Patent Publication Number: US-2022233705-A1

Title: Combined therapies of activatable immune checkpoint inhibitors and conjugated activatable antibodies

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application Nos. 62/810,68, filed Feb. 26, 2019, and 62/825,228, filed Mar. 28, 2019, the contents of which are incorporated herein by reference in their entireties. 
    
    
     FIELD OF THE INVENTION 
     This invention generally relates to methods for administering and compositions combinations of conjugated activatable antibodies and activatable immune checkpoint inhibitors for the treatment of cancer. 
     REFERENCE TO SEQUENCE LISTING 
     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-060-PCT_ST25” is incorporated herein by reference. The electronic copy of the Sequence Listing was created on Feb. 13, 2020, and the disk size is 85 kilobytes. 
     BACKGROUND OF THE INVENTION 
     Antibody-based therapies have proven to be effective treatments for several diseases, including cancers, 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. 
     Strategies have been developed to provide prodrugs of therapeutic antibodies, including antibody drug conjugates. Such antibody prodrugs are administered in a relatively inactive (or significantly less active) form, which can increase the therapeutic index of the parental antibody. Once administered, the prodrug antibody 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. Some antibody prodrugs may be targeted to members of the immune checkpoint family. Some antibody prodrugs may be targeted to molecules that are highly expressed in cancer cells. Some antibody prodrugs may be conjugated to cytotoxic compounds, thus yielding a prodrug version of an antibody drug conjugate. 
     Accordingly, there is a continued need in the field of prodrugs of antibody-based therapeutics antibody and antibody drug conjugates with increased therapeutic indices. 
     SUMMARY OF THE INVENTION 
     In one aspect of the invention, provided herein is a method of treating, alleviating a symptom of, or delaying the progression of a cancer in a subject, comprising (a) administering to the subject a conjugated activatable anti-CD166 antibody, and (b) administering to the subject an activatable immune checkpoint inhibitor, wherein the conjugated activatable anti-CD166 antibody comprises (i) an activatable anti-CD166 antibody comprising an antibody or an antigen binding fragment thereof (AB1) that specifically binds to the mammalian CD166, a masking moiety (MM1) that inhibits the binding of the AB1 to the mammalian CD166 when the activatable anti-CD166 antibody is in an uncleaved state, and a cleavable moiety (CM1) coupled to the AB1, wherein the CM1 is a polypeptide that functions as a substrate for a protease, and (ii) a toxin or toxic fragment thereof conjugated to the activatable anti-CD166 antibody. In some embodiments, the immune checkpoint is selected from the group consisting of: A2AR, B7-H3 (CD276), B7-H4, BTLA (CD272), CSF-1R, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, PD-L1, PD-L2, TDO, TIGIT, TIM-3, SIGLEC7 (CD328), and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically binds to the immune checkpoint. In some embodiments, the activatable immune checkpoint inhibitor is an activatable anti-immune checkpoint antibody that comprises an antibody or an antigen binding fragment thereof (AB2) that specifically binds to the immune checkpoint, a masking moiety (MM2) that inhibits the binding of the AB2 to the immune checkpoint when the activatable anti-immune checkpoint antibody is in an uncleaved state, and a cleavable moiety (CM2) coupled to the AB2, wherein the CM2 is a polypeptide that functions as a substrate for a protease. In some embodiments, the antigen binding fragment thereof of AB1 and/or AB2 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 MM1 is linked to the CM1 such that the activatable antibody in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM1-CM1-AB1 or AB1-CM1-MM1. 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: MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1. In some embodiments, the agent is a toxin or toxic fragment thereof. In some embodiments, the agent is a microtubule inhibitor or 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, a pyrrolobenzodiazepine or a derivative thereof, and a vinca alkaloid or a derivative thereof. In some embodiments, the agent is auristatin E, monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), a duocarmycin, a maytansinoid selected from the group consisting of DM1 and DM4, or a vinca alkaloid selected from the group consisting of: vinblastine, vincristine, vindesine, vinorelbine, vincaminol, vineridine, vinburnine, vinpocetine, vincamine, apovincamine, minovincine, methoxyminovincine, minovincinine, vincadifformine, desoxyvincaminol, and vincamajine. In some embodiments, the agent is conjugated to the AB1 via a linker, which may be cleavable or non-cleavable. In some embodiments, the linker with which the agent is conjugated to the AB1 comprises an SPDB moiety, a valine-citrulline moiety, or a PEG2-vc moiety. 
     In some embodiments, the conjugated activatable anti-CD166 antibody is administered prior to, after, or concurrently with the administration of the activatable immune checkpoint inhibitor. In some embodiments, the conjugated activatable anti-CD166 antibody is administered concurrently with the administration of the activatable immune checkpoint inhibitor, wherein the concurrent administration is in a single composition or in separate compositions. In some embodiments, the conjugated activatable anti-CD166 antibody is administered about 1 day prior to the administration of the activatable immune checkpoint inhibitor. In some embodiments, the administering of the conjugated activatable anti-CD166 antibody and the administering of the activatable immune checkpoint inhibitor are administered as part of the same dosing schedule. In some embodiments, the conjugated activatable anti-CD166 antibody and/or the activatable immune checkpoint inhibitor are administered to the subject intravenously, intraperitoneally, or intratumorally. In some embodiments, the conjugated activatable anti-CD166 antibody and/or the activatable immune checkpoint inhibitor are administered to the subject by infusion therapy. In some embodiments, administration of the conjugated activatable anti-CD166 antibody to the subject comprises inducing immunogenic cell death in a target tissue of the subject. In some embodiments, administration of the conjugated activatable anti-CD166 antibody to the subject comprises inducing dendritic cell maturation and/or activation in the subject. In some embodiments, the conjugated activatable anti-CD166 antibody and/or the activatable immune checkpoint inhibitor are administered to the subject by infusion therapy. In some embodiments, the conjugated activatable anti-CD166 antibody and/or the activatable immune checkpoint inhibitor are administered at a sub-therapeutic dose. In some embodiments, the conjugated activatable anti-CD166 antibody and/or the activatable immune checkpoint inhibitor are administered at a therapeutically effective dose. In some embodiments, the treated subject exhibits a memory T cell response in a tumor rechallenge assay. In some embodiments, CD8+ T cells from the treated subject exhibit produce IFN-gamma in a tumor rechallenge assay. In some embodiments, CD4+ T cells from the treated subject exhibit produce IFN-gamma, IL-2, and/or TNF-alpha; in some embodiments, the CD4+ T cells are from a tumor of the subject. In some embodiments, the immune checkpoint is mammalian, human, and/or cynomolgus PD-1. In some embodiments, the activatable immune checkpoint inhibitor is an activatable anti-mammalian PD-1 antibody that comprises an antibody or an antigen binding fragment thereof (AB2) that specifically binds to mammalian PD-1, a masking moiety (MM2) that inhibits the binding of the AB2 to the mammalian PD-1 when the activatable anti-mammalian PD-1 antibody is in an uncleaved state, and a cleavable moiety (CM2) coupled to the AB2, wherein the CM2 is a polypeptide that functions as a substrate for a protease. In some embodiments, the immune checkpoint is mammalian, human, and/or cynomolgus PD-L1. In some embodiments, the activatable immune checkpoint inhibitor is an activatable anti-mammalian PD-L1 antibody that comprises an antibody or an antigen binding fragment thereof (AB2) that specifically binds to mammalian PD-L1, a masking moiety (MM2) that inhibits the binding of the AB2 to the mammalian PD-L1 when the activatable anti-mammalian PD-1 antibody is in an uncleaved state, and a cleavable moiety (CM2) coupled to the AB2, wherein the CM2 is a polypeptide that functions as a substrate for a protease. In some embodiments, the MM2 is linked to the CM2 such that the activatable antibody in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM2-CM2-AB2 or AB2-CM2-MM2. In some embodiments, the activatable antibody comprises a first linking peptide (LP3) and a second linking peptide (LP4), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM2-LP3-CM2-LP4-AB2 or AB2-LP3-CM2-LP4-MM2. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A, 1B, and 1C  depict exemplary results of studies of the levels of CD166 expression in various human immune cells. These results show that CD166 is highly expressed in blood myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDCs), as well as monocytes and B cells, and could be induced following stimulation of CD4+ T cells. 
         FIGS. 2A, 2B, and 2C  depict exemplary results of studies of transgenic CT26 mouse cell line expressing human CD166. 
         FIGS. 3A-3D  depict exemplary results of in vivo efficacy of the combination of anti-CD166 conjugated activatable antibody and anti-PD-1 activatable antibody in a syngeneic mouse model. 
         FIGS. 4A, 4B, and 4C  depict exemplary results of a rechallenge assay of mice, showing that mice that were protected during the rechallenge assay had established an immunological memory response resulting from the combination treatment. 
         FIGS. 5A-5G  depict exemplary results showing that depleting a tumor-bearing mouse of CD8+ T cells resulted in a lower anti-tumor in vivo efficacy of activatable anti-CD166 antibody drug conjugate in monotherapy or in combination with activatable anti-PD-1 antibody. 
         FIG. 6  depicts the extent of CD8+ T-cell depletion in the mice in these exemplary studies. 
         FIGS. 7A and 7B  depict exemplary results of the cytotoxicity of various test articles to mature dendritic cells and activated T cells. 
         FIGS. 8A and 8B  depict exemplary results of the effect of anti-CD166 antibody drug conjugate on promoting dendritic cell maturation and T-cell co-stimulation. 
         FIGS. 9A, 9B, and 9C  depict exemplary results showing that free DM4 and anti-CD166 ADC can increase signals associated with immunogenic cell death in cancer cells and CD166-expressing cells. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides activatable monoclonal antibodies that specifically bind CD166, also known as activated leukocyte cell adhesion molecule (ALCAM). In some embodiments, the activatable monoclonal antibodies are internalized by CD166-containing cells. CD166 is a cell adhesion molecule that binds CD6, a cell surface receptor that belongs to the scavenger receptor cysteine-rich (SRCR) protein superfamily (SRCRSF). CD166 is known to be associated with cell-cell and cell-matrix interactions, cell adhesion, cell migration, and T-cell activation and proliferation. Aberrant expression and/or activity of CD166 and CD166-related signaling has been implicated in the pathogenesis of many diseases and disorders, such as cancer, inflammation, and autoimmunity. For example, CD166 is highly expressed in a variety of cancer types such as, for example, prostate cancer, breast cancer, lung cancer such as NSCLC and/or SCLC, oropharyngeal cancer, cervical cancer, and head and neck cancer such as HNSCC. 
     The disclosure provides activatable anti-CD166 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 aberrant CD166 expression and/or activity. For example, the activatable anti-CD166 antibodies are used in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a cancer or other neoplastic condition. 
     The disclosure provides activatable anti-CD166 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 CD166. In some embodiments, the cells are associated with aberrant CD166 expression and/or activity. In some embodiments, the cells are associated with normal CD166 expression and/or activity. For example, the activatable anti-CD166 antibodies are used in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a cancer or other neoplastic condition. 
     The disclosure provides activatable anti-CD166 antibodies that are useful in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a disease or disorder in which diseased cells express CD166. In some embodiments, the diseased cells are associated with aberrant CD166 expression and/or activity. In some embodiments, the diseased cells are associated with normal CD166 expression and/or activity. For example, the activatable anti-CD166 antibodies are used in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a cancer or other neoplastic condition. 
     The activatable anti-CD166 antibodies include an antibody or antigen-binding fragment thereof that specifically binds CD166 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 CD166. The MM is coupled to the antibody/antigen-binding fragment via a sequence that includes a substrate for a protease (cleavable moiety, CM), for example, a protease that is co-localized with CD166 at a treatment site in a subject. 
     Definitions 
     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&#39;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 subjects. 
     As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: 
     As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active, e.g., antigen-binding, 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 (K d &gt;10 −6 ). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab, and F(ab′)2 fragments, scFvs, and a Fab expression library. 
     The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG 1 , IgG 2 , and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. 
     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. 
     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”) chains. 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 &amp; Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989). 
     As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin, an 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 may 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. 
     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 (K d ) of the interaction, wherein a smaller K d  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” (K on ) and the “off rate constant” (K off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of K off /K on  enables the cancellation of all parameters not related to affinity and is equal to the dissociation constant K d . (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 (K d ) is ≤1 μM, in some embodiments ≤100 nM, in some embodiments ≤10 nM, and in some embodiments ≤100 μM to about 1 μM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotide can include a label for detection, if desired. 
     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 α-, α-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, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, 0-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, α-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. 
     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”. 
     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. 
     In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. 
     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 may be used to define structural and functional domains in accordance with the disclosure. 
     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) may 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). 
     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. 
     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. 
     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 may be used. Examples of 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,  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). 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 subject. 
     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)). 
     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. 
     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. 
     The term subject includes human and veterinary subjects. 
     Activatable Antibodies (AAs) 
     The disclosure provides AAs that include an antibody or antigen-binding fragment thereof that specifically binds a mammalian target. In some embodiments, the target is mammalian CD166 (ALCAM). In some embodiments, the target is mammalian PD-1. In some embodiments, the target is mammalian PD-L1. 
     In some embodiments, the mammalian target is selected from the group consisting of a human target and a cynomolgus monkey target. In some embodiments, the AB specifically binds to human target or cynomolgus monkey target with a dissociation constant of less than 1 nM. In some embodiments, the mammalian target is a human target. In some embodiments, the mammalian target is a cynomolgus target. In some embodiments, the AB has one or more of the following characteristics: (a) the AB specifically binds to human target; and (b) the AB specifically binds to human target and cynomolgus monkey target. 
     In some embodiments, the AB has one or more of the following characteristics: (a) the AB specifically binds human CD166 and cynomolgus monkey CD166; (b) the AB inhibits binding of mammalian CD6 to mammalian CD166; (c) the AB inhibits binding of human CD6 to human CD166; and (d) the AB inhibits binding of cynomolgus monkey CD6 to cynomolgus monkey CD166. 
     In some embodiments, the AB has one or more of the following characteristics: (a) the AB specifically binds human PD-1 and/or cynomolgus monkey PD-1; (b) the AB specifically binds human PD-L1 and/or cynomolgus monkey PD-L1; (c) the AB inhibits binding of mammalian PD-L1 or PD-L2 to mammalian PD-1; (d) the AB inhibits binding of human PD-L1 or PD-L2 to human PD-1; and (e) the AB inhibits binding of cynomolgus monkey PD-L1 or PD-L2 to cynomolgus monkey PD-1. 
     In some embodiments, the AB blocks the ability of a natural ligand or receptor to bind to the mammalian target 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 mammalian CD6 to bind to the mammalian CD166 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 natural ligand or receptor of CD166 is CD6. In some embodiments, the natural ligand or receptor of PD-1 is PD-L1 or PD-L2. 
     In some embodiments, the AB blocks the ability of a natural ligand to bind to the mammalian target 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, 150 nM to 400 nM, 200 nM to 300 nM, 500 nM to 1000 nM. In some embodiments, the AB blocks the ability of mammalian CD6 to bind to the mammalian CD166 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, 15 nM to 75 nM, 30 nM to 80 nM, 40 nM to 150 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, 150 nM to 400 nM, 200 nM to 300 nM, 500 nM to 1000 nM. In some embodiments, the natural ligand or receptor of CD166 is CD6. In some embodiments, the natural ligand or receptor of PD-1 is PD-L1 or PD-L2. 
     In some embodiments, the AB of the present disclosure inhibits or reduces the growth, proliferation, and/or metastasis of cells expressing mammalian target. 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 target by specifically binding to target and inhibiting, blocking, and/or preventing the binding of a natural ligand or receptor to mammalian target. 
     The antibody or antigen-binding fragment thereof of the AA is 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 its target. In some embodiments, the MM is coupled via a sequence that includes a substrate for a protease, for example, a protease that is active in diseased tissue and/or a protease that is co-localized with the target at a treatment site in a subject. The activatable antibodies provided herein, also referred to herein interchangeably as AAs or activatable antibodies, 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 the target that is at least comparable to the corresponding, unmodified antibody, also referred to herein as the parental antibody. In some embodiments, the target is CD166, PD-L1, or PD-1. 
     The disclosure provides antibodies or antigen-binding fragments thereof (AB) that specifically bind its mammalian target, for use in the AAs. In some embodiments, the antibody includes an antibody or antigen-binding fragment thereof that specifically binds the target. In some embodiments, the antibody or antigen-binding fragment thereof that binds CD166 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 the target is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody. 
     Accordingly, provided herein are activatable antibodies (AAs) comprising: (1) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD166, a masking moiety (MM1) coupled to the AB, wherein the MM1 inhibits the binding of the AB to the mammalian target when the AA 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, 
     In some embodiments, the antibodies in the AAs of the disclosure (the ABs) specifically bind a CD166 target, such as, for example, mammalian CD166, and/or human CD166. In some embodiments, the antibodies in the AAs of the disclosure (the ABs) specifically bind a PD-1 target, such as, for example, mammalian PD-1, and/or human PD-1. In some embodiments, the antibodies in the AAs of the disclosure (the ABs) specifically bind a PD-L1 target, such as, for example, mammalian PD-L1, and/or human PD-L1. 
     In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to mammalian target. In some embodiments, the AB has a dissociation constant of about 10 nM or less for binding to mammalian target. In some embodiments, the AB has a dissociation constant of about 5 nM or less for binding to target. In some embodiments, the AB has a dissociation constant of about 1 nM or less for binding to target. In some embodiments, the AB has a dissociation constant of about 0.5 nM or less for binding to target. In some embodiments, the AB has a dissociation constant of about 0.1 nM or less for binding to target. 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 target. In some embodiments, the target is CD166, PD-L1, or PD-1. 
     In some embodiments, the AA in an uncleaned state specifically binds to mammalian target 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 122. /or less than or equal to 2000 nM. In some embodiments, the target is target, PD-L1, or PD-1. 
     In some embodiments, the AA in an uncleaned state specifically binds to mammalian target with a dissociation constant greater than or equal to 1 nM, greater than or equal to 5 nM, greater than or equal to 10 nM, greater than or equal to 15 nM, greater than or equal to 20 nM, greater than or equal to 25 nM, greater than or equal to 50 nM, greater than or equal to 100 nM, greater than or equal to 150 nM, greater than or equal to 250 nM, greater than or equal to 500 nM, greater than or equal to 750 nM, greater than or equal to 1000 nM, and 122. /or greater than or equal to 2000 nM. In some embodiments, the target is target, PD-L1, or PD-1. 
     In some embodiments, the AA in an uncleaned state specifically binds to the mammalian target 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. In some embodiments, the target is target, PD-L1, or PD-1. 
     In some embodiments, the AA in an activated state specifically binds to mammalian target 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. In some embodiments, the AA in an activated state specifically binds to mammalian target with a dissociation constant is greater than or equal to 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM. In some embodiments, the target is target, PD-L1, or PD-1. 
     In some embodiments, the AA in an activated state specifically binds to the mammalian target 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. 
     Exemplary activatable anti-CD166 antibodies of the invention include, for example, activatable antibodies (AAs) that include a heavy chain and a light chain that comprise, that are, or that are derived from, the heavy chain and light chain complementarity-determining regions (CDRs), full-length, and variable region amino acid sequences shown below: 
     
