is a substrate for at least two proteases selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain and matriptase. [000219] In some embodiments, the activatable antibody includes at least a first CM and a second CM. In some embodiments, the first CM and the second CM are each polypeptides of no more than 15 amino acids long. In some embodiments, the first CM and the second CM in the activatable antibody in the uncleaved state have the structural arrangement from N-terminus to C-terminus as follows: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase. In some embodiments, the first CM is cleaved by a first cleaving agent selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase in a target tissue and the second CM is cleaved by a second cleaving agent in a target tissue. In some embodiments, the other protease is selected from the group consisting of those shown in Table A. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase, and the first CM and the second CM are different substrates for the enzyme. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of those shown in Table A. In some embodiments, the first cleaving agent and the second cleaving agent are different proteases. In some embodiments, the first cleaving agent and the second cleaving agent are co-localized in the target tissue. In some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue. [000220] In some embodiments, the activatable antibody is exposed to and cleaved by a protease such that, in the activated or cleaved state, the activated antibody includes a light chain amino acid sequence that includes at least a portion of LP2 and/or CM sequence after the protease has cleaved the CM. [000221] Suitable activatable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. [000222] Suitable activatable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as a CD47 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000223] Suitable activatable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 to a CD47 antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. [000224] Suitable activatable CD47 antibodies of the disclosure also include an antibody or antigen binding fragment thereof that cross-competes for binding to (inhibits the binding of) human CD47 and/or cynomolgus monkey CD47 to a CD47 antibody comprising a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000225] In some embodiments, the activatable CD47 antibody is an activatable antibody that, in an activated state, binds CD47 comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. [000226] In some embodiments, the MM has a dissociation constant for binding to the AB that is greater than the dissociation constant of the AB to CD47. In some embodiments, the MM does not interfere or compete with the AB for binding to CD47 when the activatable antibody is in a cleaved state. In some embodiments, the MM is a polypeptide of no more than 40 amino acids in length. In some embodiments, the MM polypeptide sequence is different from that of human CD47. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-109. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 107-109. [000227] In some embodiments, the CM is a substrate for a protease that is active in diseased tissue. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808. [000228] In some embodiments, the activatable antibody comprises an antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab’)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the AB of the activatable antibody specifically binds human CD47. In some embodiments, the AB comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. [000229] In some embodiments, the AB is linked to the CM. In some embodiments, the AB is linked directly to the CM. In some embodiments, the AB is linked to the CM via a linking peptide. In some embodiments, the MM is linked to the CM such that the activatable antibody in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments, the activatable antibody comprises a linking peptide between the MM and the CM. In some embodiments, the activatable antibody comprises a linking peptide between the CM and the AB. In some embodiments, the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need not be identical to each other. In some embodiments, each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length. [000230] In some embodiments, the activatable antibody comprises the heavy chain sequence selected from the group consisting of SEQ ID NOs: 11-14 and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81-94, and 16-114. [000231] In some embodiments, the activatable antibody comprises a combination of amino acid sequences, wherein the combination of amino acid sequences is selected from a single row in Table B, wherein for a given combination, (a) the heavy chain of the AB comprises the amino acid sequences of the VH CDR sequences corresponding to the given combination in the single row listed in Table B, (b) the light chain of the AB comprises the amino acid sequences of the VL CDR sequences corresponding to the given combination in the single row listed in Table B, (c) the MM comprises the amino acid sequence of the mask sequence (MM) corresponding to the given combination in the single row listed in Table B, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) corresponding to the given combination in the single row listed in Table B. [000232] In some embodiments, the activatable antibody comprises a combination of amino acid sequences, wherein for a given combination of amino acid sequences, (a) the heavy chain of the AB comprises the amino acid sequences of the VH sequence or VH CDR sequences selected from the group consisting of: the VH sequence or VH CDR sequences listed in the corresponding column of Table C, (b) the light chain of the AB comprises the amino acid sequences of the VL sequence or VL CDR sequences selected from the group consisting of: the VL sequence or VL CDR sequences listed in the corresponding column of Table C, (c) the MM comprises the amino acid sequence of the mask sequence (MM) selected from the group consisting of: the MM sequences listed in the corresponding column of Table C, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) selected from the group consisting of: the CM sequences listed in the corresponding column of Table C. [000233] In some embodiments, the activatable CD47 antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, a MM, and a CM, wherein the activatable antibody comprises: a heavy chain sequence of SEQ ID NOs: 11-14; and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81-94, and 16-114. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29, and the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808. In some embodiments, the AB comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. [000234] In some embodiments, the activatable CD47 antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29, and a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29, and the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808. In some embodiments, the AB comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. [000235] In some embodiments, the activatable CD47 antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as an isolated antibody of the disclosure; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. [000236] In some embodiments, the CD47 activatable antibody of the disclosure comprises an isolated antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47. In some embodiments, the antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. [000237] In some embodiments, the activatable CD47 antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically cross-competes with (inhibits the binding of) an isolated antibody of the disclosure for binding to human CD47 and/or cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. [000238] In some embodiments, the CD47 activatable antibody of the disclosure comprises an isolated antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47. In some embodiments, the antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the AB comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. [000239] In some embodiments, the activatable antibody also includes an agent conjugated to the AB. In some embodiments, the agent conjugated to the AB or the AB of an activatable antibody is a therapeutic agent. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or fragment thereof. As used herein, a fragment of a toxin is a fragment that retains toxic activity. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker that includes at least one CM1-CM2 substrate sequence. In some embodiments, the agent is conjugated to the AB via a noncleavable linker. In some embodiments, the agent is conjugated to the AB via a linker that is cleavable in an intracellular or lysosomal environment. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylator, a DNA cleaving agent, a DNA cross-linker, a DNA intercalator, or other DNA damaging agent. In some embodiments, the agent is an agent selected from the group listed in Table E. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or derivative thereof. In some embodiments, the agent is a calicheamicin or derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer. [000240] In some embodiments, the activatable antibody is conjugated to one or more equivalents of an agent. In some embodiments, the activatable antibody is conjugated to one equivalent of the agent. In some embodiments, the activatable antibody is conjugated to two, three, four, five, six, seven, eight, nine, ten, or greater than ten equivalents of the agent. In some embodiments, the activatable antibody is part of a mixture of activatable antibodies having a homogeneous number of equivalents of conjugated agents. In some embodiments, the activatable antibody is part of a mixture of activatable antibodies having a heterogeneous number of equivalents of conjugated agents. In some embodiments, the mixture of activatable antibodies is such that the average number of agents conjugated to each activatable antibody is between zero to one, between one to two, between two and three, between three and four, between four and five, between five and six, between six and seven, between seven and eight, between eight and nine, between nine and ten, and ten and greater. In some embodiments, the mixture of activatable antibodies is such that the average number of agents conjugated to each activatable antibody is one, two, three, four, five, six, seven, eight, nine, ten, or greater. [000241] In some embodiments, the activatable antibody comprises one or more site- specific amino acid sequence modifications such that the number of lysine and/or cysteine residues is increased or decreased with respect to the original amino acid sequence of the activatable antibody, thus in some embodiments correspondingly increasing or decreasing the number of agents that can be conjugated to the activatable antibody, or in some embodiments limiting the conjugation of the agents to the activatable antibody in a site-specific manner. In some embodiments, the modified activatable antibody is modified with one or more non-natural amino acids in a site-specific manner, thus in some embodiments limiting the conjugation of the agents to only the sites of the non-natural amino acids. [000242] In some embodiments, the agent is an anti-inflammatory agent. [000243] In some embodiments, the activatable antibody also includes a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. [000244] In some embodiments, the activatable antibody also includes a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is joined directly to the MM of the activatable antibody. In some embodiments, the spacer is joined directly to the MM of the activatable antibody in the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB. An example of a spacer joined directly to the N-terminus of MM of the activatable antibody is QGQSGQ (SEQ ID NO: 31). Other examples of a spacer joined directly to the N-terminus of MM of the activatable antibody include QGQSGQG (SEQ ID NO: 30), QGQSG (SEQ ID NO: 32), QGQS (SEQ ID NO: 33), QGQ, QG, and Q. Other examples of a spacer joined directly to the N-terminus of MM of the activatable antibody include GQSGQG (SEQ ID NO: 34), QSGQG (SEQ ID NO: 35), SGQG (SEQ ID NO: 36), GQG, and G. In some embodiments, no spacer is joined to the N-terminus of the MM. In some embodiments, the spacer includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 31). In some embodiments, the spacer includes at least the amino acid sequence QGQSGQG (SEQ ID NO: 30). In some embodiments, the spacer includes at least the amino acid sequence QGQSG (SEQ ID NO: 32). In some embodiments, the spacer includes at least the amino acid sequence QGQS (SEQ ID NO: 33). In some embodiments, the spacer includes at least the amino acid sequence QGQ. In some embodiments, the spacer includes at least the amino acid sequence QG. In some embodiments, the spacer includes at least the amino acid residue Q. In some embodiments, the spacer includes at least the amino acid sequence GQSGQG (SEQ ID NO: 34). In some embodiments, the spacer includes at least the amino acid sequence QSGQG (SEQ ID NO: 35). In some embodiments, the spacer includes at least the amino acid sequence SGQG (SEQ ID NO: 36). In some embodiments, the spacer includes at least the amino acid sequence GQG. In some embodiments, the spacer includes at least the amino acid sequence G. In some embodiments, the spacer is absent. [000245] In some embodiments, the AB of the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to include one or more disulfide bonds. [000246] In some embodiments, activatable antibody or antigen binding fragment thereof is conjugated to an agent. In some embodiments, the activatable antibody comprises an antibody or antigen binding fragment thereof cross-competes with (inhibits the binding of) an isolated antibody that comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the activatable antibody comprises an antibody or antigen binding fragment thereof cross-competes with (inhibits the binding of) an isolated antibody that comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent. In some embodiments, the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the agent is maytansinoid DM4. In some embodiments, the agent is duocarmycin. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker with which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety. In some embodiments, the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, vc-duocarmycin, or a PEG2-vc-MMAD moiety. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. [000247] In some embodiments, the conjugated activatable antibody comprises a conjugated activatable antibody that, in an activated state, binds CD47 comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease; and an agent conjugated to the AB. In some embodiments, the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof. In some embodiments, the agent is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a duocarmycin, a pyrrolobenzodiazepine, and a pyrrolobenzodiazepine dimer. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker with which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety. In some embodiments, the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, vc-duocarmycin, or a PEG2-vc- MMAD moiety. In some embodiments, the AB of the conjugated activatable antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). In some embodiments, the AB of the conjugated activatable antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1, and a light chain variable region amino acid sequence of SEQ ID NO: 2. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16- 29. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23, 24, 25, 27, and 28. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808. In some embodiments, the activatable antibody comprises a combination of amino acid sequences, wherein the combination of amino acid sequences is selected from a single row in Table B, wherein for a given combination, (a) the heavy chain of the AB comprises the amino acid sequences of the VH CDR sequences corresponding to the given combination in the single row listed in Table B, (b) the light chain of the AB comprises the amino acid sequences of the VL CDR sequences corresponding to the given combination in the single row listed in Table B, (c) the MM comprises the amino acid sequence of the mask sequence (MM) corresponding to the given combination in the single row listed in Table B, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) corresponding to the given combination in the single row listed in Table B. In some embodiments, the activatable antibody comprises a combination of amino acid sequences, wherein for a given combination of amino acid sequences, (a) the heavy chain of the AB comprises the amino acid sequences of the VH sequence or VH CDR sequences selected from the group consisting of: the VH sequence or VH CDR sequences listed in the corresponding column of Table C, (b) the light chain of the AB comprises the amino acid sequences of the VL sequence or VL CDR sequences selected from the group consisting of: the VL sequence or VL CDR sequences listed in the corresponding column of Table C, (c) the MM comprises the amino acid sequence of the mask sequence (MM) selected from the group consisting of: the MM sequences listed in the corresponding column of Table C, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) selected from the group consisting of: the CM sequences listed in the corresponding column of Table C. In some embodiments, the activatable antibody comprises: a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4; and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81-94, and 16-114. [000248] In some embodiments, the conjugated activatable antibody comprises a conjugated activatable antibody that, in an activated state, binds to CD47, comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds human CD47 and cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease; and an agent conjugated to the AB, wherein the AB comprises: (i) the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10), or (ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2, or (iii) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14, and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81-94, and 16-114; and wherein the agent is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, and a duocarmycin. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23, 24, 25, 27, and 28. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808. In some embodiments, the agent is conjugated to the AB via a linker, and wherein the linker to which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety. In some embodiments, the linker and toxin conjugated to the AB comprises an SPDB- DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, vc-duocarmycin, or a PEG2-vc-MMAD moiety. [000249] In some embodiments, the conjugated activatable antibody comprises a conjugated activatable antibody comprising: an antibody or antigen binding fragment thereof (AB) that, in an activated state, binds CD47; and a toxin conjugated to the AB via a linker, wherein the conjugated activatable antibody comprises amino acid sequences, a linker, and a toxin selected from a single row in Table D, wherein for the given combination: (a) the AB comprises a heavy chain comprising the amino acid sequence of the heavy chain sequence or heavy chain variable domain sequence corresponding to the given combination in the single row listed in Table D, (b) the AB comprises a light chain comprising the amino acid sequence of the light chain sequence or light chain variable domain sequence corresponding to the given combination in the single row listed in Table D, and (c) the linker and the toxin comprise the linker and the toxin corresponding to the given combination in the single row listed in Table D. [000250] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain variable region amino acid sequence of SEQ ID NO: 1. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain variable region amino acid sequence of SEQ ID NO: 2. [000251] In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a heavy chain variable region amino acid sequence of SEQ ID NO: 1. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding a light variable region chain amino acid of SEQ ID NO: 2. [000252] The disclosure also provides methods for producing an activatable antibody of the disclosure by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises a nucleic acid molecule of the disclosure or a vector of the disclosure. [000253] The disclosure also provides methods of manufacturing an activatable antibody that, in an activated state, binds CD47, the method comprising: (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises an activatable antibody of the disclosure; and (b) recovering the activatable antibody. [000254] In some embodiments, the activatable antibody includes one or more polypeptides that include the combination of sequences in a given row of Table B or any combination of a mask sequence (MM), a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequences, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequences of Table C. Table B: CD47 Activatable Antibody Combinations
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Table C: CD47 Activatable Antibody Components
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
[000255] In some embodiments, an activatable antibody of the present disclosure includes one or more polypeptides that include the combination of sequences selected from Table B or Table C, where the polypeptide includes a combination of a masking sequence selected from the column titled “Mask Sequence (MM)” of Table B or Table C, a substrate sequence from the column titled “Substrate Sequence (CM)” of Table B or Table C, a light chain variable domain or light chain CDRs from the column titled “VL or VL CDRs” or “VL CDRs SEQ ID NOs” of Table B or Table C, and a heavy chain variable domain or heavy chain CDRs from the column titled “VH or VH CDRs” or “VH CDRs SEQ ID Nos” of Table B or Table C. For example, an activatable antibody of the present disclosure may include the amino acid sequences of combination no.147, which includes the masking sequence of SEQ ID NO: 17, the substrate sequence of SEQ ID NO: 412, a light chain variable domain that includes the VL CDR sequences of SEQ ID NOs: 15, 16, and 18, and a heavy chain variable domain that includes the VH CDR sequences of 11, 12, and 13. Therefore, an activatable antibody that includes at least the combination of sequences in any given row of Table B is described herein. Similarly, any combination of a mask sequence (MM), a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequences, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequences of Table C is described herein. An activatable antibody that includes at least any combination of a masking sequence, a substrate sequence, a variable heavy chain or variable heavy chain CDRs, and a variable light chain or variable light chain CDRs selected from the corresponding columns Table B or Table C is also described herein. In some exemplary embodiments, an activatable antibody that includes at least the combination of sequences in any given row of Table B or any combination of a mask sequence (MM), a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequences, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequences of Table C can be combined with one or more toxins, including a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, or a pyrrolobenzodiazepine or a derivative thereof. In some exemplary embodiments, an activatable antibody that includes at least the combination of sequences in any given row of Table B or any combination of a mask sequence (MM), a substrate sequence (CM), a light chain variable domain sequence or light chain variable domain CDR sequences, and a heavy chain variable domain sequence or heavy chain variable domain CDR sequences of Table C can be combined with one or more toxins, including auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, and/or a duocarmycin. [000256] Any of the combinations in Table B or Table C as described above can be combined with human immunoglobulin constant regions to result in fully human IgGs including IgG1, IgG2, IgG4 or mutated constant regions to result in human IgGs with altered functions such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P. The combinations described in Table B or Table C are not limited by the particular combinations shown in any given row, and thus may include any mask sequence from column 2 of Table B (or column 1 of Table C) combined with any substrate sequence from column 3 of Table B (or column 2 of Table C) combined with any VL sequence or set of VL CDR sequences from column 4 of Table B (or column 3 or Table C) combined with any VH sequence or set of VH CDR sequences from column 5 of Table B (or column 4 of Table C). In addition to the mask sequences disclosed in column 2 of Table B or column 1 of Table C, any mask sequence disclosed herein can be used in a combination. In addition to the substrate sequences disclosed in column 3 of Table B or column 2 of Table C, any CM disclosed herein can be used in a combination. In addition to the light chain variable region sequence or light chain CDR sequences disclosed in column 4 of Table B or column 3 of Table C, any light chain variable region sequence or light chain CDR sequences disclosed herein can be used in a combination. In addition to the heavy chain variable region