       
         
           
               
               
            
               
                   
                 Human αCD166 Heavy Chain CDRs 
               
               
                   
                 VH CDR1: 
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 GFSLSTYGMGVG 
               
               
                   
               
               
                   
                 VH CDR2: 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 NIWWSEDKH 
               
               
                   
               
               
                   
                 VH CDR3: 
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 IDYGNDYAFTY 
               
               
                   
               
               
                   
                 Human αCD166 Light Chain CDRs 
               
               
                   
                 VL CDR1: 
               
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 RSSKSLLHSNGITYLY 
               
               
                   
               
               
                   
                 VL CDR1: 
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 RSSQSLLHSNGITYLY 
               
               
                   
               
               
                   
                 VL CDR2: 
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 QMSNLAS 
               
               
                   
               
               
                   
                 VL CDR2: 
               
               
                   
                 (SEQ ID NO: 7) 
               
               
                   
                 QMSNRAS 
               
               
                   
               
               
                   
                 VL CDR3: 
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
                 AQNLELPYT 
               
               
                   
               
               
                   
                 Human αCD166 Heavy Chain 
               
               
                   
                 HuCD166_HcC 
               
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTYGMG 
               
               
                   
               
               
                   
                 VGWIRQPPGKALEWLANIWWSEDKHYSPSLKSRLT 
               
               
                   
               
               
                   
                 ITKDTSKNQVVLTITNVDPVDTATYYCVQIDYGND 
               
               
                   
               
               
                   
                 YAFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS 
               
               
                   
               
               
                   
                 GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP 
               
               
                   
               
               
                   
                 AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP 
               
               
                   
               
               
                   
                 SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF 
               
               
                   
               
               
                   
                 LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
               
               
                   
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ 
               
               
                   
               
               
                   
                 DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP 
               
               
                   
               
               
                   
                 QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW 
               
               
                   
               
               
                   
                 ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR 
               
               
                   
               
               
                   
                 WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                   
                 Human αCD166 Heavy Chain 
               
               
                   
                 HuCD166_HcC Des-HC 
               
               
                   
                 (SEQ ID NO: 10) 
               
               
                   
                 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTYGMG 
               
               
                   
               
               
                   
                 VGWIRQPPGKALEWLANIWWSEDKHYSPSLKSRLT 
               
               
                   
               
               
                   
                 ITKDTSKNQVVLTITNVDPVDTATYYCVQIDYGND 
               
               
                   
               
               
                   
                 YAFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS 
               
               
                   
               
               
                   
                 GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP 
               
               
                   
               
               
                   
                 AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP 
               
               
                   
               
               
                   
                 SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF 
               
               
                   
               
               
                   
                 LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN 
               
               
                   
               
               
                   
                 WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ 
               
               
                   
               
               
                   
                 DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP 
               
               
                   
               
               
                   
                 QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW 
               
               
                   
               
               
                   
                 ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR 
               
               
                   
               
               
                   
                 WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 
               
               
                   
               
               
                   
                 Human αCD166 Heavy Chain Variable 
               
               
                   
                 Region 
               
               
                   
                 HuCD166_HcC-VH 
               
               
                   
                 (SEQ ID NO: 11) 
               
               
                   
                 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTYGMG 
               
               
                   
               
               
                   
                 VGWIRQPPGKALEWLANIWWSEDKHYSPSLKSRLT 
               
               
                   
               
               
                   
                 ITKDTSKNQVVLTITNVDPVDTATYYCVQIDYGND 
               
               
                   
               
               
                   
                 YAFTYWGQGTLVTVSS 
               
               
                   
               
               
                   
                 Human αCD166 Light Chain 
               
               
                   
                 HuCD166_Lc1 
               
               
                   
                 (SEQ ID NO: 12) 
               
               
                   
                 DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGI 
               
               
                   
               
               
                   
                 TYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGS 
               
               
                   
               
               
                   
                 GSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQ 
               
               
                   
               
               
                   
                 GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL 
               
               
                   
               
               
                   
                 LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD 
               
               
                   
               
               
                   
                 STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV 
               
               
                   
               
               
                   
                 TKSFNRGEC 
               
               
                   
               
               
                   
                 Human αCD166 Light Chain 
               
               
                   
                 Variable Region 
               
               
                   
                 HuCD166_Lc1-VL 
               
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGI 
               
               
                   
               
               
                   
                 TYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGS 
               
               
                   
               
               
                   
                 GSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGQ 
               
               
                   
               
               
                   
                 GTKLEIK 
               
            
           
         
       
     
     In some embodiments, the serum half-life of the AA is longer than that of the corresponding antibody; e.g., the pK of the AA is longer than that of the corresponding antibody. In some embodiments, the serum half-life of the AA is similar to that of the corresponding antibody. In some embodiments, the serum half-life of the AA is at least 15 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 12 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 11 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 10 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 9 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 8 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the AA is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 16 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 14 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 6 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the AA is at least 3 hours when administered to an organism. 
     Exemplary Activatable Antibodies 
     In exemplary embodiments, the AAs of the disclosure comprise any one or more of the following sequences: 
     
       
         
           
               
               
            
               
                   
                 Human anti-CD166 Heavy Chain 
               
               
                   
                 (HuCD166_HcC)-Amino Acid Sequence 
               
               
                   
                 (provided above) 
               
               
                   
                 SEQ ID NO: 9 
               
               
                   
               
               
                   
                 Human anti-CD166 Heavy Chain variable 
               
               
                   
                 region (HuCD166_HcC-VH)- 
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 (provided above) 
               
               
                   
                 SEQ ID NO: 11 
               
               
                   
               
               
                   
                 Human anti-CD166 Heavy Chain 
               
               
                   
                 (HuCD166_HcC)-Des-HC-Amino Acid 
               
               
                   
                 Sequence (provided above) 
               
               
                   
                 SEQ ID NO: 10 
               
               
                   
               
               
                   
                 Human anti-CD166 Light Chain (HuCD166_Lc1)- 
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 (provided above) 
               
               
                   
                 SEQ ID NO: 12 
               
               
                   
               
               
                   
                 Human anti-CD166 Light Chain variable region 
               
               
                   
                 (HuCD166_Lcl-VL)-Amino 
               
               
                   
                 Acid Sequence (provided above) 
               
               
                   
                 SEQ ID NO: 13 
               
               
                   
               
               
                   
                 Human αCD166 Light Chain 
               
               
                   
                 (spacer-MM-LP1-CM-LP2-Ab) Amino Acid 
               
               
                   
                 Sequence 
               
               
                   
                 [spacer (SEQ ID NO: 14)] 
               
               
                   
                 [huCD166Lc1_7614.6_3001 (SEQ ID NO: 15)] 
               
               
                   
                 SEQ ID NO: 16 
               
               
                   
                 [QGQSGQG][LCHPAVLSAWESCSSGGGS 
               
               
                   
               
               
                   
                 SGGSAVGLLAPPGGLSGRSDNHGGSDIVMTQSPLS 
               
               
                   
               
               
                   
                 LPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKP 
               
               
                   
               
               
                   
                 GQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLKI 
               
               
                   
               
               
                   
                 SRVEAEDVGVYYCAQNLELPYTFGQGTKLEIKRTV 
               
               
                   
               
               
                   
                 AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK 
               
               
                   
               
               
                   
                 VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT 
               
               
                   
               
               
                   
                 LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 9 Human αCD166 Light Chain 
               
               
                   
                 (MM-LP1-CM-LP2-Ab) Amino Acid Sequence 
               
               
                   
                 huCD166Lc1_7614.6_3001 
               
               
                   
                 SEQ ID NO: 15 
               
               
                   
                 LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLS 
               
               
                   
               
               
                   
                 GRSDNHGGSDIVMTQSPLSLPVTPGEPASISCRSS 
               
               
                   
               
               
                   
                 KSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLAS 
               
               
                   
               
               
                   
                 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQN 
               
               
                   
               
               
                   
                 LELPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLK 
               
               
                   
               
               
                   
                 SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE 
               
               
                   
               
               
                   
                 SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV 
               
               
                   
               
               
                   
                 THQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Human αCD166 Light Chain variable 
               
               
                   
                 region (spacer-MM-LP1-CM-LP2-Ab) 
               
               
                   
                 [spacer (SEQ ID NO: 14)][huCD166Lc1_ 
               
               
                   
                 7614.6_3001 (SEQ ID NO: 17)] 
               
               
                   
                 SEQ ID NO: 18 
               
               
                   
                 [QGQSGQG][LCHPAVLSAWESCSSGGGS 
               
               
                   
               
               
                   
                 SGGSAVGLLAPPGGLSGRSDNHGGSDIVMTQSPLS 
               
               
                   
               
               
                   
                 LPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKP 
               
               
                   
               
               
                   
                 GQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLKI 
               
               
                   
               
               
                   
                 SRVEAEDVGVYYCAQNLELPYTFGQGTKLEIK] 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Human αCD166 Light Chain variable 
               
               
                   
                 region (MM-LP1-CM-LP2-Ab) 
               
               
                   
                 huCD166Lc1_7614.6_3001 
               
               
                   
                 SEQ ID NO: 17 
               
               
                   
                 LCHPAVLSAWESCSSGGGSSGGSAVGLLAPPGGLS 
               
               
                   
               
               
                   
                 GRSDNHGGSDIVMTQSPLSLPVTPGEPASISCRSS 
               
               
                   
               
               
                   
                 KSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLAS 
               
               
                   
               
               
                   
                 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQN 
               
               
                   
               
               
                   
                 LELPYTFGQGTKLEIK 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Spacer 
               
               
                   
                 SEQ ID NO: 14 
               
               
                   
                 QGQSGQG 
               
               
                   
                 Masking Moiety 7614.6 
               
               
                   
                 SEQ ID NO: 19 
               
               
                   
                 LCHPAVLSAWESCSS 
               
               
                   
               
               
                   
                 Cleavable Moiety 3001 
               
               
                   
                 SEQ ID NO: 20 
               
               
                   
                 AVGLLAPPGGLSGRSDNH 
               
               
                   
               
               
                   
                 Linking peptide 1 (LP1) 
               
               
                   
                 SEQ ID NO: 21 
               
               
                   
                 GGGSSGGS 
               
               
                   
               
               
                   
                 Linking Peptide 2 (LP2) 
               
               
                   
                 GGS 
               
               
                   
               
               
                   
                 Human αPD-1 Heavy Chain CDRs 
               
               
                   
                 VH CDR1: 
               
               
                   
                 (SEQ ID NO: 51) 
               
               
                   
                 GFTFSGYAMS 
               
               
                   
               
               
                   
                 VH CDR2: 
               
               
                   
                 (SEQ ID NO: 52) 
               
               
                   
                 YISNSGGNAH 
               
               
                   
               
               
                   
                 VH CDR3: 
               
               
                   
                 (SEQ ID NO: 53) 
               
               
                   
                 EDYGTSPFVY 
               
               
                   
               
               
                   
                 Human αPD-1 Light Chain CDRs 
               
               
                   
                 VL CDR1: 
               
               
                   
                 (SEQ ID NO: 54) 
               
               
                   
                 RASESVDAYGISFMN 
               
               
                   
               
               
                   
                 VL CDR2: 
               
               
                   
                 (SEQ ID NO: 55) 
               
               
                   
                 AASNQGS 
               
               
                   
               
               
                   
                 VL CDR3: 
               
               
                   
                 (SEQ ID NO: 56) 
               
               
                   
                 QQSKDVPWT 
               
               
                   
               
               
                   
                 Human αPD-1 Heavy Chain 
               
               
                   
                 A1.5 hIgG4 5228P 
               
               
                   
                 (SEQ ID NO: 57) 
               
               
                   
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMS 
               
               
                   
               
               
                   
                 WVRQAPGKGLEWVAYISNSGGNAHYADSVKGRFTI 
               
               
                   
               
               
                   
                 SRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSP 
               
               
                   
               
               
                   
                 FVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES 
               
               
                   
               
               
                   
                 TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV 
               
               
                   
               
               
                   
                 LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN 
               
               
                   
               
               
                   
                 TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK 
               
               
                   
               
               
                   
                 PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG 
               
               
                   
               
               
                   
                 VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG 
               
               
                   
               
               
                   
                 KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL 
               
               
                   
               
               
                   
                 PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ 
               
               
                   
               
               
                   
                 PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN 
               
               
                   
               
               
                   
                 VFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                   
                 Human αPD-1 Heavy Chain 
               
               
                   
                 A1.5 hIgG4 5228P Des-HC 
               
               
                   
                 (SEQ ID NO: 58) 
               
               
                   
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMS 
               
               
                   
               
               
                   
                 WVRQAPGKGLEWVAYISNSGGNAHYADSVKGRFTI 
               
               
                   
               
               
                   
                 SRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSP 
               
               
                   
               
               
                   
                 FVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES 
               
               
                   
               
               
                   
                 TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV 
               
               
                   
               
               
                   
                 LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN 
               
               
                   
               
               
                   
                 TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK 
               
               
                   
               
               
                   
                 PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG 
               
               
                   
               
               
                   
                 VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG 
               
               
                   
               
               
                   
                 KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL 
               
               
                   
               
               
                   
                 PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ 
               
               
                   
               
               
                   
                 PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN 
               
               
                   
               
               
                   
                 VFSCSVMHEALHNHYTQKSLSLSLG 
               
               
                   
               
               
                   
                 Human αPD-1 Heavy Chain Variable Region 
               
               
                   
                 A1.5 hIgG4 5228P-VH 
               
               
                   
                 (SEQ ID NO: 60) 
               
               
                   
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMS 
               
               
                   
               
               
                   
                 WVRQAPGKGLEWVAYISNSGGNAHYADSVKGRFTI 
               
               
                   
               
               
                   
                 SRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSP 
               
               
                   
               
               
                   
                 FVYWGQGTLVTVSS 
               
               
                   
               
               
                   
                 Human αPD-1 Light Chain 
               
               
                   
                 A1.5 
               
               
                   
                 (SEQ ID NO: 59) 
               
               
                   
                 DIQLTQSPSSLSASVGDRVTITCRASESVDAYGIS 
               
               
                   
               
               
                   
                 FMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSG 
               
               
                   
               
               
                   
                 SGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQG 
               
               
                   
               
               
                   
                 TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL 
               
               
                   
               
               
                   
                 NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS 
               
               
                   
               
               
                   
                 TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT 
               
               
                   
               
               
                   
                 KSFNRGEC 
               
               
                   
               
               
                   
                 Human αPD-1 Light Chain Variable Region 
               
               
                   
                 A1.5 
               
               
                   
                 (SEQ ID NO: 61) 
               
               
                   
                 DIQLTQSPSSLSASVGDRVTITCRASESVDAYGIS 
               
               
                   
               
               
                   
                 FMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSG 
               
               
                   
               
               
                   
                 SGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQG 
               
               
                   
               
               
                   
                 TKLEIK 
               
               
                   
               
               
                   
                 Human αPD-1 Light Chain 
               
               
                   
                 (spacer-MM-LP1-CM-LP2-Ab) AA Sequence 
               
               
                   
                 [spacer (SEQ ID NO: 14)][A1.5-PD34-2011 
               
               
                   
                 (SEQ ID NO: 63)] 
               
               
                   
                 SEQ ID NO: 62 
               
               
                   
                 [QGQSGQG][TSYCSIEHYPCNTHEIGGG 
               
               
                   
               
               
                   
                 SSGGSISSGLLSGRSDNPGGGSDIQLTQSPSSLSA 
               
               
                   
               
               
                   
                 SVGDRVTITCRASESVDAYGISFMNWFQQKPGKAP 
               
               
                   
               
               
                   
                 KLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQ 
               
               
                   
               
               
                   
                 PEDFATYYCQQSKDVPWTFGQGTKLEIKRTVAAPS 
               
               
                   
               
               
                   
                 VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK 
               
               
                   
               
               
                   
                 VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA 
               
               
                   
               
               
                   
                 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC] 
               
               
                   
               
               
                   
                 Human αPD-1 Light Chain 
               
               
                   
                 (MM-LP1-CM-LP2-Ab) AA Sequence 
               
               
                   
                 A1.5-PD34-2011 
               
               
                   
                 SEQ ID NO: 63 
               
               
                   
                 TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDN 
               
               
                   
               
               
                   
                 PGGGSDIQLTQSPSSLSASVGDRVTITCRASESVD 
               
               
                   
               
               
                   
                 AYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSR 
               
               
                   
               
               
                   
                 FSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW 
               
               
                   
               
               
                   
                 TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS 
               
               
                   
               
               
                   
                 VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ 
               
               
                   
               
               
                   
                 DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL 
               
               
                   
               
               
                   
                 SSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Human αPD-1 Light Chain variable region 
               
               
                   
                 (spacer-MM-LP1-CM-LP2-Ab) 
               
               
                   
                 [spacer (SEQ ID NO: 14)] 
               
               
                   
               
               
                   
                 [A1.5-PD34-2011 (SEQ ID NO: 65)] 
               
               
                   
                 SEQ ID NO: 64 
               
               
                   
                 [QGQSGQG][TSYCSIEHYPCNTHEIGGG 
               
               
                   
               
               
                   
                 SSGGSISSGLLSGRSDNPGGGSDIQLTQSPSSLSA 
               
               
                   
               
               
                   
                 SVGDRVTITCRASESVDAYGISFMNWFQQKPGKAP 
               
               
                   
               
               
                   
                 KLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQ 
               
               
                   
               
               
                   
                 PEDFATYYCQQSKDVPWTFGQGTKLEIK] 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Human αPD-1 Light Chain variable region 
               
               
                   
                 (MM-LP1-CM-LP2-Ab) 
               
               
                   
                 A1.5-PD34-2011 
               
               
                   
                 SEQ ID NO: 65 
               
               
                   
                 TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDN 
               
               
                   
               
               
                   
                 PGGGSDIQLTQSPSSLSASVGDRVTITCRASESVD 
               
               
                   
               
               
                   
                 AYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSR 
               
               
                   
               
               
                   
                 FSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW 
               
               
                   
               
               
                   
                 TFGQGTKLEIK 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Spacer 
               
               
                   
                 SEQ ID NO: 14 
               
               
                   
                 QGQSGQG 
               
               
                   
               
               
                   
                 Masking Moiety PD34 
               
               
                   
                 SEQ ID NO: 66 
               
               
                   
                 TSYCSIEHYPCNTHE 
               
               
                   
               
               
                   
                 Cleavable Moiety 2011 
               
               
                   
                 SEQ ID NO: 67 
               
               
                   
                 ISSGLLSGRSDNP 
               
               
                   
               
               
                   
                 Linking peptide 1 (LP1) 
               
               
                   
                 SEQ ID NO: 21 
               
               
                   
                 GGGSSGGS 
               
               
                   
               
               
                   
                 Linking Peptide 2 (LP2) 
               
               
                   
                 SEQ ID NO: 36 
               
               
                   
                 GGGS 
               
               
                   
               
               
                   
                 Human αPD-L1 Heavy Chain CDRs 
               
               
                   
                 VH CDR1: 
               
               
                   
                 (SEQ ID NO: 68) 
               
               
                   
                 SYAIVIS 
               
               
                   
               
               
                   
                 VH CDR2: 
               
               
                   
                 (SEQ ID NO: 69) 
               
               
                   
                 SSIWRNGIVTVYADS 
               
               
                   
               
               
                   
                 VH CDR3: 
               
               
                   
                 (SEQ ID NO: 70) 
               
               
                   
                 WSAAFDY 
               
               
                   
               
               
                   
                 Human αPD-L1 Light Chain CDRs 
               
               
                   
                 VL CDR1: 
               
               
                   
                 (SEQ ID NO: 71) 
               
               
                   
                 RASQSISSYLN 
               
               
                   
               
               
                   
                 VL CDR2: 
               
               
                   
                 (SEQ ID NO: 72) 
               
               
                   
                 AASSLQS 
               
               
                   
                 or 
               
               
                   
                 (SEQ ID NO: 86) 
               
               
                   
               
               
                   
                 YASTLQS 
               
               
                   
               
               
                   
                 VL CDR3: 
               
               
                   
                 (SEQ ID NO: 73) 
               
               
                   
                 DNGYPST 
               
               
                   
               
               
                   
                 Human αPD-L1 Heavy Chain 
               
               
                   
                 (SEQ ID NO: 74) 
               
               
                   
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAIV 
               
               
                   
               
               
                   
                 ISWVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRF 
               
               
                   
               
               
                   
                 TISRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAF 
               
               
                   
               
               
                   
                 DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                   
               
               
                   
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                   
               
               
                   
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT 
               
               
                   
               
               
                   
                 KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP 
               
               
                   
               
               
                   
                 KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV 
               
               
                   
               
               
                   
                 EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK 
               
               
                   
               
               
                   
                 EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP 
               
               
                   
               
               
                   
                 PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 
               
               
                   
               
               
                   
                 ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV 
               
               
                   
               
               
                   
                 FSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                   
                 Human αPD-1 Heavy Chain 
               
               
                   
                 Des-HC 
               
               
                   
                 (SEQ ID NO: 75) 
               
               
                   
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMS 
               
               
                   
               
               
                   
                 WVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTI 
               
               
                   
               
               
                   
                 SRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDY 
               
               
                   
               
               
                   
                 WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA 
               
               
                   
               
               
                   
                 LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS 
               
               
                   
               
               
                   
                 SGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV 
               
               
                   
               
               
                   
                 DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKD 
               
               
                   
               
               
                   
                 TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV 
               
               
                   
               
               
                   
                 HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY 
               
               
                   
               
               
                   
                 KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS 
               
               
                   
               
               
                   
                 QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN 
               
               
                   
               
               
                   
                 NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS 
               
               
                   
               
               
                   
                 CSVMHEALHNHYTQKSLSLSLG 
               
               
                   
               
               
                   
                 Human αPD-L1 Heavy Chain Variable Region 
               
               
                   
                 (SEQ ID NO: 77) 
               
               
                   
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMS 
               
               
                   
               
               
                   
                 WVRQAPGKGLEWVSSIWRNGIVTVYADSVKGRFTI 
               
               
                   
               
               
                   
                 SRDNSKNTLYLQMNSLRAEDTAVYYCAKWSAAFDY 
               
               
                   
               
               
                   
                 WGQGTLVTVSS 
               
               
                   
               
               
                   
                 Human αPD-L1 Light Chain 
               
               
                   
                 (SEQ ID NO: 76) 
               
               
                   
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNW 
               
               
                   
               
               
                   
                 YQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD 
               
               
                   
               
               
                   
                 FTLTISSLQPEDFATYYCQQDNGYPSTFGGGTKVE 
               
               
                   
               
               
                   
                 IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY 
               
               
                   
               
               
                   
                 PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL 
               
               
                   
               
               
                   
                 SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN 
               
               
                   
               
               
                   
                 RGEC 
               
               
                   
               
               
                   
                 Human αPD-L1 Light Chain Variable Region 
               
               
                   
                 (SEQ ID NO: 78) 
               
               
                   
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY 
               
               
                   
               
               
                   
                 QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT 
               
               
                   
               
               
                   
                 LTISSLQPEDFATYYCQQDNGYPSTFGGGTKVEIKR 
               
               
                   
               
               
                   
                 Human αPD-L1 Light Chain 
               
               
                   
                 (spacer-MM-LP1-CM-LP2-Ab) AA Sequence 
               
               
                   
                 [spacer (SEQ ID NO: 85)][αPD-L1-PL07- 
               
               
                   
                 2001 (SEQ ID NO: 80)] 
               
               
                   
                 SEQ ID NO: 79 
               
               
                   
                 [QGQSGS][GIALCPSHFCQLPQTGGGSS 
               
               
                   
               
               
                   
                 GGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSL 
               
               
                   
               
               
                   
                 SASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL 
               
               
                   
               
               
                   
                 LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE 
               
               
                   
               
               
                   
                 DFATYYCQQDNGYPSTFGGGTKVEIKRTVAAPSVF 
               
               
                   
               
               
                   
                 IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD 
               
               
                   
               
               
                   
                 NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY 
               
               
                   
               
               
                   
                 EKHKVYACEVTHQGLSSPVTKSFNRGEC] 
               
               
                   
               
               
                   
                 Human αPD-L1 Light Chain 
               
               
                   
                 (MM-LP1-CM-LP2-Ab) 
               
               
                   
                 AA Sequence αPD-L1-PL07-2001 
               
               
                   
                 SEQ ID NO: 80 
               
               
                   
                 GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLS 
               
               
                   
               
               
                   
                 GRSDNHGGSDIQMTQSPSSLSASVGDRVTITCRAS 
               
               
                   
               
               
                   
                 QSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR 
               
               
                   
               
               
                   
                 FSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYPS 
               
               
                   
               
               
                   
                 TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS 
               
               
                   
               
               
                   
                 VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ 
               
               
                   
               
               
                   
                 DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL 
               
               
                   
               
               
                   
                 SSPVTKSFNRGEC 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Human αPD-L1 Light Chain variable region 
               
               
                   
                 (spacer-MM-LP1-CM-LP2-Ab) 
               
               
                   
                 [spacer (SEQ ID NO: 85)][αPD-L1-PL07-2001 
               
               
                   
                 (SEQ ID NO: 82)] 
               
               
                   
                 SEQ ID NO: 81 
               
               
                   
                 [QGQSGS][GIALCPSHFCQLPQTGGGSS 
               
               
                   
               
               
                   
                 GGSGGSGGISSGLLSGRSDNHGGSDIQMTQSPSSL 
               
               
                   
               
               
                   
                 SASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL 
               
               
                   
               
               
                   
                 LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE 
               
               
                   
               
               
                   
                 DFATYYCQQDNGYPSTFGGGTKVEIKRTVAAPSVF 
               
               
                   
               
               
                   
                 IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD 
               
               
                   
               
               
                   
                 NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY 
               
               
                   
               
               
                   
                 EKHKVYACEVTHQGLSSPVTKSFNRGEC] 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Human αPD-L1 Light Chain variable region 
               
               
                   
                 (MM-LP1-CM-LP2-Ab) αPD-L1-PL07-2001 
               
               
                   
                 SEQ ID NO: 82 
               
               
                   
                 GIALCPSHFCQLPQTGGGSSGGSGGSGGISSGLLS 
               
               
                   
               
               
                   
                 GRSDNHGGSDIQMTQSPSSLSASVGDRVTITCRAS 
               
               
                   
               
               
                   
                 QSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR 
               
               
                   
               
               
                   
                 FSGSGSGTDFTLTISSLQPEDFATYYCQQDNGYPS 
               
               
                   
               
               
                   
                 TFGGGTKVEIKR 
               
               
                   
               
               
                   
                 Amino Acid Sequence 
               
               
                   
                 Spacer 
               
               
                   
                 SEQ ID NO: 85 
               
               
                   
                 QGQSGS 
               
               
                   