sequence or heavy chain CDR sequences disclosed in column 5 of Table B or column 4 of Table C, any heavy chain variable region sequence or heavy chain CDR sequences disclosed herein can be used in a combination. [000257] In some embodiments, the serum half-life of the activatable antibody is longer than that of the corresponding antibody, e.g., the pK of the activatable antibody is longer than that of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to that of the corresponding antibody. In some embodiments, the serum half- life of the activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism. [000258] The disclosure also provides methods of producing an activatable CD47 antibody polypeptide by culturing a cell under conditions that lead to expression of the polypeptide, wherein the cell comprises an isolated nucleic acid molecule encoding an antibody and/or an activatable antibody described herein, and/or vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing an antibody and/or activatable antibody by culturing a cell under conditions that lead to expression of the antibody and/or activatable antibody, wherein the cell comprises an isolated nucleic acid molecule encoding an antibody and/or an activatable antibody described herein, and/or vectors that include these isolated nucleic acid sequences. [000259] The invention also provides a method of manufacturing activatable antibodies that in an activated state binds CD47 by (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or an antigen binding fragment thereof (AB) that specifically binds CD47, (i) wherein the CM is a polypeptide that functions as a substrate for a protease; and (ii) wherein the CM is positioned in the activatable antibody such that, when the activatable antibody is in an uncleaved state, the MM interferes with specific binding of the AB to CD47 and in a cleaved state the MM does not interfere or compete with specific binding of the AB to CD47; and (b) recovering the activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein. [000260] In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM- MM. In some embodiments, the activatable antibody comprises a linking peptide between the MM and the CM. In some embodiments, the activatable antibody comprises a linking peptide between the CM and the AB. In some embodiments, the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need not be identical to each other. In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer. [000261] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 116) and (GGGS)n (SEQ ID NO: 117), where n is an integer of at least one. [000262] In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 118), GGSGG (SEQ ID NO: 119), GSGSG (SEQ ID NO: 120), GSGGG (SEQ ID NO: 121), GGGSG (SEQ ID NO: 122), and GSSSG (SEQ ID NO: 123). [000263] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 124), GSSGGSGGSGG (SEQ ID NO: 125), GSSGGSGGSGGS (SEQ ID NO: 126), GSSGGSGGSGGSGGGS (SEQ ID NO: 127), GSSGGSGGSG (SEQ ID NO: 128), GSSGGSGGSGS (SEQ ID NO: 129), or GGGSSGGS (SEQ ID NO: 134). [000264] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 130), GSSGT (SEQ ID NO: 131) or GSSG (SEQ ID NO: 132). [000265] In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer, where LP1 comprises the amino acid sequence GGGSSGGS (SEQ ID NO: 134) and the LP2 comprises the amino acid sequence GGS. [000266] In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer, where LP1 comprises the amino acid sequence GGGSSGGS (SEQ ID NO: 134) and the LP2 comprises the amino acid sequence GGGS (SEQ ID NO: 130). Conjugated CD47 Antibodies and Activatable Antibodies [000267] In some embodiments, the CD47 antibodies and activatable antibodies described herein also include an agent conjugated to the antibody/activatable antibody. In some embodiments, the conjugated agent is a therapeutic agent, such as an anti-inflammatory and/or an antineoplastic agent. In such embodiments, the agent is conjugated to a carbohydrate moiety of the antibody/activatable antibody, for example, in some embodiments, where the carbohydrate moiety is located outside the antigen-binding region of the antibody or antigen-binding fragment in the activatable antibody. In some embodiments, the agent is conjugated to a sulfhydryl group of the antibody or antigen-binding fragment in the antibody/activatable antibody. [000268] In some embodiments, the agent is a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). [000269] In some embodiments, the agent 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 embodiments, detectable moieties are attached by spacer molecules. [000270] The disclosure also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Suitable cytotoxic agents include, for example, dolastatins and derivatives thereof (e.g. auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is monomethyl auristatin E (MMAE) or monomethyl auristatin D (MMAD). In some embodiments, the agent is an agent selected from the group listed in Table E. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or derivative thereof. In some embodiments, the agent is a calicheamicin or derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer. [000271] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. [000272] In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent. In some embodiments, the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the agent is maytansinoid DM4. In some embodiments, the agent is duocarmycin. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker with which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety. In some embodiments, the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, vc-duocarmycin, or a PEG2-vc-MMAD moiety. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. [000273] In some embodiments, the conjugated activatable antibody comprises an antibody comprising: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB comprises: (i) the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10), or (ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence selected of SEQ ID NO: 2; (b) an agent conjugated to the AB, wherein the agent is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, and a duocarmycin. [000274] The CD47 antibodies and activatable antibodies of the disclosure have at least one point of conjugation for an agent, but in the methods and compositions provided herein less than all possible points of conjugation are available for conjugation to an agent. In some embodiments, the one or more points of conjugation are sulfur atoms involved in disulfide bonds. In some embodiments, the one or more points of conjugation are sulfur atoms involved in interchain disulfide bonds. In some embodiments, the one or more points of conjugation are sulfur atoms involved in interchain sulfide bonds, but not sulfur atoms involved in intrachain disulfide bonds. In some embodiments, the one or more points of conjugation are sulfur atoms of cysteine or other amino acid residues containing a sulfur atom. Such residues may occur naturally in the antibody structure or can be incorporated into the antibody by site-directed mutagenesis, chemical conversion, or mis-incorporation of non-natural amino acids. [000275] Also provided are methods of preparing a conjugate of a CD47 antibody/CD47 activatable antibody having one or more interchain disulfide bonds in the AB and one or more intrachain disulfide bonds in the MM, and a drug reactive with free thiols is provided. The method generally includes partially reducing interchain disulfide bonds in the activatable antibody with a reducing agent, such as, for example, TCEP; and conjugating the drug reactive with free thiols to the partially reduced antibody/activatable antibody. As used herein, the term partial reduction refers to situations where an act antibody/activatable antibody is contacted with a reducing agent and less than all disulfide bonds, e.g., less than all possible sites of conjugation are reduced. In some embodiments, less than 99%, 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. [000276] In yet other embodiments, a method of reducing and conjugating an agent, e.g., a drug, to a CD47 antibody/CD47 activatable antibody resulting in selectivity in the placement of the agent is provided. The method generally includes partially reducing the CD47 antibody/CD47 activatable antibody with a reducing agent such that any conjugation sites in the masking moiety or other non-AB portion of the CD47 antibody/CD47 activatable antibody are not reduced, and conjugating the agent to interchain thiols in the AB. The conjugation site(s) are selected so as to allow desired placement of an agent to allow conjugation to occur at a desired site. The reducing agent is, for example, TCEP. The reduction reaction conditions such as, for example, the ratio of reducing agent to antibody/activatable antibody, the length of incubation, the temperature during the incubation, the pH of the reducing reaction solution, etc., are determined by identifying the conditions that produce a conjugated antibody/activatable antibody e.g. under conditions that produce a conjugated activatable antibody in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in an uncleaved state. The ratio of reduction agent to antibody/activatable antibody will vary depending on the antibody/activatable antibody. In some embodiments, the ratio of reducing agent to antibody/activatable antibody 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 about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments, the ratio is in a range of from about 5:1 to 1:1. In some embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In some embodiments, the ratio is in a range of from about 4:1 to 1:1. In some embodiments, the ratio is in a range from about 4:1 to 1.5:1. In some embodiments, the ratio is in a range from about 8:1 to about 1:1. In some embodiments, the ratio is in a range of from about 2.5:1 to 1:1. [000277] In some embodiments, the CD47 antibody undergoes conjugation first and is then further modified to include a CM and MM (resulting in an activatable antibody). In some embodiments, the CD47 activatable antibody is conjugated. [000278] In some embodiments, a method of reducing interchain disulfide bonds in the AB of an activatable CD47 antibody and conjugating an agent, e.g., a thiol-containing agent such as a drug, to the resulting interchain thiols to selectively locate agent(s) on the AB is provided. The method generally includes partially reducing the AB with a reducing agent to form at least two interchain thiols without forming all possible interchain thiols in the activatable antibody; and conjugating the agent to the interchain thiols of the partially reduced AB. For example, the AB of the activatable antibody is partially reduced for about 1 hour at about 37°C at a desired ratio of reducing agent:activatable antibody. In some embodiments, the ratio of reducing agent to activatable antibody 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 about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments, the ratio is in a range of from about 5:1 to 1:1. In some embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In some embodiments, the ratio is in a range of from about 4:1 to 1:1. In some embodiments, the ratio is in a range from about 4:1 to 1.5:1. In some embodiments, the ratio is in a range from about 8:1 to about 1:1. In some embodiments, the ratio is in a range of from about 2.5:1 to 1:1. [000279] The thiol-containing reagent can be, for example, cysteine or N-acetyl cysteine. The reducing agent can be, for example, TCEP. In some embodiments, the reduced activatable antibody can be purified prior to conjugation, using for example, column chromatography, dialysis, or diafiltration. Alternatively, the reduced antibody is not purified after partial reduction and prior to conjugation. [000280] The invention also provides partially reduced antibodies/activatable antibodies in which at least one interchain disulfide bond in the antibody/activatable antibody has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the antibody/activatable antibody, wherein the activatable antibody includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to CD47, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the CD47 target, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments the MM is coupled to the AB via the CM. In some embodiments, one or more intrachain disulfide bond(s) of the antibody/activatable antibody is not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bond(s) of the MM within the antibody/activatable antibody is not disturbed by the reducing agent. In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP. [000281] In yet other embodiments, a method of reducing and conjugating an agent, e.g., a drug, to a CD47 antibody/CD47 activatable antibody resulting in selectivity in the placement of the agent by providing an activatable CD47 antibody with a defined number and positions of lysine and/or cysteine residues. In some embodiments, the defined number of lysine and/or cysteine residues is higher or lower than the number of corresponding residues in the amino acid sequence of the parent antibody or activatable antibody. In some embodiments, the defined number of lysine and/or cysteine residues may result in a defined number of agent equivalents that can be conjugated to the CD47 antibody or activatable CD47 antibody. In some embodiments, the defined number of lysine and/or cysteine residues may result in a defined number of agent equivalents that can be conjugated to the CD47 antibody or activatable CD47 antibody in a site-specific manner. In some embodiments, the modified activatable antibody is modified with one or more non-natural amino acids in a site-specific manner, thus in some embodiments limiting the conjugation of the agents to only the sites of the non-natural amino acids. In some embodiments, the CD47 antibody or activatable CD47 antibody with a defined number and positions of lysine and/or cysteine residues can be partially reduced with a reducing agent as discussed herein such that any conjugation sites in the masking moiety or other non-AB portion of the activatable antibody are not reduced, and conjugating the agent to interchain thiols in the AB. [000282] The disclosure also provides partially reduced activatable antibodies in which at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the activatable antibody includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to the target, e.g., CD47, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the target, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for at least one protease. In some embodiments, the MM is coupled to the AB via the CM. In some embodiments, one or more intrachain disulfide bond(s) of the activatable antibody is not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bond(s) of the MM within the activatable antibody is not disturbed by the reducing agent. In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP. [000283] In some embodiments, the agent is linked to the AB using a maleimide caproyl- valine-citrulline linker or a maleimide PEG-valine-citrulline linker. In some embodiments, the agent is linked to the AB using a maleimide caproyl-valine-citrulline linker. In some embodiments, the agent is linked to the AB using a maleimide PEG-valine-citrulline linker In some embodiments, the agent is monomethyl auristatin D (MMAD) linked to the AB using a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as “vc-MMAD.” In some embodiments, the agent is monomethyl auristatin E (MMAE) linked to the AB using a maleimide PEG-valine-citrulline-para- aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as “vc- MMAE.” In some embodiments, the agent is linked to the AB using a maleimide PEG-valine- citrulline linker In some embodiments, the agent is monomethyl auristatin D (MMAD) linked to the AB using a maleimide bis-PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and this linker payload construct is referred to herein as “PEG2-vc-MMAD.” The structures of vc- MMAD, vc-MMAE, and PEG2-vc-MMAD are shown below: vc-MMAD:
Figure imgf000112_0001
vc-MMAE:
Figure imgf000113_0001
PEG2-vc-MMAD:
Figure imgf000113_0002
[000284] The disclosure also provides conjugated activatable antibodies that include an activatable antibody linked to monomethyl auristatin D (MMAD) payload, wherein the activatable antibody includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the target, and cleavable moiety (CM) coupled to the AB, and the CM is a polypeptide that functions as a substrate for at least one MMP protease. [000285] In some embodiments, the MMAD-conjugated activatable antibody can be conjugated using any of several methods for attaching agents to ABs: (a) attachment to the carbohydrate moieties of the AB, or (b) attachment to sulfhydryl groups of the AB, or (c) attachment to amino groups of the AB, or (d) attachment to carboxylate groups of the AB. [000286] In some embodiments, the MMAD payload is conjugated to the AB via a linker. In some embodiments, the MMAD payload is conjugated to a cysteine in the AB via a linker. In some embodiments, the MMAD payload is conjugated to a lysine in the AB via a linker. In some embodiments, the MMAD payload is conjugated to another residue of the AB via a linker, such as those residues disclosed herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non- cleavable linker. In some embodiments, the linker is selected from the group consisting of the linkers shown in Tables 7 and 8. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide caproyl-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide PEG-valine-citrulline linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide caproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the activatable antibody and the MMAD payload are linked via a maleimide PEG-valine- citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the MMAD payload is conjugated to the AB using the partial reduction and conjugation technology disclosed herein. [000287] In some embodiments, the polyethylene glycol (PEG) component of a linker of the present disclosure is formed from 2 ethylene glycol monomers, 3 ethylene glycol monomers, 4 ethylene glycol monomers, 5 ethylene glycol monomers, 6 ethylene glycol monomers, 7 ethylene glycol monomers 8 ethylene glycol monomers, 9 ethylene glycol monomers, or at least 10 ethylene glycol monomers. In some embodiments of the present disclosure, the PEG component is a branched polymer. In some embodiments of the present disclosure, the PEG component is an unbranched polymer. In some embodiments, the PEG polymer component is functionalized with an amino group or derivative thereof, a carboxyl group or derivative thereof, or both an amino group or derivative thereof and a carboxyl group or derivative thereof. [000288] In some embodiments, the PEG component of a linker of the present disclosure is an amino-tetra-ethylene glycol-carboxyl group or derivative thereof. In some embodiments, the PEG component of a linker of the present disclosure is an amino-tri-ethylene glycol-carboxyl group or derivative thereof. In some embodiments, the PEG component of a linker of the present disclosure is an amino-di-ethylene glycol-carboxyl group or derivative thereof. In some embodiments, an amino derivative is the formation of an amide bond between the amino group and a carboxyl group to which it is conjugated. In some embodiments, a carboxyl derivative is the formation of an amide bond between the carboxyl group and an amino group to which it is conjugated. In some embodiments, a carboxyl derivative is the formation of an ester bond between the carboxyl group and an hydroxyl group to which it is conjugated. [000289] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94/11026). [000290] Table E lists some of the exemplary pharmaceutical agents that can be employed in the herein described disclosure but in no way is meant to be an exhaustive list. Table E: Exemplary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTS Auristatins Turbostatin Auristatin E Phenstatins Monomethyl auristatin D (MMAD) Hydroxyphenstatin Monomethyl auristatin E (MMAE) Spongistatin 5 Desmethyl auristatin E (DMAE) Spongistatin 7 Auristatin F Halistatin 1 Monomethyl auristatin F (MMAF) Halistatin 2 Desmethyl auristatin F (DMAF) Halistatin 3 Auristatin derivatives, e.g., amides thereof Modified Bryostatins Auristatin tyramine Halocomstatins Auristatin quinoline Pyrrolobenzimidazoles (PBI) Dolastatins Cibrostatin6 Dolastatin derivatives Doxaliform Dolastatin 16 DmJ Anthracyclins analogues Dolastatin 16 Dpv Maytansinoids, e.g. DM-1; DM-4 Maytansinoid derivatives Cemadotin analogue (CemCH2-SH) Duocarmycin Pseudomonas toxin A (PE38) variant Duocarmycin derivatives Pseudomonas toxin A (ZZ-PE38) variant Alpha-amanitin ZJ-16 Anthracyclines OSW-1 Doxorubicin 4-Nitrobenzyloxycarbonyl Derivatives of O6-Benzylguanine Daunorubicin Topoisomerase inhibitors Bryostatins Hemiasterlin Camptothecin Cephalotaxine Camptothecin derivatives Homoharringtonine 7-substituted Camptothecin Pyrrolobenzodiazepine dimers (PBDs) 10, 11- Pyrrolobenzodiazepenes Difluoromethylenedioxycamptothecin Combretastatins Functionalized pyrrolobenzodiazepenes Debromoaplysiatoxin Functionalized pyrrolobenzodiazepene dimers Kahalalide-F Calicheamicins Discodermolide Podophyllotoxins Ecteinascidins Taxanes Vinca alkaloids ANTIVIRALS CONJUGATABLE DETECTION Acyclovir REAGENTS Vira A Fluorescein and derivatives thereof Symmetrel Fluorescein isothiocyanate (FITC) ANTIFUNGALS RADIOPHARMACEUTICALS Nystatin 125I 131I ADDITIONAL ANTI-NEOPLASTICS 89Zr Adriamycin 111In Cerubidine 123I Bleomycin 131I Alkeran 99mTc Velban 201Tl Oncovin 133Xe Fluorouracil 11C Methotrexate 62Cu Thiotepa 18F Bisantrene 68Ga Novantrone 13N Thioguanine 15O Procarabizine 38K Cytarabine 82Rb 99mTc (Technetium) ANTI-BACTERIALS Aminoglycosides HEAVY METALS Streptomycin Barium Neomycin Gold Kanamycin Platinum Amikacin Gentamicin ANTI-MYCOPLASMALS Tobramycin Tylosine Streptomycin B Spectinomycin Spectinomycin Ampicillin Sulfanilamide Polymyxin Chloramphenicol [000291] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies 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). [000292] Coupling can be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. In some embodiments, the binding is, however, 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 coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun.133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987). [000293] In some embodiments, in addition to the compositions and methods provided herein, the activatable antibody can also be modified for site-specific conjugation through modified amino acid sequences inserted or otherwise included in the activatable antibody sequence. These modified amino acid sequences are designed to allow for controlled placement and/or dosage of the conjugated agent within a conjugated activatable antibody. For example, the activatable antibody can be engineered to include cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not negatively impact protein folding and assembly, nor alter antigen binding. In some embodiments, the activatable antibody can be engineered to include or otherwise introduce one or more non-natural amino acid residues within the activatable antibody to provide suitable sites for conjugation. In some embodiments, the activatable antibody can be engineered to include or otherwise introduce enzymatically activatable peptide sequences within the activatable antibody sequence. [000294] Suitable linkers 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 antibody by way of an oligopeptide linker. In some embodiments, 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 #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC. Additional linkers include, but are not limited to, SMCC ((succinimidyl 4- (N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC (sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate), SPDB (N-succinimidyl-4-(2-pyridyldithio) butanoate), or sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfo butanoate). [000295] The linkers 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. [000296] In some embodiments, the linkers are cleavable. In some embodiments, the linkers are non-cleavable. In some embodiments, two or more linkers are present. The two or more linkers are all the same, i.e., cleavable or non-cleavable, or the two or more linkers are different, i.e., at least one cleavable and at least one non-cleavable. [000297] The present disclosure utilizes several methods for attaching agents to ABs: (a) attachment to the carbohydrate moieties of the AB, or (b) attachment to sulfhydryl groups of the AB, or (c) attachment to amino groups of the AB, or (d) attachment to carboxylate groups of the AB. According to the disclosure, ABs can be covalently attached to an agent through an intermediate linker having at least two reactive groups, one to react with AB and one to react with the agent. The linker, which may include any compatible organic compound, can be chosen such that the reaction with AB (or agent) does not adversely affect AB reactivity and selectivity. Furthermore, the attachment of linker to agent might not destroy the activity of the agent. Suitable linkers for reaction with oxidized antibodies or oxidized antibody fragments include those containing an amine selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups. Such reactive functional groups may exist as part of the structure of the linker, or can be introduced by suitable chemical modification of linkers not containing such groups. [000298] According to the present disclosure, suitable