               
               
                   
                 Masking Moiety PLO7 
               
               
                   
                 SEQ ID NO: 83 
               
               
                   
                 GIALCPSHFCQLPQT 
               
               
                   
               
               
                   
                 Cleavable Moiety 2001 
               
               
                   
                 SEQ ID NO: 84 
               
               
                   
                 ISSGLLSGRSDNH 
               
               
                   
               
               
                   
                 Linking peptide 1 (LP1) 
               
               
                   
                 SEQ ID NO: 21 
               
               
                   
                 GGGSSGGS 
               
               
                   
               
               
                   
                 Linking Peptide 2 (LP2) 
               
               
                   
                 GGS 
               
            
           
         
       
     
     In an exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD166, wherein the AB comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 9 and a light chain comprising an amino acid sequence of SEQ ID NO: 11; (b) a masking moiety (MM) coupled to the AB, wherein the MM inhibits the binding of the AB to the mammalian CD166 when the AA is in an uncleaved state, wherein the MM comprises the amino acid sequence of SEQ ID NO: 19; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease, and wherein the CM comprises the amino acid sequence of SEQ ID NO: 20. 
     In an exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD166, wherein the AB comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 9 and a light chain comprising an amino acid sequence of SEQ ID NO: 16, and is conjugated to DM4 via spdb linker (this exemplary conjugated AA is herein referred to as “spacer-7614.6-3001-HcCD166-SPDB-DM4”), also referred to as “Combination 55”. The linker toxin SPDB-DM4 is also known as N-succinimidyl 4-(2-pyridyldithio) butanoate-N2′-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine. 
     In another exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian CD166, wherein the AB comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 9 and a light chain comprising an amino acid sequence of SEQ ID NO: 15, and is further conjugated to DM4 via spdb linker this exemplary conjugated AA is herein referred to as “7614.6-3001-HcCD166-SPDB-DM4”, also referred to as “Combination 60”). 
     In an exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-1, wherein the AB comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58 and a light chain comprising an amino acid sequence of SEQ ID NO: 59; (b) a masking moiety (MM) coupled to the AB, wherein the MM inhibits the binding of the AB to the mammalian PD-1 when the AA is in an uncleaved state, wherein the MM comprises the amino acid sequence of SEQ ID NO: 66; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease, and wherein the CM comprises the amino acid sequence of SEQ ID NO: 67. 
     In an exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-1, wherein the AB comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58 and a light chain comprising an amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63. In an exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-1, wherein the AB comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65. 
     In an exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-L1, wherein the AB comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 74 or SEQ ID NO: 75 and a light chain comprising an amino acid sequence of SEQ ID NO: 76; (b) a masking moiety (MM) coupled to the AB, wherein the MM inhibits the binding of the AB to the mammalian PD-L1 when the AA is in an uncleaved state, wherein the MM comprises the amino acid sequence of SEQ ID NO: 83; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease, and wherein the CM comprises the amino acid sequence of SEQ ID NO: 84. 
     In an exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-L1, wherein the AB comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 74 or SEQ ID NO: 75 and a light chain comprising an amino acid sequence of SEQ ID NO: 79 or SEQ ID NO: 80. In an exemplary embodiment, the AA comprises: (a) an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-L1, wherein the AB comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. 
     Masking Moieties (MM) 
     The activatable 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 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 antibodies remain masked until proteolytically activated at the site of disease. Starting with an antibody as a parental therapeutic antibody, the activatable anti-CD166 antibodies of the invention were engineered by coupling the antibody to an inhibitory mask (masking moiety, MM1) through a linker that incorporates a protease substrate (CM). 
     Accordingly, the activatable antibodies provided herein include a masking moiety (MM). In some embodiments, the MM1 is an amino acid sequence that is coupled or otherwise attached to the antibody and is positioned within the activatable antibody construct such that the MM1 reduces the ability of the antibody to specifically bind its target. 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. 
     In some embodiments, in the presence of the target, the MM reduces the ability of the AB to bind its target 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. 
     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. 
     In some embodiments, the MM polypeptide sequence is different from that of the target of the AB. 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 the target of the AB and is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of the AB. 
     In one exemplary embodiment, the AAs provided herein comprise an MM, whose amino acid sequence is set forth: 
     
       
         
           
               
               
            
               
                   
                 Masking Moiety 7614.6 
               
               
                   
                 (SEQ ID NO: 19) 
               
               
                   
                 LCHPAVLSAWESCSS 
               
            
           
         
       
     
     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. 
     The K d  of the AB modified with a MM towards the 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, 25-50, 50-250, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 500-2,500, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 2,500-5,000, 5,000-50,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 50,000-5,000,000, 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 target. Conversely, the binding affinity of the AB modified with a MM towards the 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, 25-50, 50-250, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 500-2,500, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 2,500-5,000, 5,000-50,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 50,000-5,000,000, 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 target. 
     In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind its target such that the dissociation constant (K d ) of the AB when coupled to the MM towards its target is at least two times greater than the K d  of the AB when not coupled to the MM towards its target. 
     In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind its target such that the dissociation constant (K d ) of the AB when coupled to the MM towards its target is at least five times greater than the K d  of the AB when not coupled to the MM towards its target. 
     In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind its target such that the dissociation constant (K d ) of the AB when coupled to the MM towards its target is at least 10 times greater than the K d  of the AB when not coupled to the MM towards its target. 
     In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind its target such that the dissociation constant (K d ) of the AB when coupled to the MM towards its target is at least 20 times greater than the K d  of the AB when not coupled to the MM towards its target. 
     In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind its target such that the dissociation constant (K d ) of the AB when coupled to the MM towards its target is at least 40 times greater than the K d  of the AB when not coupled to the MM towards its target. 
     In some embodiments, the coupling of the MM1 to the AB reduces the ability of the AB to bind its target such that the dissociation constant (K d ) of the AB when coupled to the MM1 towards its target is at least 100 times greater than the K d  of the AB when not coupled to the MM towards its target. 
     In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind its target such that the dissociation constant (K d ) of the AB when coupled to the MM1 towards its target is at least 1000 times greater than the K d  of the AB when not coupled to the MM1 towards its target. 
     In some embodiments, the coupling of the MM1 to the AB reduces the ability of the AB to bind its target such that the dissociation constant (K d ) of the AB when coupled to the MM1 towards its target is at least 10,000 times greater than the K d  of the AB when not coupled to the MM1 towards its target. 
     The dissociation constant (K d ) of the MM1 towards the AB is generally greater than the K d  of the AB towards the target. The K d  of the MM1 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 K d  of the AB towards the target. Conversely, the binding affinity of the MM towards the AB is generally lower than the binding affinity of the AB towards the 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 target. 
     In some embodiments, the dissociation constant (Kd) of the MM1 towards the AB is approximately equal to the Kd of the AB towards the 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 target. 
     In some embodiments, the dissociation constant (Kd) of the MM1 towards the AB is less than the dissociation constant of the AB towards the target. 
     In some embodiments, the dissociation constant (Kd) of the MM1 towards the AB is greater than the dissociation constant of the AB towards the target. 
     In some embodiments, the MM1 has a Kd for binding to the AB that is no more than the Kd for binding of the AB to the target. 
     In some embodiments, the MM1 has a Kd for binding to the AB that is less than the Kd for binding of the AB to the target. 
     In some embodiments, the MM1 has a Kd for binding to the AB that is approximately equal to the Kd for binding of the AB to the target. 
     In some embodiments, the MM1 has a Kd for binding to the AB that is no less than the Kd for binding of the AB to the target. 
     In some embodiments, the MM1 has a Kd for binding to the AB that is greater than the Kd for binding of the AB to the target. 
     In some embodiments, the dissociation constant (K d ) of the MM1 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, 25-50, 50-250, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 25-500, 500-2,500, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 2,500-5,000, 5,000-50,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 50,000-5,000,000, 100,000-1,000,000, or 100,000-10,000,000 fold greater than the Kd for binding of the AB to the target. In some embodiments, the MM1 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. 
     In some embodiments, the MM1 has an affinity for binding to the AB that is less than the affinity of binding of the AB to the target. 
     In some embodiments, the MM1 has an affinity for binding to the AB that is no more than the affinity of binding of the AB to the target. 
     In some embodiments, the MM1 has an affinity for binding to the AB that is approximately equal of the affinity of binding of the AB to the target. 
     In some embodiments, the MM1 has an affinity for binding to the AB that is no less than the affinity of binding of the AB to the target. 
     In some embodiments, the MM1 has an affinity for binding to the AB that is greater than the affinity of binding of the AB to the target. 
     In some embodiments, the MM1 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. In some embodiments, the MM1 has an affinity for binding to the AB that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-25, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, 25-250, 50-500, or 100-1,000 fold less than the affinity of binding of the AB to the target. In some embodiments, the MM1 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 MM1 not covalently linked to the AB and at equimolar concentration to the AB does not inhibit the binding of the AB to the target. 
     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 MM1 or the specific binding of the parental AB to the target. When compared to the binding of the AB not modified with an MM1 or the binding of the parental AB to the target the AB&#39;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 MM1 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 MM1 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 by 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. 
     When an AB is coupled to or modified by a MM, the MM1 ‘masks’ 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. 
     An AB coupled to or modified with an MM1 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 may be desirable to insert one or more linkers, e.g., flexible linkers, into the composition so as to provide for flexibility. 
     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. 
     Cleavable Moieties (CM) 
     The activatable antibodies provided herein include a cleavable moiety (CM). In some embodiments, the CM includes an amino acid sequence that is a substrate for a protease, usually an extracellular protease. Suitable substrates can be identified using any of a variety of known techniques. For example, peptide substrates are identified using the methods described in U.S. Pat. No. 7,666,817 by Daugherty et al.; in U.S. Pat. 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). 
     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 AA when the AA is exposed to the protease. In some embodiments, the protease is co-localized with the target in a tissue, and the protease cleaves the CM in the AA when the AA is exposed to the protease.  FIG. 1  depicts activatable antibody drug conjugates being preferentially activated in the tumor microenvironment, where tumor-specific proteases are present. 
     In some embodiments, the AAs include an AB that is modified by an MM and also includes one or more cleavable moieties (CM). Such AAs exhibit activatable/switchable binding, to the AB&#39;s target. AAs 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. 
     In some embodiments, the CM is a polypeptide of up to 15 amino acids in length. 
     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. 
     In some embodiments, the CM is a CM1-CM2 substrate whose amino acid sequence is set forth: 
     
       
         
           
               
               
            
               
                   
                 Cleavable Moiety 3001 (Substrate 3001) 
               
               
                   
                 (SEQ ID NO: 20) 
               
               
                   
                 AVGLLAPPGGLSGRSDNH 
               
            
           
         
       
     
     The elements of the AAs 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 AA 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&#39;s ability to specifically bind its target by the MM. 
     The K d  of the AB modified with a MM and a CM towards the 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, 25-50, 50-250, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 25-500, 500-2,500, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 2,500-5,000, 5,000-50,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 50,000-5,000,000, 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 and a CM or of the parental AB towards the target. Conversely, the binding affinity of the AB modified with a MM1 and a CM towards the 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, 25-50, 50-250, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 25-500, 500-2,500, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 2,500-5,000, 5,000-50,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 50,000-5,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the AB not modified with an MM1 and a CM or of the parental AB towards the target. 
     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&#39;s ability to bind the target when modified with an MM1 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. 
     As used herein, the term “cleaved state” refers to the condition of the AAs following modification of the CM by at least one protease. The term “uncleaved state”, as used herein, refers to the condition of the AAs 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 AA 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 AA 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 AAs by a covalent bond (e.g., a disulfide bond between cysteine residues). 
     By activatable or switchable is meant that the AA exhibits a first level of binding to a target when the AA 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 AA 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 AA 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. 
     The CM and AB of the AAs 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 AAs 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. 
     In some embodiments AAs 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. 
     In general, an AA can be designed by selecting an AB of interest and constructing the remainder of the AA so that, when conformationally constrained, the MM1 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. 
     AAs 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 AA in the presence of at least one protease capable of cleaving the CM of the AAs to (b) a minimum detected level of target protein binding to an AA in the absence of the protease. The dynamic range of an AA can be calculated as the ratio of the dissociation constant of an AA cleaving agent (e.g., enzyme) treatment to the dissociation constant of the AAs cleaving agent treatment. The greater the dynamic range of an activatable antibody, the better the switchable phenotype of the activatable antibody. AAs having relatively higher dynamic range values (e.g., greater than 1) exhibit more desirable switching phenotypes such that target protein binding by the AAs 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 AAs than in the absence of a cleaving agent. 
     The CM is specifically cleaved by at least one protease at a rate of about 0.001-1500×10 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 1500×10 4  M −1 S −1 . In some embodiments, the CM is specifically cleaved at a rate of about 100,000 M −1 S −1 . In some embodiments, the CM is specifically cleaved at a rate from about 1×10E2 to about 1×10E6 M −1 S −1  (i.e., from about 1×10 2  to about 1×10 6  M −1 S −1 ). 
     For specific cleavage by an enzyme, contact between the enzyme and CM is made. When the AA comprising an AB coupled to a MM1 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. 
     Structural Configurations of the Activatable Antibodies 
     The AAs of the present disclosure can be provided in a variety of structural configurations. Exemplary formulae for AAs are provided below. It is specifically contemplated that the N- to C-terminal order of the AB, MM and CM may be reversed within an activatable antibody. It is also specifically contemplated that the CM and MM1 may overlap in amino acid sequence, e.g., such that the CM is contained within the MM. 
     For example, AAs 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 may be positioned N-terminal or C-terminal to the AAs elements. 
     In many embodiments it may be desirable to insert one or more linkers, e.g., flexible linkers, into the AA 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 AA constructs may benefit from introduction of one or more amino acids to provide for a flexible linker. In addition, as described below, where the AA 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. 
     In some embodiments, the AA comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the AA 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, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GGS) n , (GSGGS), (SEQ ID NO: 22) and (GGGS) n  (SEQ ID NO: 23), where n is an integer of at least one. 
     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: 24), GGSGG (SEQ ID NO: 25), GSGSG (SEQ ID NO: 26), GSGGG (SEQ ID NO: 27), GGGSG (SEQ ID NO: 28), and GSSSG (SEQ ID NO: 29). 
     In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 30), GSSGGSGGSGG (SEQ ID NO: 31), GSSGGSGGSGGS (SEQ ID NO: 32), GSSGGSGGSGGSGGGS (SEQ ID NO: 33), GSSGGSGGSG (SEQ ID NO: 34), or GSSGGSGGSGS (SEQ ID NO: 35). 
     In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 36), GSSGT (SEQ ID NO: 37) or GSSG (SEQ ID NO: 38). 
     In some embodiments, the AB has a dissociation constant of about 100 nM or less for binding to its target. 
     For example, in certain embodiments an AA 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)-LP1-(CM)-(AB) 
       (MM)-(CM)-LP2-(AB) 
       (MM)-LP1-(CM)-LP2-(AB) 
     wherein MM, CM, and AB are as defined above; wherein LP1 and LP2 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 may be positioned N-terminal or C-terminal to the AAs 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)). 
     In some embodiments, the AA 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. 
     Linkers suitable for use in compositions described herein are generally ones that provide flexibility of the modified AB or the AAs 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 may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. 
     Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example: (GS)n, (GSGGS)n (SEQ ID NO: 22) and (GGGS)n (SEQ ID NO: 23), 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: 24), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 25), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 26), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 27), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 28), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 29), and the like. The ordinarily skilled artisan will recognize that design of an AAs 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 AAs structure. 
     In some embodiments, the AA also includes a signal peptide. In some embodiments, the signal peptide is conjugated to the AA via a spacer. In some embodiments, the spacer is conjugated to the AA 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 AA 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 AA is QGQSGQ (SEQ ID NO: 39). Other examples of a spacer joined directly to the N-terminus of MM of the AA include QGQSGQG (SEQ ID NO: 14), QGQSG (SEQ ID NO: 40), QGQS (SEQ ID NO: 41), QGQ, QG, and Q. Other examples of a spacer joined directly to the N-terminus of MM of the AA include GQSGQG (SEQ ID NO: 87), QSGQG (SEQ ID NO: 88), SGQG (SEQ ID NO: 117), 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: 39). In some embodiments, the spacer includes at least the amino acid sequence QGQSGQG (SEQ ID NO: 14). In some embodiments, the spacer includes at least the amino acid sequence QGQSG (SEQ ID NO: 40). In some embodiments, the spacer includes at least the amino acid sequence QGQS (SEQ ID NO: 41). 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: 42). In some embodiments, the spacer includes at least the amino acid sequence QSGQG (SEQ ID NO: 43). In some embodiments, the spacer includes at least the amino acid sequence SGQG (SEQ ID NO: 44). 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. 
     Conjugated Activatable Antibodies 
     The AA compositions and methods provided herein enable the attachment of one or more agents to one or more cysteine residues (e.g. cysteine, lysine) in the AB without compromising the activity (e.g., the masking, activating or binding activity) of the activatable anti-target 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 anti-target 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 anti-target antibody. The compositions and methods provided herein produce conjugated activatable anti-target antibodies in which the MM retains the ability to effectively and efficiently mask the AB of the AA in an uncleaved state. The compositions and methods provided herein produce conjugated activatable anti-target antibodies in which the AA is still activated, i.e., cleaved, in the presence of a protease that can cleave the CM. 
     In some embodiments, the AAs described herein also include an agent conjugated to the 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 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 activatable antibody. 
     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). 
     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. 
     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 1. 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 an exemplary embodiment, the agent is DM4. 
     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: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In an exemplary embodiment, the agent is conjugated to the AA via lysine. In an exemplary embodiment an SPDB-DM4 is attached to an activatable antibody through the epsilon-amino group of a lysine on the AA, e.g. The epsilon-amino group of the lysine. 
     In an exemplary embodiment, the agent is DM4 and the linker-DM is as follows: 
     