linkers for attachment to reduced ABs include those having certain reactive groups capable of reaction with a sulfhydryl group of a reduced antibody or fragment. Such reactive groups include, but are not limited to: reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercuribenzoate groups and groups capable of Michael-type addition reactions (including, for example, maleimides and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc.16: 3097-3110). [000299] According to the present disclosure, suitable linkers for attachment to neither oxidized nor reduced Abs include those having certain functional groups capable of reaction with the primary amino groups present in unmodified lysine residues in the Ab. Such reactive groups include, but are not limited to, NHS carboxylic or carbonic esters, sulfo-NHS carboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonic esters, pentafluorophenyl carboxylic or carbonic esters, acyl imidazoles, isocyanates, and isothiocyanates. [000300] According to the present disclosure, suitable linkers for attachment to neither oxidized nor reduced Abs include those having certain functional groups capable of reaction with the carboxylic acid groups present in aspartate or glutamate residues in the Ab, which have been activated with suitable reagents. Suitable activating reagents include EDC, with or without added NHS or sulfo-NHS, and other dehydrating agents utilized for carboxamide formation. In these instances, the functional groups present in the suitable linkers would include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides. [000301] The agent can be attached to the linker before or after the linker is attached to the AB. In certain applications it may be desirable to first produce an AB-linker intermediate in which the linker is free of an associated agent. Depending upon the particular application, a specific agent may then be covalently attached to the linker. In some embodiments, the AB is first attached to the MM, CM and associated linkers and then attached to the linker for conjugation purposes. [000302] Branched Linkers: In specific embodiments, branched linkers that have multiple sites for attachment of agents are utilized. For multiple site linkers, a single covalent attachment to an AB would result in an AB-linker intermediate capable of binding an agent at a number of sites. The sites can be aldehyde or sulfhydryl groups or any chemical site to which agents can be attached. [000303] In some embodiments, higher specific activity (or higher ratio of agents to AB) can be achieved by attachment of a single site linker at a plurality of sites on the AB. This plurality of sites can be introduced into the AB by either of two methods. First, one may generate multiple aldehyde groups and/or sulfhydryl groups in the same AB. Second, one may attach to an aldehyde or sulfhydryl of the AB a "branched linker" having multiple functional sites for subsequent attachment to linkers. The functional sites of the branched linker or multiple site linker can be aldehyde or sulfhydryl groups, or can be any chemical site to which linkers can be attached. Still higher specific activities can be obtained by combining these two approaches, that is, attaching multiple site linkers at several sites on the AB. [000304] Cleavable Linkers: Peptide linkers that are susceptible to cleavage by enzymes of the complement system, such as but not limited to u-plasminogen activator, tissue plasminogen activator, trypsin, plasmin, or another enzyme having proteolytic activity can be used in one embodiment of the present disclosure. According to one method of the present disclosure, an agent is attached via a linker susceptible to cleavage by complement. The antibody is selected from a class that can activate complement. The antibody-agent conjugate, thus, activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, an agent is attached via a linker susceptible to cleavage by enzymes having a proteolytic activity such as a u-plasminogen activator, a tissue plasminogen activator, plasmin, or trypsin. These cleavable linkers are useful in conjugated activatable antibodies that include an extracellular toxin, e.g., by way of non-limiting example, any of the extracellular toxins shown in Table E. [000305] Non-limiting examples of cleavable linker sequences are provided in Table F. Table F: Exemplary Linker Sequences for Conjugation Types of Cleavable Sequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinase PRFKIIGG (SEQ ID NO: 615) PRFRIIGG (SEQ ID NO: 616) TGFb SSRHRRALD (SEQ ID NO: 617) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 618) Staphylokinase SSSFDKGKYKKGDDA (SEQ ID NO: 619) SSSFDKGKYKRGDDA (SEQ ID NO: 620) Factor Xa cleavable sequences IEGR (SEQ ID NO: 621) IDGR (SEQ ID NO: 622) GGSIDGR (SEQ ID NO: 623) MMP cleavable sequences Gelatinase A PLGLWA (SEQ ID NO: 624) Collagenase cleavable sequences Calf skin collagen (a1(I) chain) GPQGIAGQ (SEQ ID NO: 625) Calf skin collagen (a2(I) chain) GPQGLLGA (SEQ ID NO: 626) Bovine cartilage collagen (a1(II) chain) GIAGQ (SEQ ID NO: 627) Human liver collagen (a1(III) chain) GPLGIAGI (SEQ ID NO: 628) Human a2M GPEGLRVG (SEQ ID NO: 629) Human PZP YGAGLGVV (SEQ ID NO: 630) AGLGVVER (SEQ ID NO: 631) AGLGISST (SEQ ID NO: 632) Rat a1M EPQALAMS (SEQ ID NO: 633) QALAMSAI (SEQ ID NO: 634) Rat a2M AAYHLVSQ (SEQ ID NO: 635) MDAFLESS (SEQ ID NO: 636) Rat a1I3(2J) ESLPVVAV (SEQ ID NO: 637) Rat a1I3(27J) SAPAVESE (SEQ ID NO: 638) Human fibroblast collagenase DVAQFVLT (SEQ ID NO: 639) (autolytic cleavages) VAQFVLTE (SEQ ID NO: 640) AQFVLTEG (SEQ ID NO: 641) PVQPIGPQ (SEQ ID NO: 642) [000306] In addition, agents can be attached via disulfide bonds (for example, the disulfide bonds on a cysteine molecule) to the AB. Since many tumors naturally release high levels of glutathione (a reducing agent) this can reduce the disulfide bonds with subsequent release of the agent at the site of delivery. In some embodiments, the reducing agent that would modify a CM would also modify the linker of the conjugated activatable antibody. [000307] Spacers and Cleavable Elements: In some embodiments, it may be necessary to construct the linker in such a way as to optimize the spacing between the agent and the AB of the activatable antibody. This can be accomplished by use of a linker of the general structure: W – (CH2)n – Q wherein W is either --NH--CH2-- or --CH2--; Q is an amino acid, peptide; and n is an integer from 0 to 20. [000308] In some embodiments, the linker may comprise a spacer element and a cleavable element. The spacer element serves to position the cleavable element away from the core of the AB such that the cleavable element is more accessible to the enzyme responsible for cleavage. Certain of the branched linkers described above may serve as spacer elements. [000309] Throughout this discussion, it should be understood that the attachment of linker to agent (or of spacer element to cleavable element, or cleavable element to agent) need not be particular mode of attachment or reaction. Any reaction providing a product of suitable stability and biological compatibility is acceptable. [000310] Serum Complement and Selection of Linkers: According to one method of the present disclosure, when release of an agent is desired, an AB that is an antibody of a class that can activate complement is used. The resulting conjugate retains both the ability to bind antigen and activate the complement cascade. Thus, according to this embodiment of the present disclosure, an agent is joined to one end of the cleavable linker or cleavable element and the other end of the linker group is attached to a specific site on the AB. For example, if the agent has an hydroxy group or an amino group, it can be attached to the carboxy terminus of a peptide, amino acid or other suitably chosen linker via an ester or amide bond, respectively. For example, such agents can be attached to the linker peptide via a carbodimide reaction. If the agent contains functional groups that would interfere with attachment to the linker, these interfering functional groups can be blocked before attachment and deblocked once the product conjugate or intermediate is made. The opposite or amino terminus of the linker is then used either directly or after further modification for binding to an AB that is capable of activating complement. [000311] Linkers (or spacer elements of linkers) can be of any desired length, one end of which can be covalently attached to specific sites on the AB of the activatable antibody. The other end of the linker or spacer element can be attached to an amino acid or peptide linker. [000312] Thus, when these conjugates bind to antigen in the presence of complement the amide or ester bond that attaches the agent to the linker will be cleaved, resulting in release of the agent in its active form. These conjugates, when administered to a subject, will accomplish delivery and release of the agent at the target site, and are particularly effective for the in vivo delivery of pharmaceutical agents, antibiotics, antimetabolites, antiproliferative agents and the like as presented in but not limited to those in Table E. [000313] Linkers for Release without Complement Activation: In yet another application of targeted delivery, release of the agent without complement activation is desired since activation of the complement cascade will ultimately lyse the target cell. Hence, this approach is useful when delivery and release of the agent should be accomplished without killing the target cell. Such is the goal when delivery of cell mediators such as hormones, enzymes, corticosteroids, neurotransmitters, genes or enzymes to target cells is desired. These conjugates can be prepared by attaching the agent to an AB that is not capable of activating complement via a linker that is mildly susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, antigen-antibody complexes will form quickly whereas cleavage of the agent will occur slowly, thus resulting in release of the compound at the target site. [000314] Biochemical Cross Linkers: In some embodiments, the activatable antibody can be conjugated to one or more therapeutic agents using certain biochemical cross-linkers. Cross- linking reagents form molecular bridges that tie together functional groups of two different molecules. To link two different proteins in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation. [000315] Peptidyl linkers cleavable by lysosomal proteases are also useful, for example, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labile linkers cleavable in the low-pH environment of the lysosome can be used, for example: bis-sialyl ether. Other suitable linkers include cathepsin-labile substrates, particularly those that show optimal function at an acidic pH. [000316] Exemplary hetero-bifunctional cross-linkers are referenced in Table G. Table G: Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length after cross-linking Linker Reactive Toward Advantages and Applications (Angstroms) SMPT Primary amines Greater stability 11.2 Å Sulfhydryls SPDP Primary amines Thiolation 6.8 Å Sulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extended spacer arm 15.6 Å Sulfhydryls Sulfo-LC-SPDP Primary amines Extender spacer arm 15.6 Å Sulfhydryls Water-soluble SMCC Primary amines Stable maleimide reactive 11.6 Å group Sulfhydryls Enzyme-antibody conjugation Hapten-carrier protein conjugation Sulfo-SMCC Primary amines Stable maleimide reactive 11.6 Å group Sulfhydryls Water-soluble Enzyme-antibody conjugation MBS Primary amines Enzyme-antibody conjugation 9.9 Å Sulfhydryls Hapten-carrier protein conjugation Sulfo-MBS Primary amines Water-soluble 9.9 Å Sulfhydryls SIAB Primary amines Enzyme-antibody conjugation 10.6 Å Sulfhydryls Sulfo-SIAB Primary amines Water-soluble 10.6 Å Sulfhydryls SMPB Primary amines Extended spacer arm 14.5 Å Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5 Å Sulfhydryls Water-soluble EDE/Sulfo-NHS Primary amines Hapten-Carrier conjugation 0 Carboxyl groups ABH Carbohydrates Reacts with sugar groups 11.9 Å Nonselective [000317] Non-Cleavable Linkers or Direct Attachment: In some embodiments of the disclosure, the conjugate can be designed so that the agent is delivered to the target but not released. This can be accomplished by attaching an agent to an AB either directly or via a non- cleavable linker. [000318] These non-cleavable linkers may include amino acids, peptides, D-amino acids or other organic compounds that can be modified to include functional groups that can subsequently be utilized in attachment to ABs by the methods described herein. A-general formula for such an organic linker could be W – (CH2)n – Q wherein W is either --NH--CH2-- or --CH2--; Q is an amino acid, peptide; and n is an integer from 0 to 20. [000319] Non-Cleavable Conjugates: In some embodiments, a compound can be attached to ABs that do not activate complement. When using ABs that are incapable of complement activation, this attachment can be accomplished using linkers that are susceptible to cleavage by activated complement or using linkers that are not susceptible to cleavage by activated complement. [000320] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556. [000321] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab’ fragments of the antibody of the present disclosure can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. CD47 Antibody Drug Conjugates and CD47 Activatable Antibody Drug Conjugates [000322] In some embodiments, the antibody drug conjugates (ADCs) and activatable antibody drug conjugates (AADCs) can include one or more polypeptides that include the combination of a light chain sequence or a light chain variable domain sequence, and a heavy chain sequence or a heavy chain variable domain sequence, a linker, and a toxin in a given row of Table D or any combination of a light chain sequence or a light chain variable domain sequence, and a heavy chain sequence or a heavy chain variable domain sequence, a linker, and a toxin of Table D. For example, Combination No.1 comprises the heavy chain of SEQ ID No.1, the light chain of SEQ ID No.160, a vc linker, conjugated to MMAD toxin. Table D: CD47 Activatable ADC Combinations
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
[000323] Any of the combinations in Table D that list a heavy chain and light chain variable region can be combined with human immunoglobulin constant regions to result in fully human IgGs including IgG1, IgG2, IgG4 or mutated constant regions to result in human IgGs with altered functions such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P. The combinations described in Table D are not limited by the particular combinations shown in any given row, and thus can include any heavy chain sequence or heavy chain variable region sequence from column 2 of Table D combined with any light chain sequence or light chain variable region sequence from column 3 of Table D combined with any linker from column 4 combined with any toxin from column 5. In addition to the heavy chain sequences or heavy chain variable region sequences listed in column 2, any heavy chain sequence or heavy chain variable region sequence disclosed herein can be used in a combination. In addition to the light chain sequences or light chain variable region sequences listed in column 3, any light chain sequence or light chain variable region sequence disclosed herein can be used in a combination. In addition to the linkers listed in column 4, any linker disclosed herein can be used in a combination. In addition to the toxins listed in column 5, any toxin disclosed herein can be used in a combination. Multispecific Antibodies and Activatable Antibodies [000324] In some embodiments, the activatable CD47 antibody and/or conjugated activatable CD47 antibody is monospecific. In some embodiments, the activatable CD47 antibody and/or conjugated activatable CD47 antibody is multispecific, e.g., by way of non- limiting example, bispecific or trifunctional. [000325] In some embodiments, the activatable CD47 antibody and/or conjugated activatable CD47 antibody is formulated as part of a pro-Bispecific T Cell Engager (BITE) molecule. In some embodiments, the activatable CD47 antibody and/or conjugated activatable CD47 antibody is formulated as part of a pro-Chimeric Antigen Receptor (CAR) modified T cell or other engineered receptor. [000326] The disclosure accordingly also provides multispecific CD47 antibodies. The multispecific antibodies provided herein are multispecific antibodies that recognize CD47 and at least one or more different antigens or epitopes. [000327] The disclosure also provides multispecific CD47 activatable antibodies. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize CD47 and at least one or more different antigens or epitopes and that include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody such that coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target. In some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM) that functions as a substrate for at least one protease. The activatable multispecific antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to a target that is at least comparable to the corresponding, unmodified multispecific antibody. [000328] In some embodiments, the activatable antibody or antigen-binding fragment thereof is incorporated in a multispecific activatable antibody or antigen-binding fragment thereof, where at least one arm of the multispecific activatable antibody specifically binds CD47. In some embodiments, the activatable antibody or antigen-binding fragment thereof is incorporated in a bispecific antibody or antigen-binding fragment thereof, where at least one arm of the bispecific activatable antibody specifically binds CD47. [000329] In some embodiments, the antibody or antigen-binding fragment thereof is incorporated in a multispecific antibody or antigen-binding fragment thereof, where at least one arm of the multispecific antibody or antigen-binding fragment thereof specifically binds CD47. In some embodiments, the antibody or antigen-binding fragment thereof is incorporated in a bispecific antibody or antigen-binding fragment thereof, where at least one arm of the bispecific antibody or antigen-binding fragment thereof specifically binds CD47. [000330] In some embodiments, at least one arm of the multispecific antibody or antigen- binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 1. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a light chain variable region amino acid sequence of SEQ ID NO: 2. [000331] In some embodiments, at least one arm of the multispecific antibody or antigen- binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., a bispecific antibody or antigen- binding fragment thereof, comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 1. In some embodiments, at least one arm of the multispecific antibody or antigen-binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a light chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 2. [000332] In some embodiments, at least one arm of the multispecific antibody or antigen- binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one complementarity determining region (CDR) sequence is selected from the group consisting of a VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000333] In some embodiments, at least one arm of the multispecific antibody or antigen- binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GFTFSNYWMN (SEQ ID NO: 37) or GFTFSNYWMD (SEQ ID NO: 38); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000334] In some embodiments, at least one arm of the multispecific antibody or antigen- binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000335] In some embodiments, at least one arm of the multispecific antibody or antigen- binding fragment thereof, e.g., a bispecific antibody or antigen-binding fragment thereof, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence comprising the amino acid sequence GFTFSNYWMN (SEQ ID NO: 37) or GFTFSNYWMD (SEQ ID NO: 38); a VH CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10). [000336] In some embodiments, the multispecific antibodies/activatable antibodies are designed to engage immune effector cells, also referred to herein as immune-effector cell engaging multispecific activatable antibodies. In some embodiments, the multispecific antibodies/activatable antibodies are designed to engage leukocytes, also referred to herein as leukocyte engaging multispecific activatable antibodies. In some embodiments, the multispecific antibodies/activatable antibodies are designed to engage T cells, also referred to herein as T-cell engaging multispecific antibodies/activatable antibodies. In some embodiments, the multispecific antibodies/activatable antibodies engage a surface antigen on a leukocyte, such as on a T cell, on a natural killer (NK) cell, on a myeloid mononuclear cell, on a macrophage, and/or on another immune effector cell. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a mononuclear cell, such as a myeloid mononuclear cell. In some embodiments, the multispecific activatable antibodies are designed to bind or otherwise interact with more than one target and/or more than one epitope, also referred to herein as multi-antigen targeting activatable antibodies. As used herein, the terms “target” and “antigen” are used interchangeably. [000337] In some embodiments, immune effector cell engaging multispecific activatable antibodies of the disclosure include a targeting antibody or antigen-binding fragment thereof that binds CD47 and an immune effector cell engaging antibody or antigen-binding portion thereof, where at least one of the targeting antibody or antigen-binding fragment thereof and/or the immune effector cell engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the immune effector cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, immune effector cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47. In some embodiments, the immune effector cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, immune effector cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47. In some embodiments, the non-immune effector cell engaging antibody is a cancer targeting antibody. In some embodiments the non-immune cell effector antibody is an IgG. In some embodiments the immune effector cell engaging antibody is a scFv. In some embodiments the CD47-targeting antibody (e.g., non-immune cell effector antibody) is an IgG and the immune effector cell engaging antibody is a scFv. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a myeloid mononuclear cell. [000338] In some embodiments, T-cell engaging multispecific activatable antibodies of the disclosure include a CD47-targeting antibody or antigen-binding fragment thereof and a T-cell engaging antibody or antigen-binding portion thereof, where at least one of the CD47-targeting antibody or antigen-binding fragment thereof and/or the T-cell engaging antibody or antigen- binding portion thereof is masked. In some embodiments, the T-cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, T-cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47. In some embodiments, the T- cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen- binding fragment thereof (AB1) that binds a first, T-cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen- binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47. [000339] In some embodiments of an immune effector cell engaging multispecific antibody/activatable antibody, one antigen is CD47, and another antigen is typically a stimulatory or inhibitory receptor present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. In some embodiments, the antigen is a stimulatory receptor present on the surface of a T cell or NK cell; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments, the antigen is an inhibitory receptor present on the surface of a T-cell; examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIRs. The antibody domain conferring specificity to the T-cell surface antigen may also be substituted by a ligand or ligand domain that binds to a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PDL1, PDL2, or TNFSF9. [000340] In some embodiments, the T-cell engaging multispecific activatable antibody includes an anti-CD3 epsilon (CD3e, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, where at least one of the anti-CD3e scFv and/or the targeting antibody or antigen-binding portion thereof is masked. In some embodiments, the CD3e scFv includes a first antibody or antigen-binding fragment thereof (AB1) that binds CD3e, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind CD3e. In some embodiments, the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47. In some embodiments, the CD3e scFv includes a first antibody or antigen-binding fragment thereof (AB1) that binds CD3e, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind CD3e, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds CD47, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind CD47. [000341] In some embodiments, the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies include at least a first antibody or antigen-binding fragment thereof that binds a first target and/or first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and/or a second epitope. In some embodiments, the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind two or more different targets. In some embodiments, the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind two or more different epitopes on the same target. In some embodiments, the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind a combination of two or more different targets and two or more different epitopes on the same target. [000342] In some embodiments, a multispecific activatable antibody comprising an IgG has the IgG variable domains masked. In some embodiments, a multispecific activatable antibody comprising a scFv has the scFv domains masked. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the IgG variable domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the scFv domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where each of the IgG variable domains and the scFv domains is coupled to its own masking moiety. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for another target antigen. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for an epitope of a target antigen and another antibody domain has specificity for another epitope of the target antigen. [000343] In a multispecific activatable antibody, a scFv can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, to the carboxyl terminus of the light chain of an IgG activatable antibody, or to the carboxyl termini of both the heavy and light chains of an IgG activatable antibody. In a multispecific activatable antibody, a scFv can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, to the amino terminus of the light chain of an IgG activatable antibody, or to the amino termini of both the heavy and light chains of an IgG activatable antibody. In a multispecific activatable antibody, a scFv can be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of the IgG. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of at least one scFv. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of an IgG and a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of at least one scFv. [000344] The disclosure provides examples of multispecific activatable antibody structures which include, but are not limited to, the following: (VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*- L3-VL*-L2-CM-L1-MM)2; (VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1- MM)2; (MM-L1-CM-L2-VL-CL)2:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*)2; (MM-L1-CM-L2- VL-CL)2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*)2; (VL-CL)2:(MM-L1-CM-L2-VL*-L3-VH*- L4-VH-CH1-CH2-CH3)2; (VL-CL)2:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL-CL)2:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (MM-L1-CM-L2-VL- CL)2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1- MM)2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)2:(VH-CH1-CH2- CH3)2; (MM-L1-CM-L2-VL*-L3-VH*-L4-VL-CL)2:(VH-CH1-CH2-CH3)2; (MM-L1-CM-L2- VH*-L3-VL*-L4-VL-CL)2:(VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1- MM)2: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*- L2-CM-L1-MM)2: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*- L3-VH*-L2-CM-L1-MM)2: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL- L4-VL*-L3-VH*-L2-CM-L1-MM)2: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*)2: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL- CL-L4-VH*-L3-VL*)2: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4- VL*-L3-VH*)2: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*- L3-VH*)2: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3- VL*-L2-CM-L1-MM)2: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VH*-L3-VL*- L2-CM-L1-MM)2: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2; (VL-CL-L4-VL*-L3-VH*-L2-CM- L1-MM)2: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)2; or (VL-CL-L4-VL*-L3-VH*-L2-CM-L1- MM)2: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2, wherein: VL and VH represent the light and heavy variable domains of the first specificity, contained in the IgG; VL* and VH* represent the variable domains of the second specificity, contained in the scFv; L1 is a linker peptide connecting the masking moiety (MM) and the cleavable moiety (CM); L2 is a linker peptide connecting the cleavable moiety (CM), and the antibody; L3 is a linker peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the antibody of the first specificity to the antibody of the second specificity; CL is the light-chain constant domain; and CH1, CH2, CH3 are the heavy chain constant domains. The first and second specificities can be toward any antigen or epitope. [000345] In some embodiments of a T-cell engaging multispecific antibody/activatable antibody, one antigen is CD47, and another antigen is typically a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain conferring specificity to the T-cell surface antigen may also be substituted by a ligand or ligand domain that binds to a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/or other immune effector cell receptor. [000346] In some embodiments, the targeting antibody is a CD47 antibody disclosed herein. In some embodiments, the targeting antibody can be in the form an activatable antibody. In some embodiments, the scFv(s) can be in the form of a Pro-scFv (see, e.g., WO 2009/025846, WO 2010/081173). [000347] In some embodiments, the scFv is specific for binding CD3e, and comprises or is derived from an antibody or fragment thereof that binds CD3e, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4). [000348] In some embodiments, the anti-CTLA-4 scFv includes the amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKV EIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQ APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYW YFDLWGRGTLVTVSSAS (SEQ ID NO: 643) [000349] In some embodiments, the anti-CTLA-4 scFv includes the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 643. [000350] In some embodiments, the anti-CD3 ^ scFv includes the amino acid sequence: GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRP GQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC LDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGS GTKLEINR (SEQ ID NO: 644) [000351] In some embodiments, the anti-CD3 ^ scFv includes the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 644. [000352] In some embodiments, the scFv is specific for binding one or more T-cells, one or more NK-cells and/or one or more macrophages. In some embodiments, the scFv is specific for binding a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. [000353] In some embodiments, the multispecific antibody/activatable antibody also includes an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is conjugated to the multispecific antibody/activatable antibody via a linker. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylator or DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table E. In some embodiments, the agent is a dolastatin. In some embodiments, the agent is an auristatin or derivative thereof. In some embodiments, the agent is auristatin E or a derivative thereof. In some embodiments, the agent is monomethyl auristatin E (MMAE). In some embodiments, the agent is monomethyl auristatin D (MMAD). In some embodiments, the agent is a maytansinoid or maytansinoid derivative. In some embodiments, the agent is DM1 or DM4. In some embodiments, the agent is a duocarmycin or derivative thereof. In some embodiments, the agent is a calicheamicin or derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine. In some embodiments, the agent is a pyrrolobenzodiazepine dimer. [000354] In some embodiments, the multispecific antibody/activatable antibody also includes a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. [000355] In some embodiments, the multispecific antibody/activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the multispecific activatable antibody can be engineered to include one or more disulfide bonds. [000356] The disclosure also provides an isolated nucleic acid molecule encoding a multispecific antibody/activatable antibody described herein, as well as vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing a multispecific antibody/activatable antibody by culturing a cell under conditions that lead to expression of the antibody/activatable antibody, wherein the cell comprises such a nucleic acid molecule. In some embodiments, the cell comprises such a vector. [000357] The disclosure also provides a method of manufacturing multispecific CD47 antibodies of the disclosure by (a) culturing a cell comprising a nucleic acid construct that encodes the multispecific antibody under conditions that lead to expression of the multispecific antibody. [000358] The disclosure also provides a method of manufacturing multispecific activatable CD47 antibodies of the disclosure by (a) culturing a cell comprising a nucleic acid construct that encodes the multispecific activatable antibody under conditions that lead to expression of the multispecific activatable antibody, and (b) recovering the multispecific activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein. [000359] The disclosure also provides multispecific activatable antibodies and/or multispecific activatable antibody compositions that include at least a first antibody or antigen- binding fragment thereof (AB1) that specifically binds a first target or first epitope and a second antibody or antigen-biding fragment thereof (AB2) that binds a second target or a second epitope, where at least AB1 is coupled or otherwise attached to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind its target. In some embodiments, the MM1 is coupled to AB1 via a first cleavable moiety (CM1) sequence that includes a substrate for a protease, for example, a protease that is co-localized with the target of AB1 at a treatment site or a diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to the target of AB1 that is at least comparable to the corresponding, unmodified multispecific antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein. [000360] The disclosure also provides compositions and methods that include a multispecific activatable antibody that includes at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), where at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that decreases the ability of AB1 to bind its target. In some embodiments, each AB is coupled to a MM that decreases the ability of its corresponding AB to each target. For example, in bispecific activatable antibody embodiments, AB1 is coupled to a first masking moiety (MM1) that decreases the ability of AB1 to bind its target, and AB2 is coupled to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in such embodiments, AB1 is coupled to a first masking moiety (MM1) that decreases the ability of AB1 to bind its target, AB2 is coupled to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target, AB3 is coupled to a third masking moiety (MM3) that decreases the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein. [000361] In some embodiments, the multispecific activatable antibody further includes at least one cleavable moiety (CM) that is a substrate for a protease, where the CM links a MM to an AB. For example, in some embodiments, the multispecific activatable antibody includes at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), where at least the first AB in the multispecific activatable antibody is coupled via a first cleavable moiety (CM1) to a masking moiety (MM1) that decreases the ability of AB1 to bind its target. In some bispecific activatable antibody embodiments, AB1 is coupled via CM1 to MM1, and AB2 is coupled via a second cleavable moiety (CM2) to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in some of these embodiments, AB1 is coupled via CM1 to MM1, AB2 is coupled via CM2 to MM2, and AB3 is coupled via a third cleavable moiety (CM3) to a third masking moiety (MM3) that decreases the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein. Activatable Antibodies Having Non-Binding Steric Moieties or Binding Partners for Non-Binding Steric Moieties [000362] The disclosure also provides activatable antibodies that include non-binding steric moieties (NB) or binding partners (BP) for non-binding steric moieties, where the BP recruits or otherwise attracts the NB to the activatable antibody. The activatable antibodies provided herein include, for example, an activatable antibody that includes a non-binding steric moiety (NB), a cleavable linker (CL) and antibody or antibody fragment (AB) that binds a target; an activatable antibody that includes a binding partner for a non-binding steric moiety (BP), a CL and an AB; and an activatable antibody that includes a BP to which an NB has been recruited, a CL and an AB that binds the target. Activatable antibodies in which the NB is covalently linked to the CL and AB of the activatable antibody or is associated by interaction with a BP that is covalently linked to the CL and AB of the activatable antibody are referred to herein as “NB-containing activatable antibodies.” By activatable or switchable is meant that the activatable antibody exhibits a first level of binding to a target when the activatable antibody is in an inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target when the activatable antibody is in an uninhibited, unmasked and/or cleaved state (i.e., a second conformation, i.e., activated antibody), where the second level of target binding is greater than the first level of target binding. The activatable antibody compositions can exhibit increased bioavailability and more favorable biodistribution compared to conventional antibody therapeutics. [000363] In some embodiments, activatable antibodies provide for reduced toxicity and/or adverse side effects that could otherwise result from binding of the at non-treatment sites and/or non-diagnostic sites if the AB were not masked or otherwise inhibited from binding to such a site. [000364] CD47 activatable antibodies that include a non-binding steric moiety (NB) can be made using the methods set forth in PCT Publication No. WO 2013/192546, the contents of which are hereby incorporated by reference in their entirety. Therapeutic Uses of Antibodies, and Activatable Antibodies [000365] The invention provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating a CD47-mediated disease in a subject by administering a therapeutically effective amount of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody described herein to a subject in need thereof. [000366] There is provided a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody for use in preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating a CD47-mediated disease. [000367] The invention also provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating cancer in a subject by administering a therapeutically effective amount of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody described herein to a subject in need thereof. [000368] There is provided a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody for use in preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating cancer. [000369] The invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with aberrant expression and/or activity of CD47 in a subject using antibodies/activatable antibodies that bind CD47, particularly activatable antibodies that bind and neutralize or otherwise inhibit at least one biological activity of CD47 and/or CD47-mediated signaling. [000370] Thus, there is provided antibodies/activatable antibodies that bind CD47 for use in treating, preventing and/or delaying the onset or progression of, ameliorating, or alleviating a symptom associated with aberrant expression and/or activity of CD47. [000371] The invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with the presence, growth, proliferation, metastasis, and/or activity of cells which are expressing CD47 or aberrantly expressing CD47 in a subject using antibodies/activatable antibodies that bind CD47, particularly activatable antibodies that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are expressing or aberrantly expressing CD47. [000372] The invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with the presence, growth, proliferation, metastasis, and/or activity of cells which are expressing CD47 in a subject using antibodies/activatable antibodies that bind CD47, particularly antibodies/activatable antibodies that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are expressing CD47. [000373] The invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with the presence, growth, proliferation, metastasis, and/or activity of cells which are aberrantly expressing CD47 in a subject using antibodies/activatable antibodies that bind CD47, particularly antibodies/activatable antibodies that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are aberrantly expressing CD47. [000374] CD47 is known to be expressed in a variety of cancers, such as, by way of non- limiting example, adenocarcinoma, bile duct (biliary) cancer, bladder cancer, breast cancer, e.g., triple-negative breast cancer and Her2-negative breast cancer; carcinoid cancer; cervical cancer; cholangiocarcinoma; colorectal; endometrial; esophageal cancer; glioma; head and neck cancer, e.g., head and neck squamous cell cancer; leukemia; liver cancer; lung cancer, e.g., NSCLC, SCLC; lymphoma; melanoma; osopharyngeal cancer; ovarian cancer; pancreatic cancer; prostate cancer, e.g., metastatic castration-resistant prostate carcinoma; renal cancer; skin cancer; squamous cell cancer, stomach cancer; testis cancer; thyroid cancer; and urothelial cancer. [000375] In some embodiments, the cancer is associated with a CD47-expressing tumor. In some embodiments, the cancer is due to a CD47-expressing tumor. [000376] A CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody can be administered to a patient suffering cancer of any stage, from early to metastatic. The terms subject and patient are used interchangeably herein. [000377] In some embodiments, the subject is a mammal, such as a human, non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal in the care of a veterinarian. [000378] The CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder associated with aberrant CD47 expression and/or activity. A subject suffering from or susceptible to a disease or disorder associated with aberrant CD47 expression and/or activity is identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic condition are identified using any of a variety of clinical and/or laboratory tests such as, physical examination and blood, urine and/or stool analysis to evaluate health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests such as physical examination and/or bodily fluid analysis, e.g., blood, urine and/or stool analysis, to evaluate health status. [000379] Administration of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with aberrant CD47 expression and/or activity is considered successful if any of a variety of laboratory or clinical objectives is achieved. For example, administration of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with aberrant CD47 expression and/or activity is considered successful if one or more of the symptoms associated with the disease or disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state. Administration of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with aberrant CD47 expression and/or activity is considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worse, state. [000380] In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder, such as subjects suffering from cancer or other neoplastic condition, wherein the subject’s diseased cells are expressing CD47. In some embodiments, the diseased cells are associated with aberrant CD47 expression and/or activity. In some embodiments, the diseased cells are associated with normal CD47 expression and/or activity. A subject suffering from or susceptible to a disease or disorder wherein the subject’s diseased cells express CD47 is identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic condition are identified using any of a variety of clinical and/or laboratory tests such as, physical examination and blood, urine and/or stool analysis to evaluate health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests such as physical examination and/or bodily fluid analysis, e.g., blood, urine and/or stool analysis, to evaluate health status. [000381] In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder associated with cells expressing CD47 or the presence, growth, proliferation, metastasis, and/or activity of such cells, such as subjects suffering from cancer or other neoplastic conditions. In some embodiments, the cells are associated with aberrant CD47 expression and/or activity. In some embodiments, the cells are associated with normal CD47 expression and/or activity. A subject suffering from or susceptible to a disease or disorder associated with cells that express CD47 is identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic condition are identified using any of a variety of clinical and/or laboratory tests such as, physical examination and blood, urine and/or stool analysis to evaluate health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests such as physical examination and/or bodily fluid analysis, e.g., blood, urine and/or stool analysis, to evaluate health status. [000382] Administration of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with cells expressing CD47 is considered successful if any of a variety of laboratory or clinical objectives is achieved. For example, administration a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with cells expressing CD47 is considered successful if one or more of the symptoms associated with the disease or disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state. Administration of a CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody to a patient suffering from a disease or disorder associated with cells expressing CD47 is considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worse, state. [000383] In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is administered during and/or after treatment in combination with one or more additional agents such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent. In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) are administered simultaneously. For example, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) are administered sequentially. [000384] The disclosure also provides methods of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in which diseased cells express CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof. In some embodiments, the disorder or disease is cancer. [000385] The disclosure also provides methods of treating, alleviating a symptom of, or delaying the progression of a disorder or disease associated with cells expressing CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof. In some embodiments, the disorder or disease associated with cells expressing CD47 is cancer. In some embodiments, the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2-negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non-small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer. In some embodiments, the expression and/or activity of the mammalian CD47 is aberrant. In some embodiments, the method comprises administering an additional agent. In some embodiments, the additional agent is a therapeutic agent. [000386] The disclosure also provides methods of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing mammalian CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof. In some embodiments, the expression and/or activity of the mammalian CD47 is aberrant. In some embodiments, the method comprises administering an additional agent. In some embodiments, the additional agent is a therapeutic agent. [000387] The disclosure also provides methods of inhibiting, blocking, or preventing the binding of a natural ligand to mammalian CD47, comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof. In some embodiments, the expression and/or activity of the mammalian CD47 is aberrant. In some embodiments, the method comprises administering an additional agent. In some embodiments, the additional agent is a therapeutic agent. [000388] The disclosure also provides methods of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in which diseased cells express CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof. In some embodiments, the disorder or disease is cancer. [000389] The disclosure also provides methods of treating, alleviating a symptom of, or delaying the progression of a disorder or disease associated with cells expressing CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof. In some embodiments, the disorder or disease associated with cells expressing CD47 is cancer. In some embodiments, the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2-negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non-small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer. In some embodiments, the expression and/or activity of the mammalian CD47 is aberrant. In some embodiments, the method comprises administering an additional agent. In some embodiments, the additional agent is a therapeutic agent. [000390] The disclosure also provides methods of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing mammalian CD47 comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof. In some embodiments, the expression and/or activity of the mammalian CD47 is aberrant. In some embodiments, the method comprises administering an additional agent. In some embodiments, the additional agent is a therapeutic agent. [000391] The disclosure also provides methods of inhibiting, blocking, or preventing the binding of a natural ligand to mammalian CD47, comprising administering a therapeutically effective amount of an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure or a pharmaceutical composition comprising an antibody/conjugated antibody/activatable antibody/conjugated activatable antibody of the disclosure to a subject in need thereof. In some embodiments, the expression and/or activity of the mammalian CD47 is aberrant. In some embodiments, the method comprises administering an additional agent. In some embodiments, the additional agent is a therapeutic agent. Formulations [000392] It will be appreciated that administration of therapeutic entities in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol.32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm.203(1-2):1-60 (2000), Charman WN Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts. J Pharm Sci.89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol.52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists. [000393] Therapeutic formulations of the disclosure, which include a CD47 antibody and/or activatable CD47 antibody, such as by way of non-limiting example, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody, are used to prevent, treat or otherwise ameliorate a disease or disorder associated with aberrant target expression and/or activity. For example, therapeutic formulations of the disclosure, which include an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody, are used to treat or otherwise ameliorate a cancer or other neoplastic condition, inflammation, an inflammatory disorder, and/or an autoimmune disease. In some embodiments, the cancer is a solid tumor or a hematologic malignancy where the target is expressed. In some embodiments, the cancer is a solid tumor where the target is expressed. In some embodiments, the cancer is a hematologic malignancy where the target is expressed. In some embodiments, the target is expressed on parenchyma (e.g., in cancer, the portion of an organ or tissue that often carries out function(s) of the organ or tissue). In some embodiments, the target is expressed on a cell, tissue, or organ. In some embodiments, the target is expressed on stroma (i.e., the connective supportive framework of a cell, tissue, or organ). In some embodiments, the target is expressed on an osteoblast. In some embodiments, the target is expressed on the endothelium (vasculature). In some embodiments, the target is expressed on a cancer stem cell. In some embodiments, the agent to which the antibody and/or the activatable antibody is conjugated is a microtubule inhibitor. In some embodiments, the agent to which the antibody and/or the activatable antibody is conjugated is a nucleic acid damaging agent. [000394] Efficaciousness of prevention, amelioration or treatment is determined in association with any known method for diagnosing or treating the disease or disorder associated with target expression and/or activity, such as, for example, aberrant target expression and/or activity. Prolonging the survival of a subject or otherwise delaying the progression of the disease or disorder associated with target expression and/or activity, e.g., aberrant target expression and/or activity, in a subject indicates that the antibody, conjugated antibody, activatable antibody and/or conjugated activatable antibody confers a clinical benefit. [000395] An antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody can be administered in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol.4), 1991, M. Dekker, New York. [000396] In some embodiments where antibody fragments are used, the smallest fragment that specifically binds to the binding domain of the target protein is selected. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compounds as necessary for the particular indication being treated, for example, in some embodiments, those with complementary activities that do not adversely affect each other. In some embodiments, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth- inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [000397] The active ingredients can also 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. [000398] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. [000399] Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, 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 (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and g ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (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. Combination Treatments [000400] In some embodiments, the CD47 antibodies, conjugated CD47 antibodies, activatable CD47 antibodies and/or conjugated activatable CD47 antibodies described herein are used in conjunction with one or more additional agents or a combination of additional agents. Suitable additional agents include current pharmaceutical and/or surgical therapies for an intended application, such as, for example, cancer. For example, the CD47 antibodies, conjugated CD47 antibodies, activatable CD47 antibodies and/or conjugated activatable CD47 antibodies can be used in conjunction with an additional chemotherapeutic agent, anti-neoplastic agent, anti-inflammatory agent, an immunosuppressive agent, an alkylating agent, an anti- metabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and/or any other nucleic acid damaging agent. [000401] In some embodiments, the additional agent(s) is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine. [000402] In some embodiments, the additional agent(s) is a checkpoint inhibitor, a kinase inhibitor, an agent targeting inhibitors in the tumor microenvironment, and/or a T cell or NK agonist. In some embodiments, the additional agent(s) is radiation therapy, alone or in combination with another additional agent(s) such as a chemotherapeutic or anti-neoplastic agent. In some embodiments, the additional agent(s) is a vaccine, an oncovirus, and/or a DC- activating agent such as, by way of non-limiting example, a toll-like receptor (TLR) agonist and/or a-CD40. In some embodiments, the additional agent(s) is a tumor-targeted antibody designed to kill the tumor via ADCC or via direct conjugation to a toxin (e.g., an antibody drug conjugate (ADC). [000403] In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of CTLA-4, LAG-3, PD-1, CD47, TIGIT, TIM-3, B7H4, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an a-CSF1R inhibitor, an a-CCR4 inhibitor, a TGF-beta, a myeloid-derived suppressor cell, or a T-regulatory cell. In some embodiments, the agonist is selected from the group consisting of Ox40, GITR, CD137, ICOS, CD27, and HVEM. [000404] In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a CD47 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an a-CSF1R inhibitor. In some embodiments, the inhibitor is an a-CCR4 inhibitor. In some embodiments, the inhibitor is a TGF-beta. In some embodiments, the inhibitor is a myeloid-derived suppressor cell. In some embodiments, the inhibitor is a T-regulatory cell. [000405] In some embodiments, the agonist is Ox40. In some embodiments, the agonist is GITR. In some embodiments, the agonist is CD137. In some embodiments, the agonist is ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM. [000406] In some embodiments, the CD47 antibody, conjugated antibody, activatable antibody and/or conjugated activatable antibody is administered during and/or after treatment in combination with one or more additional agents such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent. In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are formulated into a single therapeutic composition, and the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and additional agent are administered simultaneously. Alternatively, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are administered simultaneously, or the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are administered at different times during a treatment regimen. For example, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is administered prior to the administration of the additional agent, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is administered subsequent to the administration of the additional agent, or the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are administered in an alternating fashion. As described herein, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and additional agent are administered in single doses or in multiple doses. [000407] In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) are administered simultaneously. For example, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent(s) are administered sequentially, or the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody and the additional agent are administered at different times during a treatment regimen. [000408] In some embodiments, the CD47 antibody, conjugated CD47 antibody, activatable CD47 antibody and/or conjugated activatable CD47 antibody is administered during and/or after treatment in combination with one or more additional agents such as, by way of non- limiting example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent, such as an alkylating agent, an anti-metabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and/or any other nucleic acid damaging agent. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., Abraxane®). In some embodiments, the additional agent is an anti-metabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as platinum- based chemotherapy, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeted agent, such as a kinase inhibitor, e.g., sorafenib or erlotinib. In some embodiments, the additional agent is a targeted agent, such as another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteosome inhibitor, such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immune modulating agent, such as lenolidominde or IL- 2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent considered standard of care by those skilled in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those skilled in the art. [000409] In some embodiments, the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activatable antibody or antigen-binding fragment thereof and/or another conjugated activatable antibody or antigen-binding fragment thereof. In some embodiments the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen- binding fragment thereof, another activatable antibody or antigen-binding fragment thereof and/or another conjugated activatable antibody or antigen-binding fragment thereof against the same target as the first antibody or antigen-binding fragment thereof, the first conjugated antibody or antigen-binding fragment thereof, activatable antibody or antigen-binding fragment thereof and/or a conjugated activatable antibody or antigen-binding fragment thereof, e.g., against CD47. In some embodiments the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another activatable antibody or antigen-binding fragment thereof and/or another conjugated activatable antibody or antigen-binding fragment thereof against a target different than the target of the first antibody or antigen-binding fragment thereof, the first conjugated antibody or antigen-binding fragment thereof, activatable antibody or antigen-binding fragment thereof and/or a conjugated activatable antibody or antigen-binding fragment thereof. [000410] As a non-limiting example, the antibody, antigen-binding fragment and/or the AB of an activatable antibody (of the additional agent) is a binding partner for any target listed in Table H. Table H: Exemplary Targets
Figure imgf000159_0001
Figure imgf000160_0001
[000411] As a non-limiting example, the antibody, antigen-binding fragment and/or the AB of an activatable antibody (of the additional agent) is or is derived from an antibody listed in Table J. Table J: Exemplary sources for Abs
Figure imgf000160_0002
Figure imgf000161_0001
Figure imgf000162_0001
[000412] In some embodiments, the additional antibody or antigen binding fragment thereof, conjugated antibody or antigen binding fragment thereof, activatable antibody or antigen binding fragment thereof, and/or conjugated activatable antibody or antigen binding fragment thereof is a monoclonal antibody, domain antibody, single chain, 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. In some embodiments, the additional antibody or antigen binding fragment thereof, conjugated antibody or antigen binding fragment thereof, activatable antibody or antigen binding fragment thereof, and/or conjugated activatable antibody or antigen binding fragment thereof is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody. Detection, Diagnostics, Imaging, Patient Selection [000413] The invention also provides methods and kits for using the antibodies/conjugated antibodies/activatable antibodies/conjugated activatable antibodies provided herein in a variety of diagnostic and/or prophylactic indications. For example, the invention provides methods and kits for detecting the presence or absence of a cleaving agent and a target of interest in a subject or a sample by (i) contacting a subject or sample with a CD47 activatable antibody, wherein the CD47 activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the CD47 activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to CD47, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, when the AB is in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to CD47, and when the AB is in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to CD47; and (ii) measuring a level of activated CD47 activatable antibody in the subject or sample, wherein a detectable level of activated CD47 activatable antibody in the subject or sample indicates that the cleaving agent and CD47 are present in the subject or sample and wherein no detectable level of activated CD47 activatable antibody in the subject or sample indicates that the cleaving agent, CD47 or both the cleaving agent and CD47 are absent in the subject or sample. As provided herein, the CD47 activatable antibody can bind both human and cynomolgus CD47. [000414] In some embodiments, the activatable CD47 antibody is an activatable CD47 antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable CD47 antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label. [000415] In some embodiments of these methods and kits, the activatable CD47 antibody includes a detectable label. In some embodiments of these methods and kits, the detectable label includes an imaging agent, a contrasting agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of these methods and kits, the imaging agent comprises a radioisotope. In some embodiments of these methods and kits, the radioisotope is indium or technetium. In some embodiments of these methods and kits, the contrasting agent comprises iodine, gadolinium or iron oxide. In some embodiments of these methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or b- galactosidase. In some embodiments of these methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments of these methods and kits, the luminescent label comprises an N-methylacrydium derivative. In some embodiments of these methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments of these methods and kits, the ligand-based label comprises biotin, avidin, streptavidin or one or more haptens. [000416] In some embodiments of these methods and kits, the subject is a mammal. In some embodiments of these methods, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal. In some embodiments, the subject is a rodent. [000417] In some embodiments of these methods and kits, the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method. [000418] In some embodiments of the methods and kits, the method is used to identify or otherwise refine a patient population suitable for treatment with a CD47 activatable antibody of the disclosure, followed by treatment by administering that activatable CD47 antibody and/or conjugated activatable CD47 antibody to a subject in need thereof. For example, patients that test positive for both the target (e.g., CD47) and a protease that cleaves the substrate in the cleavable moiety (CM) of CD47 activatable antibody being tested in these methods are identified as suitable candidates for treatment with such a CD47 activatable antibody comprising such a CM, and the patient is then administered a therapeutically effective amount of the activatable CD47 antibody and/or conjugated activatable CD47 antibody that was tested. Likewise, patients that test negative for either or both of the target (e.g., CD47) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with other CD47 activatable antibodies until a suitable CD47 activatable antibody for treatment is identified (e.g., a CD47 activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the patient is then administered a therapeutically effective amount of the activatable CD47 antibody and/or conjugated for which the patient tested positive. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein. [000419] In some embodiments, the antibody, the conjugated antibody, activatable antibody and/or conjugated activatable antibody contains a detectable label. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the disclosure can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, immunochemical staining, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol.42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti- analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. [000420] The antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies of the disclosure are also useful in a variety of diagnostic and prophylactic formulations. In one embodiment, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to patients that are at risk of developing one or more of the aforementioned disorders. A patient’s or organ’s predisposition to one or more of the aforementioned disorders can be determined using genotypic, serological or biochemical markers. [000421] In some embodiments of the disclosure, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to mitigate or reverse the effects of the clinical indication. [000422] An antibody, a conjugated antibody, an activatable antibody, and/or a conjugated activatable antibody of the disclosure is also useful in the detection of a target in patient samples and accordingly are useful as diagnostics. For example, the antibodies and/or activatable antibodies, and conjugated versions thereof, of the disclosure are used in in vitro assays, e.g., ELISA, to detect target levels in a patient sample. [000423] In one embodiment, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody of the disclosure is immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized antibody, conjugated antibody, activatable antibody and/or conjugated activatable antibody serves as a capture antibody for any target that may be present in a test sample. Prior to contacting the immobilized antibody and/or activatable antibody, and/or conjugated versions thereof, with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte. [000424] Subsequently the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen. Such a sample is, e.g., a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology. After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The labeled second antibody serves as a detecting antibody. The level of detectable label is measured, and the concentration of target antigen in the test sample is determined by comparison with a standard curve developed from the standard samples. [000425] It will be appreciated that based on the results obtained using the antibodies and activatable antibodies of the disclosure, and conjugated versions thereof, in an in vitro diagnostic assay, it is possible to stage a disease in a subject based on expression levels of the target antigen. For a given disease, samples of blood are taken from subjects diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic treatment of the disease. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of concentrations of the antigen that may be considered characteristic of each stage is designated. [000426] An antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody can also be used in diagnostic and/or imaging methods. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, activatable antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM. Such activatable antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of an activatable antibody) in a given cell or tissue of a given host organism. Such accumulation of activated antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses target to which the activated antibody binds. [000427] For example, the CM can be selected to be substrate for at least one protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like. The AB can be one that binds a target antigen. Using methods as disclosed herein, or when appropriate, methods familiar to one skilled in the art, a detectable label (e.g., a fluorescent label or radioactive label or radiotracer) can be conjugated to an AB or other region of an antibody and/or activatable antibody. Suitable detectable labels are discussed in the context of the above screening methods and additional specific examples are provided below. Using an AB specific to a protein or peptide of the disease state, along with at least one protease whose activity is elevated in the disease tissue of interest, activatable antibodies will exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated antibodies in the disease tissue is enhanced relative to non-disease tissues. [000428] In another example, activatable antibodies can be used to detect the presence or absence of a cleaving agent in a sample. For example, where the activatable antibodies contain a CM susceptible to cleavage by an enzyme, the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample. In another example, where the activatable antibodies contain a CM susceptible to cleavage by reducing agent, the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the activatable antibodies can be detectably labeled, and can be bound to a support (e.g., a solid support, such as a slide or bead). The detectable label can be positioned on a portion of the activatable antibody that is not released following cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage has occurred. The assay can be conducted by, for example, contacting the immobilized, detectably labeled activatable antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants. The presence or absence of the cleaving agent (e.g., enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the activatable antibodies prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the activatable antibody by the cleaving agent in the sample. [000429] Such detection methods can be adapted to also provide for detection of the presence or absence of a target that is capable of binding the AB of the activatable antibodies when cleaved. Thus, the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest. The presence or absence of the cleaving agent can be detected by the presence of and/or an increase in detectable label of the activatable antibodies as described above, and the presence or absence of the target can be detected by detection of a target-AB complex e.g., by use of a detectably labeled anti-target antibody. [000430] Activatable antibodies are also useful in in situ imaging for the validation of activatable antibody activation, e.g., by protease cleavage, and binding to a particular target. In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this technique, it is possible to confirm both binding to a given target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label). [000431] These techniques are useful with any frozen cells or tissue derived from a disease site (e.g. tumor tissue) or healthy tissues. These techniques are also useful with fresh cell or tissue samples. [000432] In these techniques, an activatable antibody is labeled with a detectable label. The detectable label can be a fluorescent dye, (e.g. a fluorophore, Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), an Alexa Fluor® label), a near infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin and an amplification reagent such as streptavidin, or an enzyme (e.g. horseradish peroxidase or alkaline phosphatase). [000433] Detection of the label in a sample that has been incubated with the labeled, activatable antibody indicates that the sample contains the target and contains a protease that is specific for the CM of the activatable antibody. In some embodiments, the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and/or by using an agent that is specific for the protease, for example, an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase; see e.g., International Publication Number WO 2010/129609, published 11 November 2010. The same approach of using broad spectrum protease inhibitors such as those described herein, and/or by using a more selective inhibitory agent can be used to identify a protease that is specific for the CM of the activatable antibody. In some embodiments, the presence of the target can be confirmed using an agent that is specific for the target, e.g., another antibody, or the detectable label can be competed with unlabeled target. In some embodiments, unlabeled activatable antibody could be used, with detection by a labeled secondary antibody or more complex detection system. [000434] Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the activatable antibody. [000435] These techniques are also useful in kits and/or as reagents for the detection, identification or characterization of protease activity in a variety of cells, tissues, and organisms based on the protease-specific CM in the activatable antibody. [000436] The disclosure provides methods of using the antibodies and/or activatable antibodies in a variety of diagnostic and/or prophylactic indications. For example, the disclosure provides methods of detecting presence or absence of a cleaving agent and a target of interest in a subject or a sample by (i) contacting a subject or sample with an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C- terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to the target; and (ii) measuring a level of activated activatable antibody in the subject or sample, wherein a detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent and the target are present in the subject or sample and wherein no detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or sample. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label. [000437] The disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or sample with an activatable antibody in the presence of a target of interest, e.g., the target, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to the target; and (ii) measuring a level of activated activatable antibody in the subject or sample, wherein a detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent is present in the subject or sample and wherein no detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or sample. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label. [000438] The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample, where the kits include at least an activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C- terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to the target; and (ii) measuring a level of activated activatable antibody in the subject or sample, wherein a detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent is present in the subject or sample and wherein no detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or sample. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label. [000439] The disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or sample with an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, an antigen binding domain (AB) that specifically binds the target, and a detectable label, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C- terminus as follows: MM-CM-AB or AB-CM-MM; wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; wherein, in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to the target; and wherein the detectable label is positioned on a portion of the activatable antibody that is released following cleavage of the CM; and (ii) measuring a level of detectable label in the subject or sample, wherein a detectable level of the detectable label in the subject or sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or sample and wherein no detectable level of the detectable label in the subject or sample indicates that the cleaving agent is present in the subject or sample. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label. [000440] The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody (e.g., an activatable antibody to which a therapeutic agent is conjugated) described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample. [000441] The disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody in the presence of the target, and (ii) measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample. Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the detectable label is attached to the masking moiety. In some embodiments, the detectable label is attached to the cleavable moiety N-terminal to the protease cleavage site. In some embodiments, a single antigen binding site of the AB is masked. In some embodiments wherein an antibody of the disclosure has at least two antigen binding sites, at least one antigen binding site is masked and at least one antigen binding site is not masked. In some embodiments all antigen binding sites are masked. In some embodiments, the measuring step includes use of a secondary reagent comprising a detectable label. [000442] The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample with an activatable antibody in the presence of the target, and measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample. Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM- MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the detectable label is attached to the masking moiety. In some embodiments, the detectable label is attached to the cleavable moiety N-terminal to the protease cleavage site. In some embodiments, a single antigen binding site of the AB is masked. In some embodiments wherein an antibody of the disclosure has at least two antigen binding sites, at least one antigen binding site is masked and at least one antigen binding site is not masked. In some embodiments all antigen binding sites are masked. In some embodiments, the measuring step includes use of a secondary reagent comprising a detectable label. [000443] The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample at a detectable level. [000444] The disclosure provides methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM and (ii) measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample. A reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%. Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label. [000445] The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample. A reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%. [000446] The disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM; and (ii) measuring a level of detectable label in the subject or biological sample, wherein a detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample. A reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%. Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label. [000447] The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent of interest in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM, wherein a detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent, the target, or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample. A reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%. [000448] In some embodiments of these methods and kits, the activatable antibody includes a detectable label. In some embodiments of these methods and kits, the detectable label includes an imaging agent, a contrasting agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of these methods and kits, the imaging agent comprises a radioisotope. In some embodiments of these methods and kits, the radioisotope is indium or technetium. In some embodiments of these methods and kits, the contrasting agent comprises iodine, gadolinium or iron oxide. In some embodiments of these methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or b- galactosidase. In some embodiments of these methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments of these methods and kits, the luminescent label comprises an N-methylacrydium derivative. In some embodiments of these methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments of these methods and kits, the ligand-based label comprises biotin, avidin, streptavidin or one or more haptens. [000449] In some embodiments of these methods, the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method. [000450] In some embodiments, in situ imaging and/or in vivo imaging are useful in methods to identify which patients to treat. For example, in in situ imaging, the activatable antibodies are used to screen patient samples to identify those patients having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site. [000451] In some embodiments in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative for either or both of the target (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the patient is then administered a therapeutically effective amount of the activatable antibody for which the patient tested positive. [000452] In some embodiments in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative might be identified as suitable candidates for another form of therapy. In some embodiments, such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the patient is then administered a therapeutically effective amount of the activatable antibody for which the patient tested positive. [000453] In some embodiments of the methods and kits, the method or kit is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested in these methods are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative for both of the targets (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with other activatable antibodies until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, patients that test negative for either of the target (e.g., the target) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. In some embodiments, patients that test negative for either of the target (e.g., the target) are identified as not being suitable candidates for treatment with such an activatable antibody comprising such a CM. In some embodiments, such patients can be tested with other activatable antibodies until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label. [000454] In some embodiments, a method or kit is used to identify or otherwise refine a patient population suitable for treatment with an anti-the target activatable antibody and/or conjugated activatable antibody (e.g., activatable antibody to which a therapeutic agent is conjugated) of the disclosure, followed by treatment by administering that activatable antibody and/or conjugated activatable antibody to a subject in need thereof. For example, patients that test positive for both the targets (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody and/or conjugated activatable antibody being tested in these methods are identified as suitable candidates for treatment with such antibody and/or such a conjugated activatable antibody comprising such a CM, and the patient is then administered a therapeutically effective amount of the activatable antibody and/or conjugated activatable antibody that was tested. Likewise, patients that test negative for either or both of the target (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with other antibody and/or conjugated activatable antibody until a suitable antibody and/or conjugated activatable antibody for treatment is identified (e.g., an activatable antibody and/or conjugated activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the patient is then administered a therapeutically effective amount of the activatable antibody and/or conjugated activatable antibody for which the patient tested positive. [000455] In some embodiments of these methods and kits, the MM is a peptide having a length from about 4 to 40 amino acids. In some embodiments of these methods and kits, the activatable antibody comprises a linker peptide, wherein the linker peptide is positioned between the MM and the CM. In some embodiments of these methods and kits, the activatable antibody comprises a linker peptide, where the linker peptide is positioned between the AB and the CM. In some embodiments of these methods and kits, the activatable antibody comprises a first linker peptide (L1) and a second linker peptide (L2), wherein the first linker peptide is positioned between the MM and the CM and the second linker peptide is positioned between the AB and the CM. In some embodiments of these methods and kits, each of L1 and L2 is a peptide of about 1 to 20 amino acids in length, and wherein each of L1 and L2 need not be the same linker. In some embodiments of these methods and kits, one or both of L1 and L2 comprises a glycine-serine polymer. In some embodiments of these methods and kits, at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GSGGS)n (SEQ ID NO: 116) and (GGGS)n (SEQ ID NO: 117), where n is an integer of at least one. In some embodiments of these methods and kits, at least one of L1 and L2 comprises an amino acid sequence having the formula (GGS)n, where n is an integer of at least one. In some embodiments of these methods and kits, at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO: 118), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 119), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 120), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 121), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 122), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 123). [000456] In some embodiments of these methods and kits, the AB comprises an antibody or antibody fragment sequence selected from the cross-reactive antibody sequences presented herein. In some embodiments of these methods and kits, the AB comprises a Fab fragment, a scFv or a single chain antibody (scAb). [000457] In some embodiments of these methods and kits, the cleaving agent is a protease that is co-localized in the subject or sample with the target and the CM is a polypeptide that functions as a substrate for the protease, wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease. In some embodiments of these methods and kits, the CM is a polypeptide of up to 15 amino acids in length. In some embodiments of these methods and kits, the CM is coupled to the N-terminus of the AB. In some embodiments of these methods and kits, the CM is coupled to the C-terminus of the AB. In some embodiments of these methods and kits, the CM is coupled to the N-terminus of a VL chain of the AB. [000458] The antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies of the disclosure are used in diagnostic and prophylactic formulations. In one embodiment, an activatable antibody is administered to patients that are at risk of developing one or more of the aforementioned inflammation, inflammatory disorders, cancer or other disorders. [000459] A patient’s or organ’s predisposition to one or more of the aforementioned disorders can be determined using genotypic, serological or biochemical markers. [000460] In some embodiments of the disclosure, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody is administered to mitigate or reverse the effects of the clinical indication. [000461] Antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies of the disclosure are also useful in the detection of the target in patient samples and accordingly are useful as diagnostics. For example, the antibodies, conjugated antibodies, the activatable antibodies and/or conjugated activatable antibodies of the disclosure are used in in vitro assays, e.g., ELISA, to detect target levels in a patient sample. [000462] In one embodiment, an antibody and/or activatable antibody of the disclosure is immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized antibody and/or activatable antibody serves as a capture antibody for any target that may be present in a test sample. Prior to contacting the immobilized antibody and/or activatable antibody with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte. [000463] Subsequently the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen. Such a sample is, e.g., a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology. After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The labeled second antibody serves as a detecting antibody. The level of detectable label is measured, and the concentration of target antigen in the test sample is determined by comparison with a standard curve developed from the standard samples. [000464] It will be appreciated that based on the results obtained using the antibodies and/or activatable antibodies of the disclosure in an in vitro diagnostic assay, it is possible to stage a disease in a subject based on expression levels of the Target antigen. For a given disease, samples of blood are taken from subjects diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic treatment of the disease. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of concentrations of the antigen that may be considered characteristic of each stage is designated. [000465] Antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies can also be used in diagnostic and/or imaging methods. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, activatable antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM. Such activatable antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of an activatable antibody) in a given cell or tissue of a given host organism. Such accumulation of activated antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses target to which the activated antibody binds. [000466] For example, the CM can be selected to be a protease substrate for a protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like. The AB can be one that binds a target antigen. Using methods familiar to one skilled in the art, a detectable label (e.g., a fluorescent label or radioactive label or radiotracer) can be conjugated to an AB or other region of an activatable antibody. Suitable detectable labels are discussed in the context of the above screening methods and additional specific examples are provided below. Using an AB specific to a protein or peptide of the disease state, along with a protease whose activity is elevated in the disease tissue of interest, activatable antibodies will exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated antibodies in the disease tissue is enhanced relative to non-disease tissues. [000467] In another example, activatable antibodies can be used to detect the presence or absence of a cleaving agent in a sample. For example, where the activatable antibodies contain a CM susceptible to cleavage by an enzyme, the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample. In another example, where the activatable antibodies contain a CM susceptible to cleavage by reducing agent, the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the activatable antibodies can be detectably labeled, and can be bound to a support (e.g., a solid support, such as a slide or bead). The detectable label can be positioned on a portion of the activatable antibody that is not released following cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage has occurred. The assay can be conducted by, for example, contacting the immobilized, detectably labeled activatable antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants. The presence or absence of the cleaving agent (e.g., enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the activatable antibodies prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the activatable antibody by the cleaving agent in the sample. [000468] Such detection methods can be adapted to also provide for detection of the presence or absence of a target that is capable of binding the AB of the activatable antibodies when cleaved. Thus, the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest. The presence or absence of the cleaving agent can be detected by the presence of and/or an increase in detectable label of the activatable antibodies as described above, and the presence or absence of the target can be detected by detection of a target-AB complex e.g., by use of a detectably labeled anti-target antibody. [000469] Activatable antibodies are also useful in in situ imaging for the validation of activatable antibody activation, e.g., by protease cleavage, and binding to a particular target. In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this technique, it is possible to confirm both binding to a given target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label). [000470] These techniques are useful with any frozen cells or tissue derived from a disease site (e.g. tumor tissue) or healthy tissues. These techniques are also useful with fresh cell or tissue samples. [000471] In these techniques, an activatable antibody is labeled with a detectable label. The detectable label can be a fluorescent dye, (e.g. Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), a near infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin and an amplification reagent such as streptavidin, or an enzyme (e.g. horseradish peroxidase or alkaline phosphatase). [000472] Detection of the label in a sample that has been incubated with the labeled, activatable antibody indicates that the sample contains the target and contains a protease that is specific for the CM of the activatable antibody. In some embodiments, the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and/or by using an agent that is specific for the protease, for example, an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase; see e.g., International Publication Number WO 2010/129609, published 11 November 2010. The same approach of using broad spectrum protease inhibitors such as those described herein, and/or by using a more selective inhibitory agent can be used to identify a protease or class of proteases specific for the CM of the activatable antibody. In some embodiments, the presence of the target can be confirmed using an agent that is specific for the target, e.g., another antibody, or the detectable label can be competed with unlabeled target. In some embodiments, unlabeled activatable antibody could be used, with detection by a labeled secondary antibody or more complex detection system. [000473] Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the activatable antibody. [000474] These techniques are also useful in kits and/or as reagents for the detection, identification or characterization of protease activity in a variety of cells, tissues, and organisms based on the protease-specific CM in the activatable antibody. [000475] In some embodiments, in situ imaging and/or in vivo imaging are useful in methods to identify which patients to treat. For example, in in situ imaging, the activatable antibodies are used to screen patient samples to identify those patients having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site. [000476] In some embodiments in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative for either or both of the target and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the activatable antibody being tested). In some embodiments, such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). [000477] In some embodiments in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the activatable antibody being tested). In some embodiments, such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). Pharmaceutical Compositions [000478] The antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies of the disclosure (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody, the conjugated antibody, activatable antibody and/or conjugated activatable antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 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. [000479] Pharmaceutical compositions according to the invention can include an antibody/activatable antibody of the invention and a carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits. [000480] In some embodiments, the pharmaceutical composition comprises an antibody of the disclosure, an activatable antibody of the disclosure, a conjugated antibody or the disclosure, and/or a conjugated activatable antibody of the disclosure, and a carrier. In some embodiments, the pharmaceutical composition comprises an additional agent. In some embodiments, the additional agent is a therapeutic agent. [000481] A pharmaceutical composition of the disclosure is 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 (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include 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 can 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. [000482] 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 ^ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, 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 such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. [000483] Sterile injectable solutions can 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 filtered sterilization. Generally, dispersions are 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. [000484] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. [000485] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. [000486] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. [000487] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. [000488] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. [000489] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. [000490] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. [000491] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES EXAMPLE 1. Characterization of Anti-Human CD47 Antibodies [000492] The studies provided herein were designed to evaluate binding of anti-human CD47 antibodies of the disclosure. [000493] An anti-human CD47 monoclonal antibody of the present disclosure was obtained using mouse hybridoma technology in accordance with techniques known in the art. Mice were immunized with human CD47 and subsequent hybridomas were screened for binding to human CD47 by ELISA and subsequently confirmed to be cytotoxic in a piggyback assay and confirmed to bind cell surfaces by FACS. The mouse anti-human CD47 H4L2 monoclonal antibody of the present disclosure includes a heavy chain variable region (VH) of SEQ ID NO: 1, and a light chain variable region (VL) of SEQ ID NO: 2. In some embodiments of the present disclosure, an exemplary antibody of the present disclosure includes a heavy chain having the H4L2 heavy chain variable region of SEQ ID NO: 1 and a light chain having the H4L2 light chain variable region of SEQ ID NO: 2. [000494] The mouse anti-human CD47 monoclonal antibody H4L2 was humanized to form humanized anti-human CD47 heavy and light chains. As shown in Table 1, exemplary embodiments of these humanized antibody heavy chains of the present disclosure include H4L2- hIgG1 heavy chain with an human IgG1 Fc domain (SEQ ID NO: 11), H4L2-hIgG1-enhanced heavy chain with a modified human IgG1 Fc domain (SEQ ID NO: 12), H4L2-hIgG4 heavy chain