       
         
         
             
             
         
       
     
     The disclosure also provides conjugated AAs that include an AA linked to monomethyl auristatin D (MMAD) payload, wherein the AA includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM1) that inhibits the binding of the AB of the AA 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. 
     In some embodiments, the MMAD-conjugated AA 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. 
     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 6 and 7. In some embodiments, the AA and the MMAD payload are linked via a maleimide caproyl-valine-citrulline linker. In some embodiments, the AA and the MMAD payload are linked via a maleimide PEG-valine-citrulline linker. In some embodiments, the AA and the MMAD payload are linked via a maleimide caproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some embodiments, the AA 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. 
     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. 
     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 a hydroxyl group to which it is conjugated. 
     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  212 Bi,  131 I,  131 In,  90 Y, and  186 Re. 
     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). 
     Table 1 lists some of the exemplary pharmaceutical agents that may be employed in the herein described disclosure but in no way is meant to be an exhaustive list. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Exemplary Pharmaceutical Agents for Conjugation 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 CYTOTOXIC AGENTS 
               
               
                   
                 Auristatins 
               
               
                   
                 Auristatin E 
               
               
                   
                 Monomethyl auristatin D (MMAD) 
               
               
                   
                 Monomethyl auristatin E (MMAE) 
               
               
                   
                 Desmethyl auristatin E (DMAE) 
               
               
                   
                 Auristatin F 
               
               
                   
                 Monomethyl auristatin F (MMAF) 
               
               
                   
                 Desmethyl auristatin F (DMAF) 
               
               
                   
                 Auristatin derivatives, e.g., amides thereof 
               
               
                   
                 Auristatin tyramine 
               
               
                   
                 Auristatin quinoline 
               
               
                   
                 Dolastatins 
               
               
                   
                 Dolastatin derivatives 
               
               
                   
                 Dolastatin 16 DmJ 
               
               
                   
                 Dolastatin 16 Dpv 
               
               
                   
                 Maytansinoids, e.g. DM-1; DM-4 
               
               
                   
                 Maytansinoid derivatives 
               
               
                   
                 Duocarmycin 
               
               
                   
                 Duocarmycin derivatives 
               
               
                   
                 Alpha-amanitin 
               
               
                   
                 Anthracyclines 
               
               
                   
                 Doxorubicin 
               
               
                   
                 Daunorubicin 
               
               
                   
                 Bryostatins 
               
               
                   
                 Camptothecin 
               
               
                   
                 Camptothecin derivatives 
               
               
                   
                 7-substituted Camptothecin 
               
               
                   
                 10,11-Difluoromethylenedioxycamptothecin 
               
               
                   
                 Combretastatins 
               
               
                   
                 Debromoaplysiatoxin 
               
               
                   
                 Kahalalide-F 
               
               
                   
                 Discodermolide 
               
               
                   
                 Ecteinascidins 
               
               
                   
                 ANTIVIRALS 
               
               
                   
                 Acyclovir 
               
               
                   
                 Vira A 
               
               
                   
                 Symmetrel 
               
               
                   
                 ANTIFUNGALS 
               
               
                   
                 Nystatin 
               
               
                   
                 ADDITIONAL ANTI-NEOPLASTICS 
               
               
                   
                 Adriamycin 
               
               
                   
                 Cerubidine 
               
               
                   
                 Bleomycin 
               
               
                   
                 Alkeran 
               
               
                   
                 Velban 
               
               
                   
                 Oncovin 
               
               
                   
                 Fluorouracil 
               
               
                   
                 Methotrexate 
               
               
                   
                 Thiotepa 
               
               
                   
                 Bisantrene 
               
               
                   
                 Novantrone 
               
               
                   
                 Thioguanine 
               
               
                   
                 Procarabizine 
               
               
                   
                 Cytarabine 
               
               
                   
                 ANTI-BACTERIALS 
               
               
                   
                 Aminoglycosides 
               
               
                   
                 Streptomycin 
               
               
                   
                 Neomycin 
               
               
                   
                 Kanamycin 
               
               
                   
                 Amikacin 
               
               
                   
                 Gentamicin 
               
               
                   
                 Tobramycin 
               
               
                   
                 Streptomycin B 
               
               
                   
                 Spectinomycin 
               
               
                   
                 Ampicillin 
               
               
                   
                 Sulfanilamide 
               
               
                   
                 Polymyxin 
               
               
                   
                 Chloramphenicol 
               
               
                   
                 Turbostatin 
               
               
                   
                 Phenstatins 
               
               
                   
                 Hydroxyphenstatin 
               
               
                   
                 Spongistatin 5 
               
               
                   
                 Spongistatin 7 
               
               
                   
                 Halistatin 1 
               
               
                   
                 Halistatin 2 
               
               
                   
                 Halistatin 3 
               
               
                   
                 Modified Bryostatins 
               
               
                   
                 Halocomstatins 
               
               
                   
                 Pyrrolobenzimidazoles (PBI) 
               
               
                   
                 Cibrostatin6 
               
               
                   
                 Doxaliform 
               
               
                   
                 Anthracyclins analogues 
               
               
                   
                 Cemadotin analogue (CemCH2-SH) 
               
               
                   
                 Pseudomonas toxin A (PE38) variant 
               
               
                   
                 Pseudomonas toxin A (ZZ-PE38) variant 
               
               
                   
                 ZJ-101 
               
               
                   
                 OSW-1 
               
               
                   
                 4-Nitrobenzyloxycarbonyl Derivatives of 
               
               
                   
                 O6-Benzylguanine 
               
               
                   
                 Topoisomerase inhibitors 
               
               
                   
                 Hemiasterlin 
               
               
                   
                 Cephalotaxine 
               
               
                   
                 Homoharringtonine 
               
               
                   
                 Pyrrolobenzodiazepine dimers (PBDs) 
               
               
                   
                 Functionalized pyrrolobenzodiazepenes 
               
               
                   
                 Calicheamicins 
               
               
                   
                 Podophyllotoxins 
               
               
                   
                 Taxanes 
               
               
                   
                 Vinca alkaloids 
               
               
                   
                 CONJUGATABLE DETECTION REAGENTS 
               
               
                   
                 Fluorescein and derivatives thereof 
               
               
                   
                 Fluorescein isothiocyanate (FITC) 
               
               
                   
                 RADIOPHARMACEUTICALS 
               
               
                   
                   125 I 
               
               
                   
                   131 I 
               
               
                   
                   89 Zr 
               
               
                   
                   111 In 
               
               
                   
                   123 I 
               
               
                   
                   131 I 
               
               
                   
                   99 mTc 
               
               
                   
                   201 Tl 
               
               
                   
                   133 Xe 
               
               
                   
                   11 C 
               
               
                   
                   62 Cu 
               
               
                   
                   18 F 
               
               
                   
                   68 Ga 
               
               
                   
                   13 N 
               
               
                   
                   15 O 
               
               
                   
                   38 K 
               
               
                   
                   82 Rb 
               
               
                   
                   99 mTc (Technetium) 
               
               
                   
                 HEAVY METALS 
               
               
                   
                 Barium 
               
               
                   
                 Gold 
               
               
                   
                 Platinum 
               
               
                   
                 ANTI-MYCOPLASMALS 
               
               
                   
                 Tylosine 
               
               
                   
                 Spectinomycin 
               
               
                   
                   
               
            
           
         
       
     
     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). 
     In some embodiments, the AA is conjugated to one or more equivalents of an agent. In some embodiments, the AA is conjugated to one equivalent of the agent. In some embodiments, the AA is conjugated to two, three, four, five, six, seven, eight, nine, ten, or greater than ten equivalents of the agent. In some embodiments, the AA is part of a mixture of AAs having a homogeneous number of equivalents of conjugated agents. In some embodiments, the AA is part of a mixture of AAs having a heterogeneous number of equivalents of conjugated agents. In some embodiments, the mixture of AAs is such that the average number of agents conjugated to each AA 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 AAs is such that the average number of agents conjugated to each AA is one, two, three, four, five, six, seven, eight, nine, ten, or greater. In some embodiments, there is a mixture of AAs such that the average number of agents conjugated to each AA is between three and four. In some embodiments, there is a mixture of AAs such that such that the average number of agents conjugated to each AA is between 3.4 and 3.8. In some embodiments, there is a mixture of AAs such that such that the average number of agents conjugated to each AA is between 3.4 and 3.6. In some embodiments, the AA 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 AA in a site-specific manner. In some embodiments, the modified AA 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. 
     Compositions and Methods to Generate Conjugated Activatable Antibodies 
     The activatable anti-target antibodies have at least one point of conjugation for an agent (to produce a conjugated AA). In some embodiments, not all possible points of conjugation are used. In some embodiments, some of the natural points of contact are modified or removed to no longer be available for conjugation to an agent. In some embodiments, the one or more points of conjugation are nitrogen atoms, such as the epsilon amino group of lysine. 
     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 may 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 an activatable anti-target 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 AA with a reducing agent, such as, for example, TCEP; and conjugating the drug reactive with free thiols to the partially reduced activatable antibody. As used herein, the term partial reduction refers to situations where an activatable anti-target 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. 
     In yet other embodiments, a method of reducing and conjugating an agent, e.g., a drug, to an activatable anti-target antibody resulting in selectivity in the placement of the agent is provided. The method generally includes partially reducing the activatable anti-target antibody with a reducing agent such that any conjugation sites in the masking moiety or other non-AB portion of the AA 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 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 AA in which the MM retains the ability to effectively and efficiently mask the AB of the AA in an uncleaved state. The ratio of reduction agent to activatable anti-target antibody will vary depending on the activatable antibody. In some embodiments, the ratio of reducing agent to activatable anti-target 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. 
     In some embodiments, a method of reducing interchain disulfide bonds in the AB of an activatable anti-target 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 AA 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 AA 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. 
     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 AA 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 activatable anti-target antibodies in which at least one interchain disulfide bond in the AA has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the AA includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to target, a masking moiety (MM) that inhibits the binding of the AB of the AA 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 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 AA is not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bond(s) of the MM within the AA is not disturbed by the reducing agent. In some embodiments, the AA 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. 
     The disclosure also provides partially reduced AAs in which at least one interchain disulfide bond in the AA has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the AA includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to the target, e.g., CD166, a masking moiety (MM) that inhibits the binding of the AB of the AA 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 AA is not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bond(s) of the MM within the AA is not disturbed by the reducing agent. In some embodiments, the AA 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. 
     In yet other embodiments, a method of reducing and conjugating an agent, e.g., a drug, to an activatable anti-target antibody resulting in selectivity in the placement of the agent by providing an activatable anti-target 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 anti-target antibody or activatable anti-target 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 anti-target antibody or activatable anti-target antibody in a site-specific manner. In some embodiments, the modified A 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 anti-target antibody or activatable anti-target antibody with a defined number and positions of lysine and/or cysteine residues may 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 AA are not reduced, and conjugating the agent to interchain thiols in the AB. 
     Coupling may 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). 
     In some embodiments, in addition to the compositions and methods provided herein, the conjugated AA can also be modified for site-specific conjugation through modified amino acid sequences inserted or otherwise included in the AA 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 AA 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 AA can be engineered to include or otherwise introduce one or more non-natural amino acid residues within the AA to provide suitable sites for conjugation. In some embodiments, the AA can be engineered to include or otherwise introduce enzymatically activatable peptide sequences within the AA 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. Pat. 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). 
     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. NETS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone. In an exemplary embodiment the linker is SPDB. In another exemplary embodiment, the linker is SPDB agent is DM4. 
     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. 
     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 may 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 may be introduced by suitable chemical modification of linkers not containing such groups. 
     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. 101: 3097-3110). 
     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. 
     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. 
     The agent may 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. 
     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 may be aldehyde or sulfhydryl groups or any chemical site to which agents can be attached. 
     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 may 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 may be aldehyde or sulfhydryl groups, or may be any chemical site to which linkers may be attached. Still higher specific activities may 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 may 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 AAs that include an extracellular toxin, e.g., by way of non-limiting example, any of the extracellular toxins shown in Table 1. 
     Non-limiting examples of cleavable linker sequences are provided in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2  
               
             
            
               
                   
               
               
                 Exemplary Linker Sequences for Conjugation 
               
            
           
           
               
               
            
               
                 Types of Cleavable 
                 Amino Acid 
               
               
                 Sequences 
                 Sequence 
               
               
                   
               
               
                 Plasmin cleavable sequences 
                   
               
               
                 Pro-urokinase 
                 PRFKIIGG 
               
               
                   
                 (SEQ ID NO: 89) 
               
               
                   
               
               
                   
                 PRFRIIGG 
               
               
                   
                 (SEQ ID NO: 90) 
               
               
                   
               
               
                 TGFβ 
                 SSRHRRALD 
               
               
                   
                 (SEQ ID NO: 91) 
               
               
                   
               
               
                 Plasminogen 
                 RKSSIIIRMRDVVL 
               
               
                   