with a human IgG4 Fc domain (SEQ ID NO: 13), and H4L2-hIgG2s heavy chain with a modified human IgG2 Fc domain (SEQ ID NO: 14). Exemplary embodiments of these humanized antibody light chains of the present disclosure include H4L2-h-kappa light chain with a human light chain kappa domain (SEQ ID NO: 15). The amino acids sequences of these exemplary heavy and light chains of the present disclosure are shown below in Table 1. [000495] In some embodiments of the present disclosure, an exemplary antibody of the present disclosure includes the heavy chain H4L2-hIgG1 of SEQ ID NO: 11 with the light chain H4L2-h-kappa of SEQ ID NO: 15. In some embodiments of the present disclosure, an exemplary antibody of the present disclosure includes the heavy chain H4L2-hIgG1-enhanced of SEQ ID NO: 12 with the light chain H4L2-h-kappa of SEQ ID NO: 15. In some embodiments of the present disclosure, an exemplary antibody of the present disclosure includes the heavy chain H4L2-hIgG4 of SEQ ID NO: 13 with the light chain H4L2-h-kappa of SEQ ID NO: 15. In some embodiments of the present disclosure, an exemplary antibody of the present disclosure includes the heavy chain H4L2-hIgG2s of SEQ ID NO: 14 with the light chain H4L2-h-kappa of SEQ ID NO: 15. Table 1. Anti-Human CD47 Mouse and Humanized Antibody Sequences 1. Anti-huCD47 H4L2-hIgG1 H4 Heavy Chain (amino acid sequence) QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDKSTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) 2. Anti-huCD47 H4L2-hIgG1-enhanced H4 Heavy Chain (amino acid sequence) QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDKSTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12) 3. Anti-huCD47 H4L2-hIgG4 H4 Heavy Chain (amino acid sequence) QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDKSTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 13) 4. Anti-huCD47 H4L2-hIgG2s H4 Heavy Chain (amino acid sequence) QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDKSTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK VDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) 5. Anti-huCD47 H4L2-hkappa L2 Light Chain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC (SEQ ID NO: 15) 6. Anti-huCD47 H4L2 H4 VH domain (amino acid sequence) QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDKSTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 1) 7. Anti-huCD47 H4L2 L2 VL domain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 2) 8. Anti-huCD47 parental hybridoma M1-vh1 VH domain (amino acid sequence) QVQLQQPGAELVRPGASVKLSCRASGYTFTNYWMTWVKQRPEQGLEWIGRIDPYDVETHYNHKFQDKAIL TVDKSSSTAYMQLSSLTSEDSAVYYCARGGVGGMDYWGQGTSVTVSS (SEQ ID NO: 57) 9. Anti-huCD47 H1 VH domain (amino acid sequence) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMTWVRQAPGQGLEWMGRIDPYDVETHYAQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 58) 10. Anti-huCD47 H2 VH domain (amino acid sequence) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRVTI TVDESTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 59) 11. Anti-huCD47 H3 VH domain (amino acid sequence) QVQLVQSGAEVKKPGSSVKLSCKASGYTFTNYWMTWVRQAPGQGLEWIGRIDPYDVETHYAQKFQGRATL TVDESTSTAYMELSSLRSEDTAVYYCARGGVGGMDYWGQGTLVTVSS (SEQ ID NO: 60) 12. Anti-huCD47 parental hybridoma M1-vk1 VL domain (amino acid sequence) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGHSPNLLIYRVSKRFSGVPDRFSGS GSGTDFTLKITRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK (SEQ ID NO: 75) 13. Anti-huCD47 L1 VL domain (amino acid sequence) DIVMTQSPDSLAVSLGERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSLQAEDVAVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 76) 14. Anti-huCD47 L3 VH domain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSSGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 77) 15. Anti-huCD47 L4 VH domain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSSGQTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 78) 16. Anti-huCD47 L5 VH domain (amino acid sequence) DVVMTQSPDSLAVSLGERATINCRSSQSIVHSSGSTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGS GSGTDFTLTISSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK (SEQ ID NO: 79) [000496] Exemplary anti-CD47 antibodies of the present disclosure were assayed for their affinity to human and cynomolgus CD47 antigens using standard ELISA methods. These binding affinities were compared to anti-CD47 antibodies B6H12-IgG1 and 5F9-IgG4. As shown in Table 2, these exemplary results demonstrate that an exemplary antibody of the present disclosure binds to human CD47 at an affinity comparable to or better than that of the 5F9-IgG4 and B6H12-IgG1 anti-CD47 antibodies. These exemplary results also show that an exemplary antibody of the present disclosure binds to human and cynomolgus CD47 at comparable affinities. Table 2: Equilibrium Dissociation Constants of Humanized Anti-Human CD47 Antibodies to Human and Cynomolgus CD47 Antigen
Figure imgf000195_0001
[000497] The anti-CD47 antibodies of the present disclosure were also assayed for their ability to block the CD47-SIRPa interaction. In this exemplary microtiter plate assay performed using standard protocols, SIRPa/human Fc fusion protein in phosphate-buffered saline (PBS) was combined with CD47/mouse Fc protein in the absence or presence of increasing concentrations of the indicated anti-CD47 antibody. Plates were subsequently washed and incubated with an HRP-conjugated anti-mouse Fc secondary antibody and developed with TMB substrate. As shown in Table 3, these exemplary results demonstrate that an exemplary antibody of the present disclosure blocks the CD47-SIRPa interaction at an IC50 comparable to or better than that of the 5F9-IgG4 and B6H12-IgG1 anti-CD47 antibodies. Table 3: Blocking Assay of human CD47-SIRPa interaction
Figure imgf000196_0001
EXAMPLE 2. Mask Discovery [000498] The studies provided herein were designed to identify and characterize masking moieties for use in activatable anti-human CD47 antibodies of the disclosure. [000499] Humanized anti-human CD47 hu H4L2 monoclonal antibody of the present disclosure (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2), was used to screen a random X15 peptide library with a total diversity of 4 x 160, where X is any amino acid, using a method similar to that described in PCT International Publication Number WO 2010/081173, published 15 July 2010. The screening included magnetic-activated cell sorting (MACS) and fluorescence- activated cell sorting (FACS). Amino acid sequences of exemplary individual clones of masking peptides (MM) that were isolated and sequenced are shown in Table 4. Table 4. Anti-Human CD47 masking peptides (MM) Clone# Sequence Sequence ID No. 47.1 LYDCYALSKSYCMGL (SEQ ID NO: 16) 47.2 GDCTYTHSKHYCESV (SEQ ID NO: 17) 47.3 LECDPYMGKAMCINY (SEQ ID NO: 18) 47.4 DRAFCRFYGTCWDTW (SEQ ID NO: 19) 47.5 EYDHCQYKLKCGLYT (SEQ ID NO: 20) 47.6 TDCTYIHSKSYCQNY (SEQ ID NO: 21) 47.7 SSCQYGWRPEVCPLK (SEQ ID NO: 22) 47.8 KCSRPTHPFKNECQN (SEQ ID NO: 23) 47.9 GWDCYSISKHICNPK (SEQ ID NO: 24) 47.10 SSLRCINMLKCYPAT (SEQ ID NO: 25) 47.11 LSCYGPKLKQECTML (SEQ ID NO: 26) 47.12 PNLCTYPRQTCPFKT (SEQ ID NO: 27) 47.13 SRCYGPYLARQCLSH (SEQ ID NO: 28) 47.14 KCLNHWNNPNHTCNK (SEQ ID NO: 29) [000500] These masking peptides were used to generate anti-human CD47 activatable antibodies of the disclosure. The sequences for certain of these anti-human CD47 activatable antibodies are shown below in Table 5. In some embodiments, these anti-human CD47 activatable antibodies include cleavable moiety 2001 (ISSGLLSGRSDNH; SEQ ID NO: 406), cleavable moiety 3001 (AVGLLAPPGGLSGRSDNH; SEQ ID NO: 412), cleavable moiety 2007 (ISSGLLSGRSDIH; SEQ ID NO: 684), cleavable moiety 2008 (ISSGLLSGRSDQH; SEQ ID NO: 685), cleavable moiety 2011 (ISSGLLSGRSDNP; SEQ ID NO: 688), cleavable moiety 2012 (ISSGLLSGRSANP; SEQ ID NO: 689), cleavable moiety 2013 (ISSGLLSGRSANI; SEQ ID NO: 690), cleavable moiety 3007 (AVGLLAPPGGLSGRSDIH; SEQ ID NO: 692), cleavable moiety 3008 (AVGLLAPPGGLSGRSDQH; SEQ ID NO: 693), cleavable moiety 3011 (AVGLLAPPGGLSGRSDNP; SEQ ID NO: 696), cleavable moiety 3012 (AVGLLAPPGGLSGRSANP; SEQ ID NO: 697), cleavable moiety 3013 (AVGLLAPPGGLSGRSANI; SEQ ID NO: 698), or cleavable moiety 5007 (APRSALAHGLF; SEQ ID NO: 446), cleavable moiety 5008 (ALAHGLFAPRSF; SEQ ID NO: 447), or cleavable moiety 5011 (GLPTFVHLPRQV ; SEQ ID NO: 450) as indicated. [000501] While certain sequences shown below include the spacer sequence of SEQ ID NO: 645, those of ordinary skill in the art appreciate that the activatable anti-human CD47 antibodies of the disclosure can include any suitable spacer sequence, such as, for example, a spacer sequence selected from the group consisting of QGQSGQG (SEQ ID NO: 30), QGQSGQ (SEQ ID NO: 31), QGQSG (SEQ ID NO: 32), QGQS (SEQ ID NO: 33), QGQ, QG, GQSGQG (SEQ ID NO: 34), QSGQG (SEQ ID NO: 35), SGQG (SEQ ID NO: 36), GQG, G, or Q. In some embodiments, the activatable anti-human CD47 antibodies of the disclosure can have no spacer sequence joined to its N-terminus. Table 5. Anti-Human CD47 Activatable Antibody Sequences 8. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.1-5007 VL domain (SEQ ID NO: 41)] [QGQSGQG][LYDCYALSKSYCMGLGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 61) 9. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.2-5007 VL domain (SEQ ID NO: 42)] [QGQSGQG][GDCTYTHSKHYCESVGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 62) 10. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.3-5007 VL domain (SEQ ID NO: 43)] [QGQSGQG][LECDPYMGKAMCINYGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 63) 11. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.4-5007 VL domain (SEQ ID NO: 44)] [QGQSGQG][DRAFCRFYGTCWDTWGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 64) 12. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.5-5007 VL domain (SEQ ID NO: 45)] [QGQSGQG][EYDHCQYKLKCGLYTGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 65) 13. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.6-5007 VL domain (SEQ ID NO: 46)] [QGQSGQG][TDCTYIHSKSYCQNYGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 66) 14. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.7-5007 VL domain (SEQ ID NO: 47)] [QGQSGQG][SSCQYGWRPEVCPLKGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 67) 15. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.8-5007 VL domain (SEQ ID NO: 48)] [QGQSGQG][KCSRPTHPFKNECQNGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 68) 16. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.9-5007 VL domain (SEQ ID NO: 49)] [QGQSGQG][GWDCYSISKHICNPKGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 69) 17. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.10-5007 VL domain (SEQ ID NO: 50)] [QGQSGQG][SSLRCINMLKCYPATGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 70) 18. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.11-5007 VL domain (SEQ ID NO: 51)] [QGQSGQG][LSCYGPKLKQECTMLGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 71) 19. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.12-5007 VL domain (SEQ ID NO: 52)] [QGQSGQG][PNLCTYPRQTCPFKTGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 72) 20. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.13-5007 VL domain (SEQ ID NO: 53)] [QGQSGQG][SRCYGPYLARQCLSHGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 73) 21. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.14-5007 VL domain (SEQ ID NO: 54)] [QGQSGQG][KCLNHWNNPNHTCNKGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIK] (SEQ ID NO: 74) 22. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.1-5007 light chain (SEQ ID NO: 81)] [QGQSGQG][LYDCYALSKSYCMGLGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 101) 23. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.2-5007 Light chain (SEQ ID NO: 82)] [QGQSGQG][GDCTYTHSKHYCESVGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 102) 24. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.3-5007 Light chain (SEQ ID NO: 83)] [QGQSGQG][LECDPYMGKAMCINYGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 103) 25. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.4-5007 Light chain (SEQ ID NO: 84)] [QGQSGQG][DRAFCRFYGTCWDTWGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 104) 26. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.5-5007 Light chain (SEQ ID NO: 85)] [QGQSGQG][EYDHCQYKLKCGLYTGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 105) 27. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.6-5007 Light chain (SEQ ID NO: 86)] [QGQSGQG][TDCTYIHSKSYCQNYGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 106) 28. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.7-5007 Light chain (SEQ ID NO: 87)] [QGQSGQG][SSCQYGWRPEVCPLKGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 107) 29. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.8-5007 Light chain (SEQ ID NO: 88)] [QGQSGQG][KCSRPTHPFKNECQNGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 108) 30. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.9-5007 Light chain (SEQ ID NO: 89)] [QGQSGQG][GWDCYSISKHICNPKGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 109) 31. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.10-5007 Light chain (SEQ ID NO: 90)] [QGQSGQG][SSLRCINMLKCYPATGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 110) 32. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.11-5007 Light chain (SEQ ID NO: 91)] [QGQSGQG][LSCYGPKLKQECTMLGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 111) 33. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.12-5007 Light chain (SEQ ID NO: 92)] [QGQSGQG][PNLCTYPRQTCPFKTGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 112) 34. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.13-5007 Light chain (SEQ ID NO: 93)] [QGQSGQG][SRCYGPYLARQCLSHGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 113) 35. [Spacer (SEQ ID NO: 30)][huCD47 H4L2-47.14-5007 Light chain (SEQ ID NO: 94)] [QGQSGQG][KCLNHWNNPNHTCNKGGGSSGGSAPRSALAHGLFGGGSDVVMTQSPDSLAVSLG ERATINCRSSQSIVHSNGNTYLEWYQQKPGQPPKLLIYRVSKRFSGVPDRFSGSGSGTDFTLTI SSVQAEDVGVYYCFQGSHVPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 114) [000502] As shown in Figure 1A, the binding affinity of the anti-human CD47 activatable antibodies of the present disclosure to human CD47 polypeptide were assayed using an ELISA. The activatable antibodies of the present disclosure that were assayed in this exemplary study were (a) hu H4L2-IgG4 HC / H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 41), hu H4L2-IgG4 HC/ H4L2-47.2-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 42), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 43), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 44), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 45), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 46), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 47), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 48), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 49), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 50), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 51), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 52), hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 53), and hu H4L2-IgG4 HC/ H4L2-47.1-5007 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 54). Humanized anti-human CD47 antibodies of the present disclosure hu H4L2-IgG4 HC/ H4L2 LC (VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2) were used as a control. Masking efficiencies (i.e., the ratio of the binding affinity of the activatable antibody to the binding affinity of the parental antibody) of these masking peptides of the present disclosure were determined and shown in Table 6. These exemplary results showed that all of the exemplary masking peptides demonstrated a masking efficiency of at least 2 orders of magnitude, with 12 of the 14 exemplary masking peptides showing masking efficiencies in excess of three orders of magnitude. Table 6: Masking Efficiencies of Anti-CD47 Activatable Antibodies
Figure imgf000204_0001
Figure imgf000205_0001
[000503] As shown in Figure 1B, certain of the exemplary anti-CD47 activatable antibodies were treated with the protease matriptase (overnight at 37º C) and then assayed with ELISA to determine their binding affinity to human CD47 antigen. As shown in these exemplary results, cleavage of the activatable antibodies with a protease that can specifically cleave the cleavable moiety restored the binding affinity of the activatable antibodies such that they were essentially the same as the parental anti-CD47 antibody. These exemplary results showed that exemplary anti-CD47 activatable antibodies of present disclosure, when activated by matriptase, can specifically bind to their CD47 target with an affinity comparable to that of the parental antibody. EXAMPLE 3: Antibody-Dependent Cellular Phagocytosis (ADCP) Assays of Masking Peptides [000504] The studies provided herein were designed to assay the activatable anti-human CD47 antibodies of the present disclosure to induce antibody-dependent cellular phagocytosis (ADCP) via effecting the FcgRIIa receptor pathway. As shown in these exemplary results, intact activatable antibodies that contain masking peptides of the present disclosure showed lower ADCP activation via the FcgRIIa receptor pathway compared to the unmasked parental anti- CD47 antibody. [000505] In this assay (FcgRIIa-H ADCP Reporter Bioassay, Promega), target cells expressing a target antigen were co-cultured with an engineered effector cell line with the FcgRIIa-H receptor. In the presence of an antibody that binds the target antigen via its antigen- binding domain while simultaneously binding FcgRIIa on the effector cells via its Fc effector domain, the antibody binding results in FcgRIIa receptor clustering, intracellular signaling, and NFAT-RE-mediated luciferase activity from the effector cell line. The magnitude of luminescence resulting from the luciferase activity was measured as a proxy for ADCP. [000506] Referring to Table 7 and Figure 2, the EC50 and masking efficiencies of the indicated anti-CD47 activatable antibodies (as defined in Example 1 herein) of the present disclosure as determined in a FcgRIIa-mediated ADCP assay are shown. The amount of the indicated test article was added to the cell co-culture and the luminescence was measured. These exemplary results show that all of the exemplary anti-CD47 activatable antibodies showed masking as compared to the parental antibody (Ab). The highest observed masking efficiency was observed with anti-CD47 activatable antibodies of the present disclosure having masking peptides 47.11 and 47.12. Table 7: Masking Efficiency of CD47 Activatable Antibodies in FcgRIIa ADCP Assay
Figure imgf000206_0001
EXAMPLE 4: Antibody-Dependent Cellular Phagocytosis (ADCP) Assays of Fc Effector Domains [000507] The studies provided herein were designed to assay the activatable anti-human CD47 antibodies of the present disclosure to induce antibody-dependent cellular phagocytosis (ADCP) via effecting the FcgRIIa receptor pathway. As shown in these exemplary results, intact activatable antibodies that contain masking peptides of the present disclosure showed lower ADCP activation via the FcgRIIa receptor pathway compared to the unmasked parental anti- CD47 antibody. These exemplary results also show that antibodies and activatable antibodies of the present disclosure with IgG1 Fc effector domains showed higher levels of ADCP that those with IgG4 Fc effector domains. [000508] Referring to Table 8 and Figures 3A-3D, the IC50 and masking efficiencies of the indicated anti-CD47 activatable antibodies of the present disclosure as determined in a FcgRIIa- mediated ADCP assay, as described in Example 3. The assayed test articles were hu anti-CD47 H4L2-hIgG1 (“H4L2-IgG1”; HC of SEQ ID NO: 11, LC of SEQ ID NO: 15), hu anti-CD47 H4L2-hIgG1 enhanced (“H4L2-IgG1-en”; HC of SEQ ID NO: 12, LC of SEQ ID NO: 15), hu anti-CD47 H4L2-hIgG4 (“H4L2-IgG4”; HC of SEQ ID NO: 13, LC of SEQ ID NO: 15), hu anti- CD47 H4L2-hIgG1-47.3-5007 (“H4L2-IgG1”; HC of SEQ ID NO: 11, VL of SEQ ID NO: 83), hu anti-CD47 H4L2-hIgG1-47.10-5007 (“H4L2-IgG1”; HC of SEQ ID NO: 11, VL of SEQ ID NO: 90), and hu anti-CD47 H4L2-hIgG1-47.14-5007 (“H4L2-IgG1”; HC of SEQ ID NO: 11, VL of SEQ ID NO: 94). These antibodies of the present disclosure were tested against an anti- CD47 antibody 5F9-IgG4. The target cells tested in these assays were NCI H82 cells (Figures 3A and 3B) and Jurkat cells (Figures 3C and 3D). Table 8: FcgRIIa-Mediated ADCP Assay of CD47 Antibodies and Activatable Antibodies
Figure imgf000207_0001
[000509] These exemplary results show that the exemplary anti-CD47 antibodies based on IgG1 effector Fc domains showed higher ADCP activity compared to those with IgG4 domains. These exemplary results showed that the exemplary anti-CD47 activatable antibodies when intact showed lower ADCP activity compared to the unmasked the parental antibody (Ab). EXAMPLE 5: Antibody-Dependent Cellular Phagocytosis (ADCP) Assays of Fc Effector Domains [000510] The studies provided herein were designed to assay the activatable anti-human CD47 antibodies of the present disclosure to measure their ability to induce antibody-dependent cellular phagocytosis (ADCP). These exemplary results also show that antibodies and activatable antibodies of the present disclosure with IgG1 Fc effector domains showed higher levels of ADCP that those with IgG2s Fc effector domains. [000511] In this assay, fresh peripheral blood mononuclear cells (PBMCs) were isolated from 3 donors using Ficoll histopaque solution. The PBMCs were sorted and purified using anti- CD14 micromagnetic beads and validated with anti-CD14 APC antibody and FACS analysis. The purified cells were cultured for 14 days and differentiated macrophages were validated using CD11b PE antibody followed with FACS analysis. Approximately 25,000 macrophage cells per well were plated into a 96 well plate. Each test article was prepared in PBS at the indicated concentration an added to the plate. Approximately 1x106 target cells were washed and labeled with the fluorescent dye and added to the 96 well plate containing the macrophages. The plates were scanned at 10x imaging for every 30 min using Incucyte S3 and the intensity was measured as shown. [000512] Referring to Figures 4A-4C, the assayed test articles were hu anti-CD47 H4L2- hIgG1 (“H4L2-IgG1”; HC of SEQ ID NO: 11, LC of SEQ ID NO: 15), hu anti-CD47 H4L2- hIgG1 enhanced (“H4L2-IgG1-en”; HC of SEQ ID NO: 12, LC of SEQ ID NO: 15), and hu anti- CD47 H4L2-hIgG2s (“H4L2-IgG2s”; HC of SEQ ID NO: 14, LC of SEQ ID NO: 15). The target cells tested in these assays were Daudi cells, derived from human B lymphoblast cells. [000513] These exemplary results showed that an ADCP effect was observed with H4L2- IgG1 and H4L2-IgG1-en anti-CD47 antibodies of the present disclosure, which was great than that observed for H4L2-IgG2s of the present disclosure. EXAMPLE 6: Anti-Human CD47-AADC in vivo Efficacy in H82 and Raji Xenograft Models [000514] This Example shows that anti-human CD47 activatable antibodies of the present disclosure are efficacious in mouse xenograft models compared to a vehicle control. These exemplary results show that these anti-CD47 antibodies of the present disclosure show in vivo efficacy in this model compared to a vehicle control. [000515] FIGS.5A and 5B shows the efficacy of anti-human CD47 antibodies of the present disclosure hu anti-CD47 H4L2-hIgG1 (“H4L2-IgG1”; HC of SEQ ID NO: 11, LC of SEQ ID NO: 15), hu anti-CD47 H4L2-hIgG1 enhanced (“H4L2-IgG1 en”; HC of SEQ ID NO: 12, LC of SEQ ID NO: 15), hu anti-CD47 H4L2-hIgG4 (“H4L2-IgG4”; HC of SEQ ID NO: 13, LC of SEQ ID NO: 15), and hu anti-CD47 H4L2-hIgG2s (“H4L2-IgG2s”; HC of SEQ ID NO: 14, LC of SEQ ID NO: 15). These antibodies of the present disclosure were tested against a PBS buffer vehicle control and an anti-CD47 antibody 5F9-IgG4. The NOD/SCID mice with H82 cell line xenografts (derived from human small cell lung cancer) were dosed at 10 mg/kg every other day for 4 weeks of the indicated test article and the tumor size was measured at the indicated days. Referring to Figure 5A, these exemplary results show that the anti-CD47 antibodies of the present disclosure show anti-tumor activity that comparable to the 5F9 antibody. [000516] Referring to Figure 5B, the indicated anti-CD47 antibodies of the present disclosure were tested for in vivo efficacy in a NOD/SCID mice with CD20-expressing Raji cell xenografts (derived from human B-cell lymphoma). These antibodies of the present disclosure were tested against a PBS buffer vehicle control and the anti-CD20 antibody rituximab. The test articles were dosed at 10 mg/kg every other day for 4 weeks and the tumor size was measured at the indicated days. The test articles were also compared to rituximab, either alone or in combination with the H4L2-IgG4 antibody of the present disclosure. The exemplary results shown in Figure 5B demonstrate that the anti-CD47 antibodies of the present disclosure show anti-tumor activity higher than that of the vehicle control and rituximab, with the H4L2-IgG4 / rituximab combination showing the highest relative efficacy.

Claims

CLAIMS 1. An isolated antibody or an antigen binding fragment thereof (AB) that specifically binds human CD47 and cynomolgus monkey CD47.
2. The isolated antibody of claim 1, wherein the antibody or antigen binding fragment thereof comprises an amino acid sequence comprising amino acid sequences selected from the group consisting of: (a) the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10); (b) the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10), and (c) the VH CDR1 sequence comprising the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10).
3. The isolated antibody of claim 1 or claim 2, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 1 and 58-60, and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 2 and 76-79.
4. An isolated antibody or antigen binding fragment thereof that binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as the isolated antibody of any one of claims 1-3.
5. An isolated antibody or antigen binding fragment thereof that inhibits the binding of the isolated antibody of any one of claims 1-3 to human CD47 and/or cynomolgus monkey CD47.
6. An activatable antibody that, in an activated state, binds CD47 comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to human CD47 and cynomolgus monkey CD47; a masking moiety (MM) coupled to the AB, wherein the MM inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
7. The activatable antibody of claim 6, wherein the MM has a dissociation constant for binding to the AB that is greater than the dissociation constant of the AB to CD47.
8. The activatable antibody of claim 6 or claim 7, wherein the MM does not interfere or compete with the AB for binding to CD47 when the activatable antibody is in a cleaved state.
9. The activatable antibody of any one of claims 6-8, wherein the MM is a polypeptide of no more than 40 amino acids in length.
10. The activatable antibody of any one of claims 6-9, wherein the MM polypeptide sequence is different from that of human CD47.
11. The activatable antibody of any one of claims 6-10, wherein the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB.
12. The activatable antibody of any one of claims 6-11, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29.
13. The activatable antibody of any one of claims 6-12, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23-25, 27, and 28.
14. The activatable antibody of any one of claims 6-13, wherein the CM is a substrate for a protease that is active in diseased tissue.
15. The activatable antibody of any one of claims 6-14, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808.
16. The activatable antibody of any one of claims 6-15, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
17. The antibody of any one of claims 1-5 and 99-102 or the activatable antibody of any one of claims 6-16, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab’)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
18. The antibody of any one of claims 1-5 and 99-102 or the activatable antibody of any one of claims 6-17, wherein the AB specifically binds human CD47.
19. The activatable antibody of any one of claims 6-18, wherein AB comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10).
20. The activatable antibody of any one of claims 6-19, wherein the AB comprises a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 1 and 58-60, and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 2 and 76-79.
21. The activatable antibody of any one of claims 6-20, wherein the AB is linked to the CM.
22. The activatable antibody of any one of claims 6-21, wherein the AB is linked directly to the CM.
23. The activatable antibody of any one of claims 6-21, wherein the AB is linked to the CM via a linking peptide.
24. The activatable antibody of any one of claims 6-23, wherein the MM is linked to the CM such that the activatable antibody in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM.
25. The activatable antibody of any one of claims 6-24, wherein the activatable antibody comprises a linking peptide between the MM and the CM.
26. The activatable antibody of any one of claims 6-24, wherein the activatable antibody comprises a linking peptide between the CM and the AB.
27. The activatable antibody of any one of claims 6-26, wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
28. The activatable antibody of claim 27, wherein the two linking peptides need not be identical to each other.
29. The activatable antibody of claim 27 or claim 28, wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.
30. The activatable antibody of any one of claims 6-29, wherein the activatable antibody comprises the heavy chain sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81- 94, and 101-114.
31. The activatable antibody of any one of claims 6-29, wherein the activatable antibody comprises a combination of amino acid sequences, wherein the combination of amino acid sequences is selected from a single row in Table B, wherein for a given combination, (a) the heavy chain of the AB comprises the amino acid sequences of the VH CDR sequences corresponding to the given combination in the single row listed in Table B, (b) the light chain of the AB comprises the amino acid sequences of the VL CDR sequences corresponding to the given combination in the single row listed in Table B, (c) the MM comprises the amino acid sequence of the mask sequence (MM) corresponding to the given combination in the single row listed in Table B, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) corresponding to the given combination in the single row listed in Table B.
32. The activatable antibody of any one of claims 6-29, wherein the activatable antibody comprises a combination of amino acid sequences, wherein for a given combination of amino acid sequences, (a) the heavy chain of the AB comprises the amino acid sequences of the VH sequence or VH CDR sequences selected from the group consisting of: the VH sequence or VH CDR sequences listed in the corresponding column of Table C, (b) the light chain of the AB comprises the amino acid sequences of the VL sequence or VL CDR sequences selected from the group consisting of: the VL sequence or VL CDR sequences listed in the corresponding column of Table C, (c) the MM comprises the amino acid sequence of the mask sequence (MM) selected from the group consisting of: the MM sequences listed in the corresponding column of Table C, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) selected from the group consisting of: the CM sequences listed in the corresponding column of Table C.
33. An activatable antibody comprising an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, a MM, and a CM, wherein the activatable antibody comprises: a heavy chain sequence comprising an amino acid sequence of SEQ ID NO: 1; and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 41-54, 61-74, 81-94, and 101-114.
34. An activatable antibody comprising an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29, and a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808.
35. The activatable antibody of claim 34, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23-25, 27, and 28, and the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
36. The activatable antibody of claim 34 or 35, wherein the AB comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10).
37. The activatable antibody of any one of claims 34 to 36, wherein the AB comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8, 140-182, and 185-227.
38. An activatable anti-human CD47 antibody comprising an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically binds to the same epitope on human CD47 and/or cynomolgus monkey CD47 as the isolated antibody of any one of claims 1-5 and 99-102; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
39. An activatable anti-human CD47 antibody comprising an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB specifically cross-competes with the isolated antibody of any one of claims 1-5 and 99-102 for binding to human CD47 and/or cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
40. A conjugated antibody or conjugated activatable antibody comprising the antibody of any one of claims 1-5 and 99-102 conjugated to an agent or the activatable antibody of any one of claims 6 to 39 conjugated to an agent.
41. The conjugated antibody or conjugated activatable antibody of claim 40, wherein the agent is a toxin or fragment thereof.
42. The conjugated antibody or conjugated activatable antibody of claim 40 or claim 41, wherein the agent is a microtubule inhibitor.
43. The conjugated antibody or conjugated activatable antibody of claim 40 or claim 41, wherein the agent is a nucleic acid damaging agent.
44. The conjugated antibody or conjugated activatable antibody of claim 40 or claim 41, wherein the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof.
45. The conjugated antibody or conjugated activatable antibody of any one of claims 40-42 and 44, wherein the agent is auristatin E or a derivative thereof.
46. The conjugated antibody or conjugated activatable antibody of any one of claims 40-42 and 44, wherein the agent is monomethyl auristatin E (MMAE).
47. The conjugated antibody or conjugated activatable antibody of any one of claims 40-42 and 44, wherein the agent is monomethyl auristatin D (MMAD).
48. The conjugated antibody or conjugated activatable antibody of any one of claims 40-42 and 44, wherein the agent is a maytansinoid selected from the group consisting of DM1 and DM4.
49. The conjugated antibody or conjugated activatable antibody of any one of claims 40-42 and 44, wherein the agent is maytansinoid DM4.
50. The conjugated antibody or conjugated activatable antibody of any one of claims 40, 41, 43, and 44, wherein the agent is a duocarmycin.
51. The conjugated antibody or conjugated activatable antibody of any one of claims 40-50, wherein the agent is conjugated to the AB via a linker.
52. The conjugated antibody or conjugated activatable antibody of any one of claims 40-51, wherein the linker with which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety.
53. The conjugated antibody or conjugated activatable antibody of any one of claims 40-52, wherein the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, a vc-duocarmycin moiety, or a PEG2-vc-MMAD moiety.
54. The conjugated antibody or conjugated activatable antibody of claim 51, wherein the linker is a cleavable linker.
55. The conjugated antibody or conjugated activatable antibody of claim 51, wherein the linker is a non-cleavable linker.
56. The conjugated antibody or conjugated activatable antibody of any one of claims 40 and 51-55, wherein the agent is a detectable moiety.
57. The conjugated antibody or conjugated activatable antibody of claim 56, wherein the detectable moiety is a diagnostic agent.
58. A conjugated activatable antibody that, in an activated state, binds CD47 comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to human CD47 and cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease; and an agent conjugated to the AB.
59. The conjugated activatable antibody of claim 58, wherein the agent is selected from the group consisting of a dolastatin or a derivative thereof, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, a calicheamicin or a derivative thereof, and a pyrrolobenzodiazepine or a derivative thereof.
60. The conjugated activatable antibody of claim 58 or 59, wherein the agent is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a calicheamicin, a duocarmycin, a pyrrolobenzodiazepine, and a pyrrolobenzodiazepine dimer.
61. The conjugated activatable antibody of any one of claims 58-60, wherein the agent is conjugated to the AB via a linker.
62. The conjugated activatable antibody of any one of claims 58-61, wherein the linker with which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety.
63. The conjugated activatable antibody of any one of claims 58-62, wherein the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, a vc-duocarmycin moiety, or a PEG2-vc-MMAD moiety.
64. The conjugated activatable antibody of any one of claims 58-63, wherein the antibody or antigen binding fragment thereof comprises the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10).
65. The conjugated activatable antibody of any one of claims 58-64, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 1 and 58-60, and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 2 and 76-79.
66. The conjugated activatable antibody of any one of claims 58-65, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29.
67. The conjugated activatable antibody of any one of claims 58-66, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23- 25, 27, and 28.
68. The conjugated activatable antibody of any one of claims 58-67, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808.
69. The conjugated activatable antibody of any one of claims 58-68, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
70. The conjugated activatable antibody of any one of claims 58-69, wherein the activatable antibody comprises a combination of amino acid sequences, wherein the combination of amino acid sequences is selected from a single row in Table B, wherein for a given combination, (a) the heavy chain of the AB comprises the amino acid sequences of the VH CDR sequences corresponding to the given combination in the single row listed in Table B, (b) the light chain of the AB comprises the amino acid sequences of the VL CDR sequences corresponding to the given combination in the single row listed in Table B, (c) the MM comprises the amino acid sequence of the mask sequence (MM) corresponding to the given combination in the single row listed in Table B, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) corresponding to the given combination in the single row listed in Table B.
71. The conjugated activatable antibody of any one of claims 58-70, wherein the activatable antibody comprises a combination of amino acid sequences, wherein for a given combination of amino acid sequences, (a) the heavy chain of the AB comprises the amino acid sequences of the VH sequence or VH CDR sequences selected from the group consisting of: the VH sequence or VH CDR sequences listed in the corresponding column of Table C, (b) the light chain of the AB comprises the amino acid sequences of the VL sequence or VL CDR sequences selected from the group consisting of: the VL sequence or VL CDR sequences listed in the corresponding column of Table C, (c) the MM comprises the amino acid sequence of the mask sequence (MM) selected from the group consisting of: the MM sequences listed in the corresponding column of Table C, and (d) the CM comprises the amino acid sequence of the substrate sequence (CM) selected from the group consisting of: the CM sequences listed in the corresponding column of Table C.
72. The conjugated activatable antibody of any one of claims 58-71, wherein the activatable antibody comprises: a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and 11-14; and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 15, 41-57, 61-77, 81-97, and 101-117.
73. A conjugated antibody comprising: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD47, wherein the AB comprises: (i) the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10), or (ii) a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 1 and 58-60, and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 2 and 76-79; (b) an agent conjugated to the AB, wherein the agent is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, a duocarmycin, and a calicheamicin.
74. A conjugated activatable antibody that, in an activated state, binds to CD47, comprising: an antibody or an antigen binding fragment thereof (AB) that specifically binds to human CD47 and cynomolgus monkey CD47; a masking moiety (MM) that inhibits the binding of the AB to CD47 when the activatable antibody is in an uncleaved state; a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease; and an agent conjugated to the AB, wherein the AB comprises: (i) the VH CDR1 sequence comprising the amino acid sequence GYTFTNY (SEQ ID NO: 3) or the amino acid sequence NYWMT (SEQ ID NO: 4); a VH CDR2 sequence comprising the amino acid sequence DPYDVE (SEQ ID NO: 5) or the amino acid sequence RIDPYDVETHYAQKFQG (SEQ ID NO: 6) or the amino acid sequence RIDPYDVETHYNHK (SEQ ID NO: 39); a VH CDR3 sequence comprising the amino acid sequence GGVGGMDY (SEQ ID NO: 7); a VL CDR1 sequence comprising the amino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 8) or RSSQSIVHSSGNTYLE (SEQ ID NO: 40) or RSSQSIVHSSGQTYLE (SEQ ID NO: 55) or RSSQSIVHSSGSTYLE (SEQ ID NO: 56); a VL CDR2 sequence comprising the amino acid sequence RVSKRFS (SEQ ID NO: 9); and a VL CDR3 sequence comprising the amino acid sequence FQGSHVPRT (SEQ ID NO: 10), or (ii) a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 1 and 57-59, and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NOs: 2 and 76-79, or (iii) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 11-14, and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 15, 41-57, 61-77, 81-97, and 101-117; and wherein the agent is selected from the group consisting of auristatin E, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), maytansinoid DM4, maytansinoid DM1, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, a duocarmycin, and a calicheamicin.
75. The conjugated activatable antibody of claim 74, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-29.
76. The conjugated activatable antibody of claim 74 or claim 75, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23- 25, 27, and 28.
77. The conjugated activatable antibody of any one of claims 74-76, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-458, 680-698, 713, 714, and 789-808.
78. The conjugated activatable antibody of any one of claims 74-77, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 359, 370, 377, 382, 390, 397, 406-425, 429, 430, 446, 447, 450, 680-698, 713, 714, and 807-808.
79. The conjugated activatable antibody of any one of claims 74-78, wherein the agent is conjugated to the AB via a linker, and wherein the linker to which the agent is conjugated to the AB comprises an SPDB moiety, a vc moiety, or a PEG2-vc moiety.
80. The conjugated activatable antibody of any one of claims 74-79, wherein the linker and toxin conjugated to the AB comprises an SPDB-DM4 moiety, a vc-MMAD moiety, a vc-MMAE moiety, a vc-duocarmycin moiety, or a PEG2-vc-MMAD moiety.
81. A conjugated activatable antibody or conjugated antibody comprising: an antibody or antigen binding fragment thereof (AB) that, in an activated state, binds CD47; and a toxin conjugated to the AB via a linker, wherein the conjugated activatable antibody or the conjugated antibody comprises amino acid sequences, a linker, and a toxin selected from a single row in Table D, wherein for the given combination: (a) the AB comprises a heavy chain comprising the amino acid sequence of the heavy chain sequence or heavy chain variable domain sequence corresponding to the given combination in the single row listed in Table D, (b) the AB comprises a light chain comprising the amino acid sequence of the light chain sequence or light chain variable domain sequence corresponding to the given combination in the single row listed in Table D, and (c) the linker and the toxin comprise the linker and the toxin corresponding to the given combination in the single row listed in Table D.
82. A pharmaceutical composition comprising the antibody of any one of claims 1-5 and 99-102 the activatable antibody of any one of claims 6 to 39, or the conjugated antibody or conjugated activatable antibody of any one of claims 40 to 81; and a carrier.
83. The pharmaceutical composition of claim 82 comprising an additional agent.
84. The pharmaceutical composition of claim 83, wherein the additional agent is a therapeutic agent.
85. An isolated nucleic acid molecule encoding the isolated antibody of any one of claims 1-5 and 99-102 or the activatable antibody of any one of claims 6 to 39.
86. A vector comprising the isolated nucleic acid molecule of claim 85.
87. A method of producing an antibody or an activatable antibody by culturing a cell under conditions that lead to expression of the antibody or the activatable antibody, wherein the cell comprises the nucleic acid molecule of claim 85 or the vector of claim 86.
88. A method of manufacturing an activatable antibody that, in an activated state, binds CD47, the method comprising: (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises an activatable antibody of any one of claims 6 to 39; and (b) recovering the activatable antibody.
89. A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in which diseased cells express CD47 comprising administering a therapeutically effective amount of the antibody of any one of claims 1-5 and 99-102, the activatable antibody of any one of claims 6 to 39, the conjugated antibody or conjugated activatable antibody of any one of claims 40 to 81, or the pharmaceutical composition of any one of claims 82 to 84 to a subject in need thereof.
90. The method of claim 89, wherein the disorder or disease is cancer.
91. A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease associated with cells expressing CD47 comprising administering a therapeutically effective amount of the antibody of any one of claims 1-5 and 99-102, the activatable antibody of any one of claims 6 to 39, the conjugated antibody or conjugated activatable antibody of any one of claims 40 to 81, or the pharmaceutical composition of any one of claims 82 to 84 to a subject in need thereof.
92. The method of claim 91, wherein the disorder or disease associated with cells expressing CD47 is cancer.
93. The method of claim 90 or claim 92, wherein the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2-negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non-small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer.
94. A method of inhibiting or reducing the growth, proliferation, or metastasis of cells expressing mammalian CD47 comprising administering a therapeutically effective amount of the antibody of any one of claims 1 to 5, the activatable antibody of any one of claims 6 to 39, the conjugated antibody or conjugated activatable antibody of any one of claims 40 to 81, or the pharmaceutical composition of any one of claims 82 to 84 to a subject in need thereof.
95. A method of inhibiting, blocking, or preventing the binding of a natural ligand to mammalian CD47, comprising administering a therapeutically effective amount of the antibody of any one of claims 1-5 and 99-102, the activatable antibody of any one of claims 6 to 39, the conjugated antibody or conjugated activatable antibody of any one of claims 40 to 81, or the pharmaceutical composition of any one of claims 82 to 84 to a subject in need thereof.
96. The method of any one of claim 94 or 95, wherein the expression and/or activity of the mammalian CD47 is aberrant.
97. The method of any one of claims 89-96, wherein the method comprises administering an additional agent.
98. The method of claim 97, wherein the additional agent is a therapeutic agent.
99. The isolated antibody of any one of claims 1-5, wherein the isolated antibody or antigen binding fragment thereof, when bound to CD47, blocks the interaction of CD47 with SIRPa.
100. The isolated antibody of claim 99, wherein the CD47 is human CD47 and wherein the SIRPa is human SIRPa.
101. The isolated antibody of any one of claims 1-5, wherein the isolated antibody or antigen binding fragment thereof, when bound to CD47, induces antibody-dependent cellular phagocytosis (ADCP).
102. The isolated antibody of claim 101, wherein the ADCP is induced via binding of the Fc domain of the antibody to a Fc receptor.
103. The activatable antibody of any one of claims 6-39 or the conjugated antibody of any one of claims 40-57, 73, and 81 or the conjugated activatable antibody of any one of claims 40-72 and 74-81, wherein the AB when bound to CD47, blocks the interaction of CD47 with SIRPa.
104. The activatable antibody of claim 103, wherein the CD47 is human CD47 and wherein the SIRPa is human SIRPa.
105. The activatable antibody of any one of claims 6-39 or the conjugated antibody of any one of claims 40-57, 73, and 81 or the conjugated activatable antibody of any one of claims 40-72 and 74-81, wherein the AB when bound to CD47, induces antibody-dependent cellular phagocytosis (ADCP).
106. The isolated antibody of claim 105, wherein the ADCP is induced via binding of the Fc domain of the antibody to a Fc receptor.
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