                 (SEQ ID NO: 92) 
               
               
                   
               
               
                 Staphylokinase 
                 SSSFDKGKYKKGDDA 
               
               
                   
                 (SEQ ID NO: 93) 
               
               
                   
               
               
                   
                 SSSFDKGKYKRGDDA 
               
               
                   
                 (SEQ ID NO: 94) 
               
               
                   
               
               
                 Factor Xa cleavable sequences 
                 IEGR 
               
               
                   
                 (SEQ ID NO: 95) 
               
               
                   
               
               
                   
                 IDGR 
               
               
                   
                 (SEQ ID NO: 96) 
               
               
                   
               
               
                   
                 GGSIDGR 
               
               
                   
                 (SEQ ID NO: 97) 
               
               
                   
               
               
                 MMP cleavable sequences 
                   
               
               
                 Gelatinase A 
                 PLGLWA 
               
               
                   
                 (SEQ ID NO: 98) 
               
               
                   
               
               
                 Collagenase cleavable sequences 
                   
               
               
                 Calf skin collagen 
                 GPQGIAGQ 
               
               
                 (α1(I) chain) 
                 (SEQ ID NO: 99) 
               
               
                   
               
               
                 Calf skin collagen 
                 GPQGLLGA 
               
               
                 (α2(I) chain) 
                 (SEQ ID NO: 100) 
               
               
                   
               
               
                 Bovine cartilage collagen 
                 GIAGQ 
               
               
                 (α1(II) chain) 
                 (SEQ ID NO: 101) 
               
               
                   
               
               
                 Human liver collagen 
                 GPLGIAGI 
               
               
                 (α1(III) chain) 
                 (SEQ ID NO: 102) 
               
               
                   
               
               
                 Human α 2 M 
                 GPEGLRVG 
               
               
                   
                 (SEQ ID NO: 103) 
               
               
                   
               
               
                 Human PZP 
                 YGAGLGVV 
               
               
                   
                 (SEQ ID NO: 104) 
               
               
                   
                 AGLGVVER 
               
               
                   
                 (SEQ ID NO: 105) 
               
               
                   
                 AGLGISST 
               
               
                   
                 (SEQ ID NO: 106) 
               
               
                   
               
               
                 Rat α 1 M 
                 EPQALAMS 
               
               
                   
                 (SEQ ID NO: 107) 
               
               
                   
                 QALAMSAI 
               
               
                   
                 (SEQ ID NO: 108) 
               
               
                   
               
               
                 Rat α 2 M 
                 AAYHLVSQ 
               
               
                   
                 (SEQ ID NO: 109) 
               
               
                   
                 MDAFLESS 
               
               
                   
                 (SEQ ID NO: 110) 
               
               
                   
               
               
                 Rat α 1 I 3 (2J) 
                 ESLPVVAV 
               
               
                   
                 (SEQ ID NO: 111) 
               
               
                   
               
               
                 Rat α 1 I 3 (27J) 
                 SAPAVESE 
               
               
                   
                 (SEQ ID NO: 112) 
               
               
                   
               
               
                 Human fibroblast collagenase 
                 DVAQFVLT 
               
               
                   
                 (SEQ ID NO: 113) 
               
               
                   
               
               
                 (autolytic cleavages) 
                 VAQFVLTE 
               
               
                   
                 (SEQ ID NO: 114) 
               
               
                   
               
               
                   
                 AQFVLTEG 
               
               
                   
                 (SEQ ID NO: 115) 
               
               
                   
               
               
                   
                 PVQPIGPQ 
               
               
                   
                 (SEQ ID NO: 116) 
               
               
                   
               
            
           
         
       
     
     In addition, agents may 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. 
     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 may be accomplished by use of a linker of the general structure: 
       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.
 
     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. 
     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. 
     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 a hydroxy group or an amino group, it may 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 may 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. 
     Linkers (or spacer elements of linkers) may 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 may be attached to an amino acid or peptide linker. 
     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 1. 
     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 may 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. 
     Biochemical Cross Linkers: In some embodiments, the AA may 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. In addition, acid-labile linkers cleavable in the low-pH environment of the lysosome may 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 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Exemplary Hetero-Bifunctional Cross Linkers 
               
               
                 HETERO-BIFUNCTIONAL CROSS-LINKERS 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Spacer Arm 
               
               
                   
                   
                   
                 Length after 
               
               
                   
                   
                 Advantages and 
                 cross-linking 
               
               
                 Linker 
                 Reactive Toward 
                 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 
                 15.6 
                 Å 
               
               
                   
                   
                 arm 
               
               
                   
                 Sulfhydryls 
               
               
                 Sulfo-LC-SPDP 
                 Primary amines 
                 Extender spacer 
                 15.6 
                 Å 
               
               
                   
                   
                 arm 
               
               
                   
                 Sulfhydryls 
                 Water-soluble 
               
               
                 SMCC 
                 Primary amines 
                 Stable maleimide 
                 11.6 
                 Å 
               
               
                   
                   
                 reactive group 
               
               
                   
                 Sulfhydryls 
                 Enzyme-antibody 
               
               
                   
                   
                 conjugation 
               
               
                   
                   
                 Hapten-carrier 
               
               
                   
                   
                 protein conjugation 
               
               
                 Sulfo-SMCC 
                 Primary amines 
                 Stable maleimide 
                 11.6 
                 Å 
               
               
                   
                   
                 reactive group 
               
               
                   
                 Sulfhydryls 
                 Water-soluble 
               
               
                   
                   
                 Enzyme-antibody 
               
               
                   
                   
                 conjugation 
               
               
                 MBS 
                 Primary amines 
                 Enzyme-antibody 
                 9.9 
                 Å 
               
               
                   
                   
                 conjugation 
               
               
                   
                 Sulfhydryls 
                 Hapten-carrier 
               
               
                   
                   
                 protein conjugation 
               
               
                 Sulfo-MBS 
                 Primary amines 
                 Water-soluble 
                 9.9 
                 Å 
               
               
                   
                 Sulfhydryls 
               
               
                 SIAB 
                 Primary amines 
                 Enzyme-antibody 
                 10.6 
                 Å 
               
               
                   
                   
                 conjugation 
               
               
                   
                 Sulfhydryls 
               
               
                 Sulfo-SIAB 
                 Primary amines 
                 Water-soluble 
                 10.6 
                 Å 
               
               
                   
                 Sulfhydryls 
               
               
                 SMPB 
                 Primary amines 
                 Extended spacer 
                 14.5 
                 Å 
               
               
                   
                   
                 arm 
               
               
                   
                 Sulfhydryls 
                 Enzyme-antibody 
               
               
                   
                   
                 conjugation 
               
               
                 Sulfo-SMPB 
                 Primary amines 
                 Extended spacer 
                 14.5 
                 Å 
               
               
                   
                   
                 arm 
               
               
                   
                 Sulfhydryls 
                 Water-soluble 
               
            
           
           
               
               
               
               
            
               
                 EDE/Sulfo- 
                 Primary amines 
                 Hapten-Carrier 
                 0 
               
               
                 NHS 
                   
                 conjugation 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Carboxyl groups 
                   
                   
                   
               
               
                 ABH 
                 Carbohydrates 
                 Reacts with 
                 11.9 
                 Å 
               
               
                   
                   
                 sugar groups 
               
               
                   
                 Nonselective 
               
               
                   
               
            
           
         
       
     
     Non-Cleavable Linkers or Direct Attachment: In some embodiments of the disclosure, the conjugate may be designed so that the agent is delivered to the target but not released. This may be accomplished by attaching an agent to an AB either directly or via a non-cleavable linker. 
     These non-cleavable linkers may include amino acids, peptides, D-amino acids or other organic compounds that may 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 may be attached to ABs that do not activate complement. When using ABs that are incapable of complement activation, this attachment may be accomplished using linkers that are susceptible to cleavage by activated complement or using linkers that are not susceptible to cleavage by activated 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. Pat. No. 5,013,556. 
     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. 
     Activatable Antibodies Having Non-Binding Steric Moieties or Binding Partners for Non-Binding Steric Moieties 
     The disclosure also provides AAs 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 AAs provided herein include, for example, an AA that includes a non-binding steric moiety (NB), a cleavable linker (CL) and antibody or antibody fragment (AB) that binds a target; an AA that includes a binding partner for a non-binding steric moiety (BP), a CL and an AB; and an AA that includes a BP to which an NB has been recruited, a CL and an AB that binds the target. AAs in which the NB is covalently linked to the CL and AB of the AA or is associated by interaction with a BP that is covalently linked to the CL and AB of the AA are referred to herein as “NB-containing activatable antibodies.” By activatable or switchable is meant that the AA exhibits a first level of binding to a target when the AA is in an inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target when the AA 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 AA compositions can exhibit increased bioavailability and more favorable biodistribution compared to conventional antibody therapeutics. 
     In some embodiments, AAs 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. 
     AAs 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. 
     Production of Activatable Antibodies 
     The disclosure also provides methods of producing an activatable anti-target 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 AA described herein, and/or vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing an antibody and/or AA 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 AA described herein, and/or vectors that include these isolated nucleic acid sequences. 
     The invention also provides a method of manufacturing AAs that in an activated state binds target by (a) culturing a cell comprising a nucleic acid construct that encodes the AA under conditions that lead to expression of the activatable antibody, wherein the AA comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or an antigen binding fragment thereof (AB) that specifically binds target, (i) wherein the CM is a polypeptide that functions as a substrate for a protease; and (ii) wherein the CM is positioned in the AA such that, when the AA is in an uncleaved state, the MM1 interferes with specific binding of the AB to target and in a cleaved state the MM1 does not interfere or compete with specific binding of the AB to target; and (b) recovering the activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein. 
     The following exemplary nucleotide sequences are provided for use to make and use the AAs and conjugated AAs provided herein. Also provided are nucleotide sequences that are at least 90%, 95%, or even 99% homologous to the nucleotide sequences provided below. 
     
       
         
           
               
               
            
               
                   
                 Encoding amino acid sequence of SEQ ID NO: 9 
               
               
                   
                 Human αCD166 Heavy Chain (HuCD166_HcC)- 
               
               
                   
                 Nucleotide sequence 
               
               
                   
                 SEQ ID NO: 45 
               
               
                   
                 CAGATCACCCTGAAAGAGTCCGGCCCCACCCTGGT 
               
               
                   
               
               
                   
                 GAAACCCACCCAGACCCTGACCCTGACATGCACCT 
               
               
                   
               
               
                   
                 TCTCCGGCTTCAGCCTGTCCACCTACGGCATGGGC 
               
               
                   
               
               
                   
                 GTGGGCTGGATCAGGCAGCCTCCTGGCAAGGCCCT 
               
               
                   
               
               
                   
                 GGAATGGCTGGCCAACATCTGGTGGTCCGAGGACA 
               
               
                   
               
               
                   
                 AGCACTACTCCCCCAGCCTGAAGTCCCGGCTGACC 
               
               
                   
               
               
                   
                 ATCACCAAGGACACCTCCAAGAACCAGGTGGTGCT 
               
               
                   
               
               
                   
                 GACAATCACAAACGTGGACCCCGTGGACACCGCCA 
               
               
                   
               
               
                   
                 CCTACTACTGCGTGCAGATCGACTACGGCAACGAC 
               
               
                   
               
               
                   
                 TACGCCTTCACCTACTGGGGCCAGGGCACACTGGT 
               
               
                   
               
               
                   
                 GACAGTGTCCTCCGCCTCCACCAAGGGCCCCTCCG 
               
               
                   
               
               
                   
                 TGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCT 
               
               
                   
               
               
                   
                 GGCGGCACAGCTGCCCTGGGCTGCCTGGTGAAAGA 
               
               
                   
               
               
                   
                 CTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACT 
               
               
                   
               
               
                   
                 CTGGCGCCCTGACCAGCGGAGTGCACACCTTCCCT 
               
               
                   
               
               
                   
                 GCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTC 
               
               
                   
               
               
                   
                 CTCCGTGGTGACCGTGCCCTCCAGCTCTCTGGGCA 
               
               
                   
               
               
                   
                 CCCAGACCTACATCTGCAACGTGAACCACAAGCCC 
               
               
                   
               
               
                   
                 TCCAACACCAAGGTGGACAAGAAGGTGGAACCCAA 
               
               
                   
               
               
                   
                 GTCCTGCGACAAGACCCACACCTGTCCCCCCTGCC 
               
               
                   
               
               
                   
                 CTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTT 
               
               
                   
               
               
                   
                 CTGTTCCCCCCAAAGCCTAAGGACACCCTGATGAT 
               
               
                   
               
               
                   
                 CTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGG 
               
               
                   
               
               
                   
                 ACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAAT 
               
               
                   
               
               
                   
                 TGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA 
               
               
                   
               
               
                   
                 GACCAAGCCCAGAGAGGAACAGTACAACTCCACCT 
               
               
                   
               
               
                   
                 ACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAG 
               
               
                   
               
               
                   
                 GACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGT 
               
               
                   
               
               
                   
                 GTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGA 
               
               
                   
               
               
                   
                 CCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCT 
               
               
                   
               
               
                   
                 CAGGTGTACACACTGCCCCCTAGCCGGGAAGAGAT 
               
               
                   
               
               
                   
                 GACCAAGAATCAGGTGTCCCTGACCTGTCTGGTGA 
               
               
                   
               
               
                   
                 AAGGCTTCTACCCCTCCGATATCGCCGTGGAATGG 
               
               
                   
               
               
                   
                 GAGTCCAACGGCCAGCCCGAGAACAACTACAAGAC 
               
               
                   
               
               
                   
                 CACCCCCCCTGTGCTGGACTCCGACGGCTCATTCT 
               
               
                   
               
               
                   
                 TCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGG 
               
               
                   
               
               
                   
                 TGGCAGCAGGGCAACGTGTTCTCCTGCAGCGTGAT 
               
               
                   
               
               
                   
                 GCACGAGGCCCTGCACAACCACTACACCCAGAAGT 
               
               
                   
               
               
                   
                 CCCTGTCCCTGAGCCCCGGCAAG 
               
               
                   
               
               
                   
                 Encoding amino acid sequence of 
               
               
                   
                 SEQ ID NO: 10 
               
               
                   
                 Human αCD166 Heavy Chain 
               
               
                   
                 (HuCD166_HcC)-Des-HC Nucleotide sequence 
               
               
                   
                 SEQ ID NO: 46 
               
               
                   
                 CAGATCACCCTGAAAGAGTCCGGCCCCACCCTGGT 
               
               
                   
               
               
                   
                 GAAACCCACCCAGACCCTGACCCTGACATGCACCT 
               
               
                   
               
               
                   
                 TCTCCGGCTTCAGCCTGTCCACCTACGGCATGGGC 
               
               
                   
               
               
                   
                 GTGGGCTGGATCAGGCAGCCTCCTGGCAAGGCCCT 
               
               
                   
               
               
                   
                 GGAATGGCTGGCCAACATCTGGTGGTCCGAGGACA 
               
               
                   
               
               
                   
                 AGCACTACTCCCCCAGCCTGAAGTCCCGGCTGACC 
               
               
                   
               
               
                   
                 ATCACCAAGGACACCTCCAAGAACCAGGTGGTGCT 
               
               
                   
               
               
                   
                 GACAATCACAAACGTGGACCCCGTGGACACCGCCA 
               
               
                   
               
               
                   
                 CCTACTACTGCGTGCAGATCGACTACGGCAACGAC 
               
               
                   
               
               
                   
                 TACGCCTTCACCTACTGGGGCCAGGGCACACTGGT 
               
               
                   
               
               
                   
                 GACAGTGTCCTCCGCCTCCACCAAGGGCCCCTCCG 
               
               
                   
               
               
                   
                 TGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCT 
               
               
                   
               
               
                   
                 GGCGGCACAGCTGCCCTGGGCTGCCTGGTGAAAGA 
               
               
                   
               
               
                   
                 CTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACT 
               
               
                   
               
               
                   
                 CTGGCGCCCTGACCAGCGGAGTGCACACCTTCCCT 
               
               
                   
               
               
                   
                 GCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTC 
               
               
                   
               
               
                   
                 CTCCGTGGTGACCGTGCCCTCCAGCTCTCTGGGCA 
               
               
                   
               
               
                   
                 CCCAGACCTACATCTGCAACGTGAACCACAAGCCC 
               
               
                   
               
               
                   
                 TCCAACACCAAGGTGGACAAGAAGGTGGAACCCAA 
               
               
                   
               
               
                   
                 GTCCTGCGACAAGACCCACACCTGTCCCCCCTGCC 
               
               
                   
               
               
                   
                 CTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTT 
               
               
                   
               
               
                   
                 CTGTTCCCCCCAAAGCCTAAGGACACCCTGATGAT 
               
               
                   
               
               
                   
                 CTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGG 
               
               
                   
               
               
                   
                 ACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAAT 
               
               
                   
               
               
                   
                 TGGTACGTGGACGGCGTGGAAGTGCACAACGCCAA 
               
               
                   
               
               
                   
                 GACCAAGCCCAGAGAGGAACAGTACAACTCCACCT 
               
               
                   
               
               
                   
                 ACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAG 
               
               
                   
               
               
                   
                 GACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGT 
               
               
                   
               
               
                   
                 GTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGA 
               
               
                   
               
               
                   
                 CCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCT 
               
               
                   
               
               
                   
                 CAGGTGTACACACTGCCCCCTAGCCGGGAAGAGAT 
               
               
                   
               
               
                   
                 GACCAAGAATCAGGTGTCCCTGACCTGTCTGGTGA 
               
               
                   
               
               
                   
                 AAGGCTTCTACCCCTCCGATATCGCCGTGGAATGG 
               
               
                   
               
               
                   
                 GAGTCCAACGGCCAGCCCGAGAACAACTACAAGAC 
               
               
                   
               
               
                   
                 CACCCCCCCTGTGCTGGACTCCGACGGCTCATTCT 
               
               
                   
               
               
                   
                 TCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGG 
               
               
                   
               
               
                   
                 TGGCAGCAGGGCAACGTGTTCTCCTGCAGCGTGAT 
               
               
                   
               
               
                   
                 GCACGAGGCCCTGCACAACCACTACACCCAGAAGT 
               
               
                   
               
               
                   
                 CCCTGTCCCTGAGCCCCGGC 
               
               
                   
               
               
                   
                 Encoding amino acid sequence of 
               
               
                   
                 SEQ ID NO: 16 
               
               
                   
                 Human αCD166 Light Chain 
               
               
                   
                 (spacer-MM-LP1-CM-LP2-Ab) 
               
               
                   
                 [spacer (SEQ ID NO: 49)] 
               
               
                   
                 [huCD166Lc1_7614.6_3001 (SEQ ID NO: 48)] 
               
               
                   
                 SEQ ID NO: 47 
               
               
                   
                 [CAGGGACAGTCTGGCCAGGGC][CTGTGTCACCC 
               
               
                   
               
               
                   
                 TGCTGTGCTGTCTGCCTGGGAGTCCTGTTCCAGCG 
               
               
                   
               
               
                   
                 GCGGAGGCTCCTCTGGCGGCTCTGCTGTGGGCCTG 
               
               
                   
               
               
                   
                 CTGGCTCCACCTGGCGGCCTGTCCGGCAGATCTGA 
               
               
                   
               
               
                   
                 CAACCACGGCGGCTCCGACATCGTGATGACCCAGT 
               
               
                   
               
               
                   
                 CCCCCCTGTCCCTGCCCGTGACTCCTGGCGAGCCT 
               
               
                   
               
               
                   
                 GCCTCCATCTCCTGCCGGTCCTCCAAGTCCCTGCT 
               
               
                   
               
               
                   
                 GCACTCCAACGGCATCACCTACCTGTACTGGTATC 
               
               
                   
               
               
                   
                 TGCAGAAGCCCGGCCAGTCCCCTCAGCTGCTGATC 
               
               
                   
               
               
                   
                 TACCAGATGTCCAACCTGGCCTCCGGCGTGCCCGA 
               
               
                   
               
               
                   
                 CAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCA 
               
               
                   
               
               
                   
                 CCCTGAAGATCTCCCGGGTGGAAGCCGAGGACGTG 
               
               
                   
               
               
                   
                 GGCGTGTACTACTGCGCCCAGAACCTGGAACTGCC 
               
               
                   
               
               
                   
                 CTACACCTTCGGCCAGGGCACCAAGCTGGAAATCA 
               
               
                   
               
               
                   
                 AGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTC 
               
               
                   
               
               
                   
                 CCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGC 
               
               
                   
               
               
                   
                 CTCCGTGGTCTGCCTGCTGAACAACTTCTACCCCC 
               
               
                   
               
               
                   
                 GCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCC 
               
               
                   
               
               
                   
                 CTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGA 
               
               
                   
               
               
                   
                 GCAGGACTCCAAGGACAGCACCTACTCCCTGTCCT 
               
               
                   
               
               
                   
                 CCACCCTGACCCTGTCCAAGGCCGACTACGAGAAG 
               
               
                   
               
               
                   
                 CACAAGGTGTACGCCTGCGAAGTGACCCACCAGGG 
               
               
                   
               
               
                   
                 ACTGAGCAGCCCCGTGACCAAGTCCTTCAACCGGG 
               
               
                   
               
               
                   
                 GCGAGTGC] 
               
               
                   
               
               
                   
                 Encoding amino acid sequence of 
               
               
                   
                 SEQ ID NO: 15 Human αCD166 Light 
               
               
                   
                 Chain (MM-LP1-CM-LP2-Ab) 
               
               
                   
                 huCD166Lc1_7614.6_3001 
               
               
                   
                 SEQ ID NO: 48 
               
               
                   
                 CTGTGTCACCCTGCTGTGCTGTCTGCCTGGGAGTC 
               
               
                   
               
               
                   
                 CTGTTCCAGCGGCGGAGGCTCCTCTGGCGGCTCTG 
               
               
                   
               
               
                   
                 CTGTGGGCCTGCTGGCTCCACCTGGCGGCCTGTCC 
               
               
                   
               
               
                   
                 GGCAGATCTGACAACCACGGCGGCTCCGACATCGT 
               
               
                   
               
               
                   
                 GATGACCCAGTCCCCCCTGTCCCTGCCCGTGACTC 
               
               
                   
               
               
                   
                 CTGGCGAGCCTGCCTCCATCTCCTGCCGGTCCTCC 
               
               
                   
               
               
                   
                 AAGTCCCTGCTGCACTCCAACGGCATCACCTACCT 
               
               
                   
               
               
                   
                 GTACTGGTATCTGCAGAAGCCCGGCCAGTCCCCTC 
               
               
                   
               
               
                   
                 AGCTGCTGATCTACCAGATGTCCAACCTGGCCTCC 
               
               
                   
               
               
                   
                 GGCGTGCCCGACAGATTCTCCGGCTCTGGCTCCGG 
               
               
                   
               
               
                   
                 CACCGACTTCACCCTGAAGATCTCCCGGGTGGAAG 
               
               
                   
               
               
                   
                 CCGAGGACGTGGGCGTGTACTACTGCGCCCAGAAC 
               
               
                   
               
               
                   
                 CTGGAACTGCCCTACACCTTCGGCCAGGGCACCAA 
               
               
                   
               
               
                   
                 GCTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCG 
               
               
                   
               
               
                   
                 TGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAG 
               
               
                   
               
               
                   
                 TCCGGCACCGCCTCCGTGGTCTGCCTGCTGAACAA 
               
               
                   
               
               
                   
                 CTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGG 
               
               
                   
               
               
                   
                 TGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAA 
               
               
                   
               
               
                   
                 TCCGTCACCGAGCAGGACTCCAAGGACAGCACCTA 
               
               
                   
               
               
                   
                 CTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCG 
               
               
                   
               
               
                   
                 ACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTG 
               
               
                   
               
               
                   
                 ACCCACCAGGGACTGAGCAGCCCCGTGACCAAGTC 
               
               
                   
               
               
                   
                 CTTCAACCGGGGCGAGTGC 
               
               
                   
               
               
                   
                 Nucleotide Sequence Encoding 
               
               
                   
                 SEQ ID NO: 14 Spacer 
               
               
                   
                 SEQ ID NO: 49 
               
               
                   
                 CAGGGACAGTCTGGCCAGGGC 
               
            
           
         
       
     
     Therapeutic Use of Activatable Antibodies, and Conjugated Activatable Antibodies 
     The disclosure 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 a target in a subject using AAs that bind target, particularly AAs that bind and neutralize or otherwise inhibit at least one biological activity of target and/or target-mediated signaling. 
     The disclosure 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 target or aberrantly expressing target in a subject using AAs that bind target, particularly AAs that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are expressing or aberrantly expressing target. 
     The disclosure 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 target in a subject using AAs that bind target, particularly AAs that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are expressing target. 
     The disclosure 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 target in a subject using AAs that bind target, particularly AAs that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are aberrantly expressing target. 
     The disclosure 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 an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody described herein to a subject in need thereof. 
     The disclosure also provides AAs that bind target, particularly AAs that bind and neutralize or otherwise inhibit at least one biological activity of target and/or target signaling, for use in treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with aberrant expression and/or activity of target in a subject. 
     The disclosure also provides AAs that bind target, particularly AAs that bind, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells which are expressing or aberrantly expressing target, for use in 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 or aberrantly expressing target in a subject. 
     The disclosure also provides an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody described herein, for use in preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating cancer in a subject, wherein the antibody is for administration in a therapeutically effective amount. 
     By way of non-limiting example, the AAs of the disclosure can be used for treating, preventing and/or delaying the onset or progression of an epithelial or squamous cell cancer, a carcinoid, and/or a neuroendocrine cancer. Examples of cancers include, but are not limited to, adenocarcinoma, bile duct (biliary) cancer, bladder cancer, breast cancer, e.g., triple-negative breast cancer, Her2-negative breast cancer, estrogen receptor-positive breast cancer; carcinoid cancer; cervical cancer; cholangiocarcinoma; colorectal; endometrial; 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. 
     In some embodiments, the cancer is any epithelial or squamous cancer. In some embodiments, the cancer is prostate cancer, breast cancer, lung cancer, cervical cancer, oropharyngeal cancer, and/or head and neck cancer. 
     In some embodiments, the cancer is a bladder cancer, a bone cancer, a breast cancer, a carcinoid, a cervical cancer, a colorectal cancer, a colon cancer, an endometrial cancer, an epithelial cancer, a glioma, a head and neck cancer, a liver cancer, a lung cancer, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, a sarcoma, a skin cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, and/or a urothelial cancer. 
     In some embodiments, the cancer is selected from the group consisting of triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), Ras mutant colorectal carcinoma, a rare epithelial cancer, oropharyngeal cancer, cervical cancer, head and neck squamous cell carcinoma (HNSCC), and/or prostate cancer. In some embodiments, the cancer is associated with a target-expressing tumor. In some embodiments, the cancer is due to a target-expressing tumor. 
     An anti-target antibody, a conjugated anti-target antibody, an activatable anti-target antibody and/or a conjugated activatable anti-target antibody used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody can be administered to a subject suffering cancer of any stage, from early to metastatic. 
     In exemplary embodiments the subject is suffering from, or suspected to be suffering from breast carcinoma, castration-resistant prostate cancer (CPRC), cholangiocarcinoma, endometrial carcinoma, epithelial ovarian carcinoma, head and neck squamous cell carcinoma (HNSCC), and non-small cell lung cancer (NSCLC). 
     In exemplary embodiments the subject is suffering from, or suspected to be suffering from, a skin lesion. In some embodiments, the skin lesion is a skin metastasis. 
     As provided herein, the subject to be treated 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. 
     In some embodiments, a subject suffering from, or suspected to be suffering from a breast carcinoma, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has an estrogen receptor expressing (ER+) tumor and should have received anti-hormonal therapy and has experienced disease progression prior to being treated with the AA of the present disclosure. In some embodiments, a subject suffering from, or suspected to be suffering from a breast carcinoma, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has a triple negative breast carcinoma (TNBC) and has received ≥2 prior lines of therapy prior to being treated with the AA of the present disclosure. 
     In some embodiments, a subject suffering from, or suspected to be suffering from a castration-resistant prostate carcinoma, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has received ≥1 prior therapy, before being treated with the AA of the present disclosure. 
     In some embodiments, a subject suffering from, or suspected to be suffering from a cholangiocarcinoma, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has failed ≥1 prior line of gemcitabine-containing regimen, before being treated with the AA of the present disclosure. 
     In some embodiments, a subject suffering from, or suspected to be suffering from a endometrial carcinoma, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has received ≥1 platinum-containing regimen for extra-uterine or advanced disease, before being treated with the AA of the present disclosure. 
     In some embodiments, a subject suffering from, or suspected to be suffering from a epithelial ovarian carcinoma, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, either has a non-breast cancer (BRCA) mutation (germline or somatic), or has an unknown BRCA mutational status and has platinum-resistant or platinum refractory ovarian carcinoma. In some embodiments, a subject suffering from, or suspected to be suffering from an epithelial ovarian carcinoma, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has a BRCA mutation and is refractory to, or otherwise ineligible for, PARP inhibitors. 
     In some embodiments, a subject suffering from, or suspected to be suffering from a HNSCC, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has received ≥1 platinum-containing regimen and a PD-1/PD-L1 inhibitor (if approved for the subject&#39;s indication and locality), before being treated with the AA of the present disclosure. 
     In some embodiments, a subject suffering from, or suspected to be suffering from a NSCLC, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has received ≥1 platinum-containing regimen before being treated with the AA of the present disclosure. In some embodiments, a subject suffering from, or suspected to be suffering from a NSCLC, who receives an AA of the present disclosure, e.g. Combination 55 or Combination 60, has been previously administered a checkpoint inhibitor (if approved for the subject&#39;s indication in their locality) before being treated with the AA of the present disclosure. 
     In some embodiments, a subject who has any of the following may not be eligible to receive an AA of the present disclosure for the treatment of breast carcinoma, castration-resistant prostate cancer (CPRC), cholangiocarcinoma, endometrial carcinoma, epithelial ovarian carcinoma, HNSCC, and NSCLC: active or chronic corneal disorder, history of corneal transplantation, active herpetic keratitis, and active ocular conditions requiring ongoing treatment/monitoring; serious concurrent illness, including clinically relevant active infection; history of or current active autoimmune diseases; significant cardiac disease such as recent myocardial infarction; history of multiple sclerosis or other demyelinating disease, Eaton-Lambert syndrome (para-neoplastic syndrome), history of hemorrhagic or ischemic stroke within the last 6 months, or alcoholic liver disease; non-healing wound(s) or ulcer(s) except for ulcerative lesions caused by the underlying neoplasm; history of severe allergic or anaphylactic reactions to previous monoclonal antibody therapy; currently receiving anticoagulation therapy with warfarin; or major surgery (requiring general anesthesia) within 3 months prior to dosing. 
     Activatable anti-target antibody and/or conjugated activatable anti-target antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder associated with aberrant target expression and/or activity. A subject suffering from or susceptible to a disease or disorder associated with aberrant target 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. 
     Administration of an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody to a subject suffering from a disease or disorder associated with aberrant target expression and/or activity is considered successful if any of a variety of laboratory or clinical objectives is achieved. For example, administration of an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody to a subject suffering from a disease or disorder associated with aberrant target 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 an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody to a subject suffering from a disease or disorder associated with aberrant target 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. 
     In some embodiments, activatable anti-target antibody and/or conjugated activatable anti-target 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&#39;s diseased cells are expressing target. In some embodiments, the diseased cells are associated with aberrant target expression and/or activity. In some embodiments, the diseased cells are associated with normal target expression and/or activity. A subject suffering from or susceptible to a disease or disorder wherein the subject&#39;s diseased cells express target 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. 
     In some embodiments, activatable anti-target antibody and/or conjugated activatable anti-target antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder associated with cells expressing target 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 target expression and/or activity. In some embodiments, the cells are associated with normal target expression and/or activity. A subject suffering from or susceptible to a disease or disorder associated with cells that express target 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. 
     Administration of an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody to a subject suffering from a disease or disorder associated with cells expressing target is considered successful if any of a variety of laboratory or clinical objectives is achieved. For example, administration of an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody to a subject suffering from a disease or disorder associated with cells expressing target 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 an anti-target antibody, conjugated anti-target antibody, activatable anti-target antibody and/or conjugated activatable anti-target antibody to a subject suffering from a disease or disorder associated with cells expressing target is considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worse, state. 
     In some embodiments, activatable anti-target antibody and/or conjugated activatable anti-target 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, activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent(s) are administered simultaneously. For example, activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent(s) are administered sequentially. 
     In some embodiments, activatable anti-target antibodies and/or conjugated activatable anti-target 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 anti-target antibodies, conjugated anti-target antibodies, activatable anti-target antibodies and/or conjugated activatable anti-target antibodies can be used in conjunction with an additional chemotherapeutic or anti-neoplastic agent. 
     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. 
     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 α-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). 
     In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of CTLA-4, LAG-3, PD-1, target, 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 DO inhibitor, an α-CSF1R inhibitor, an α-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. 
     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 target 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 α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-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. 
     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. 
     In some embodiments, the AA and/or conjugated AA 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, activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent are formulated into a single therapeutic composition, and activatable anti-target antibody and/or conjugated activatable anti-target antibody and additional agent are administered simultaneously. Alternatively, activatable anti-target antibody and/or conjugated activatable anti-target antibody and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent are administered simultaneously, or activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent are administered at different times during a treatment regimen. For example, activatable anti-target antibody and/or conjugated activatable anti-target antibody is administered prior to the administration of the additional agent, activatable anti-target antibody and/or conjugated activatable anti-target antibody is administered subsequent to the administration of the additional agent, or activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent are administered in an alternating fashion. As described herein, activatable anti-target antibody and/or conjugated activatable anti-target antibody and additional agent are administered in single doses or in multiple doses. 
     In some embodiments, activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent(s) are administered simultaneously. For example, activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent(s) are administered sequentially, or activatable anti-target antibody and/or conjugated activatable anti-target antibody and the additional agent are administered at different times during a treatment regimen. 
     In some embodiments, activatable anti-target antibody and/or conjugated activatable anti-target 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. 
     In some embodiments, the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another AA or antigen-binding fragment thereof and/or another conjugated AA 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 AA or antigen-binding fragment thereof and/or another conjugated AA 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, AA or antigen-binding fragment thereof and/or a conjugated AA or antigen-binding fragment thereof, e.g., against target. In some embodiments the additional agent is another antibody or antigen-binding fragment thereof, another conjugated antibody or antigen-binding fragment thereof, another AA or antigen-binding fragment thereof and/or another conjugated AA 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, AA or antigen-binding fragment thereof and/or a conjugated AA or antigen-binding fragment thereof. 
     In some embodiments, the additional antibody or antigen binding fragment thereof, conjugated antibody or antigen binding fragment thereof, AA or antigen binding fragment thereof, and/or conjugated AA 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, AA or antigen binding fragment thereof, and/or conjugated AA or antigen binding fragment thereof is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody. 
     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&#39;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 W N “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. 
     Therapeutic formulations of the disclosure, which include an activatable anti-target antibody, such as by way of non-limiting example, AA and/or a conjugated AA, 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 AA 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 AA is conjugated is a microtubule inhibitor. In some embodiments, the agent to which the AA is conjugated is a nucleic acid damaging agent. 
     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 AA and/or conjugated AA confers a clinical benefit. 
     An AA and/or a conjugated AA 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. 
     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 compound 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. 
     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. 
     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. 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 γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT′ (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. 
     Diagnostic Uses 
     The invention also provides methods and kits for using the activatable anti-target antibodies and/or conjugated activatable anti-CD166 antibodies 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 an anti-target activatable antibody, wherein the anti-target AA comprises a masking moiety (MM1), 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 anti-target AA 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 MM1 is a peptide that inhibits binding of the AB to target, and wherein the MM1 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 target, and when the AB is in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to target; and (ii) measuring a level of activated anti-target AA in the subject or sample, wherein a detectable level of activated anti-target AA in the subject or sample indicates that the cleaving agent and target are present in the subject or sample and wherein no detectable level of activated anti-target AA in the subject or sample indicates that the cleaving agent, target or both the cleaving agent and target are absent in the subject or sample. 
     In some embodiments, the activatable anti-target antibody is an activatable anti-target antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable anti-target antibody is not conjugated to an agent. In some embodiments, the activatable anti-target antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable anti-target 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. 
     In some embodiments of these methods and kits, the activatable anti-target 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 β-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. 
     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. 
     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. 
     In some embodiments of the methods and kits, the method is used to identify or otherwise refine a patient population suitable for treatment with an anti-target AA of the disclosure, followed by treatment by administering that activatable anti-target antibody and/or conjugated activatable anti-target antibody to a subject in need thereof. For example, patients that test positive for both the target (e.g., CD166) and a protease that cleaves the substrate in the CM (CM) of the anti-target AA being tested in these methods are identified as suitable candidates for treatment with such an anti-target AA comprising such a CM, and the patient is then administered a therapeutically effective amount of the activatable anti-target antibody and/or conjugated activatable anti-target antibody that was tested. Likewise, patients that test negative for either or both of the target (e.g., CD166) and the protease that cleaves the substrate in the CM in the AA 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 anti-target AAs until a suitable anti-target AA for treatment is identified (e.g., an anti-target AA 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 anti-target 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. 
     In some embodiments, the AA and/or conjugated AA 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, N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 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. 
     Accordingly, the AAs and conjugated AAs of the disclosure are also useful in a variety of diagnostic and prophylactic formulations. In one embodiment, an AA and/or a conjugated AA is administered to subjects that are at risk of developing one or more of the aforementioned disorders. A subject&#39;s or organ&#39;s predisposition to one or more of the aforementioned disorders can be determined using genotypic, serological or biochemical markers. 
     In some embodiments of the disclosure, an AA and/or a conjugated AA is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, an AA and/or a conjugated AA is administered to mitigate or reverse the effects of the clinical indication. 
     An activatable antibody, and/or a conjugated AA of the disclosure is also useful in the detection of a target in subject 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 subject sample. 
     In one embodiment, an AA and/or a conjugated AA of the disclosure is immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized AA and/or conjugated AA serves as a capture antibody for any target that may be present in a test sample. Prior to contacting the immobilized activatable antibody, and/or conjugated versions thereof, with a subject 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. 
     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. 
     It will be appreciated that based on the results obtained using the AAs 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. 
     An AA and/or a conjugated AA 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, AAs 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 AAs 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. 
     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, AAs 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. 
     In another example, AAs can be used to detect the presence or absence of a cleaving agent in a sample. For example, where the AAs contain a CM susceptible to cleavage by an enzyme, the AAs can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample. In another example, where the AAs contain a CM susceptible to cleavage by reducing agent, the AAs can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the AAs 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 AA 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 AAs 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 AAs prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the AA by the cleaving agent in the sample. 
     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 AAs 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 AAs 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. 
     AAs are also useful in in situ imaging for the validation of AA 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). 
     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. 
     In these techniques, an AA is labeled with a detectable label. The detectable label may 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, AA 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 Nov. 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 AA 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 AAs 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 AA 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 AA 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 AA in the subject or sample, wherein a detectable level of activated AA 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 AA 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 AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA is not conjugated to an agent. In some embodiments, the AA comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of AA 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 AA in the presence of a target of interest, e.g., the target, wherein the AA 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 AA 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 MM1 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 MM1 interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM1 does not interfere or compete with specific binding of the AB to the target; and (ii) measuring a level of activated AA in the subject or sample, wherein a detectable level of activated AA in the subject or sample indicates that the cleaving agent is present in the subject or sample and wherein no detectable level of activated AA 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 AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA is not conjugated to an agent. In some embodiments, the AA comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of AA 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 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 AA 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 AA 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 AA in the subject or sample, wherein a detectable level of activated AA in the subject or sample indicates that the cleaving agent is present in the subject or sample and wherein no detectable level of activated AA 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 AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA is not conjugated to an agent. In some embodiments, the AA comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of AA 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 AA 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 AA 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 MM1 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 MM1 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 AA 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 AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA is not conjugated to an agent. In some embodiments, the AA comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of AA 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 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 AA and/or conjugated AA (e.g., an AA 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 AA and/or conjugated AA in the subject or biological sample, wherein a detectable level of activated AA 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 AA 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 AA 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 AA in the presence of the target, and (ii) measuring a level of activated AA in the subject or biological sample, wherein a detectable level of activated AA 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 AA 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 AA cannot be detected in the subject or biological sample. Such an AA 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 AA 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 AA in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an AA 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 AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA 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 CM 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. 
     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 AA and/or conjugated AA described herein for use in contacting a subject or biological sample with an AA in the presence of the target, and measuring a level of activated AA in the subject or biological sample, wherein a detectable level of activated AA 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 AA 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 AA cannot be detected in the subject or biological sample. Such an AA 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 AA 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 MM1 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 MM1 of the AA in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an AA 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 AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA 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 CM 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. 
     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 AA and/or conjugated AA described herein for use in contacting a subject or biological sample and means for detecting the level of activated AA and/or conjugated AA in the subject or biological sample, wherein the AA includes a detectable label that is positioned on a portion of the AA that is released following cleavage of the CM, wherein a detectable level of activated AA 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 AA cannot be detected in the subject or biological sample, and wherein no detectable level of activated AA in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample at a detectable level. 
     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 AA includes a detectable label that is positioned on a portion of the AA that is released following cleavage of the CM and (ii) measuring a level of activated AA in the subject or biological sample, wherein a detectable level of activated AA 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 AA cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated AA 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 AA 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 AA 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 AA in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an AA 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 AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA is not conjugated to an agent. In some embodiments, the AA comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of AA 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 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 AA and/or conjugated AA described herein for use in contacting a subject or biological sample and means for detecting the level of activated AA and/or conjugated AA in the subject or biological sample, wherein a detectable level of activated AA 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 AA cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated AA 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 AA includes a detectable label that is positioned on a portion of the AA 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 AA 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 AA includes a masking moiety (MM1), 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 AA 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 MM1 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 MM1 of the AA in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM1 of an AA 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 AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA is not conjugated to an agent. In some embodiments, the AA comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of AA 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 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 AA and/or conjugated AA described herein for use in contacting a subject or biological sample and means for detecting the level of activated AA and/or conjugated AA in the subject or biological sample, wherein the AA includes a detectable label that is positioned on a portion of the AA 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 AA 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%. 
     In some embodiments of these methods and kits, the AA 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 β-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. 
     In some embodiments of these methods and kits, the subject is a mammal. In some embodiments of these methods and kits, 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. 
     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. 
     In some embodiments, in situ imaging and/or in vivo imaging are useful in methods to identify which subjects to treat. For example, in in situ imaging, the AAs are used to screen subject samples to identify those subjects having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site. 
     In some embodiments in situ imaging is used to identify or otherwise refine a subject population suitable for treatment with an AA of the disclosure. For example, subjects that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the CM (CM) of the AA being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an AA comprising such a CM. Likewise, subjects 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 AA being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such subjects that test negative with respect to a first AA can be tested with other AAs comprising different CMs until a suitable AA for treatment is identified (e.g., an AA comprising a CM that is cleaved by the subject at the site of disease). In some embodiments, the subject is then administered a therapeutically effective amount of the AA for which the subject tested positive. 
     In some embodiments in vivo imaging is used to identify or otherwise refine a subject population suitable for treatment with an AA of the disclosure. For example, subjects that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the CM (CM) of the AA being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an AA comprising such a CM. Likewise, subjects that test negative might be identified as suitable candidates for another form of therapy. In some embodiments, such subjects that test negative with respect to a first AA can be tested with other AAs comprising different CMs until a suitable AA for treatment is identified (e.g., an AA comprising a CM that is cleaved by the subject at the site of disease). In some embodiments, the subject is then administered a therapeutically effective amount of the AA for which the subject tested positive. 
     In some embodiments of the methods and kits, the method or kit is used to identify or otherwise refine a subject population suitable for treatment with an AA of the disclosure. For example, subjects that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the CM (CM) of the AA being tested in these methods are identified as suitable candidates for treatment with such an AA comprising such a CM. Likewise, subjects that test negative for both of the targets (e.g., the target) and the protease that cleaves the substrate in the CM in the AA being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such subjects can be tested with other AAs until a suitable AA for treatment is identified (e.g., an AA comprising a CM that is cleaved by the subject at the site of disease). In some embodiments, subjects that test negative for either of the target (e.g., the target) are identified as suitable candidates for treatment with such an AA comprising such a CM. In some embodiments, subjects that test negative for either of the target (e.g., the target) are identified as not being suitable candidates for treatment with such an AA comprising such a CM. In some embodiments, such subjects can be tested with other AAs until a suitable AA for treatment is identified (e.g., an AA comprising a CM that is cleaved by the subject at the site of disease). In some embodiments, the AA is an AA to which a therapeutic agent is conjugated. In some embodiments, the AA is not conjugated to an agent. In some embodiments, the AA comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of AA 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. 
     In some embodiments, a method or kit is used to identify or otherwise refine a subject population suitable for treatment with an anti-the target AA and/or conjugated AA (e.g., AA to which a therapeutic agent is conjugated) of the disclosure, followed by treatment by administering that AA and/or conjugated AA to a subject in need thereof. For example, subjects that test positive for both the targets (e.g., the target) and a protease that cleaves the substrate in the CM (CM) of the AA and/or conjugated AA being tested in these methods are identified as suitable candidates for treatment with such antibody and/or such a conjugated AA comprising such a CM, and the subject is then administered a therapeutically effective amount of the AA and/or conjugated AA that was tested. Likewise, subjects 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 AA being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such subjects can be tested with other antibody and/or conjugated AA until a suitable antibody and/or conjugated AA for treatment is identified (e.g., an AA and/or conjugated AA comprising a CM that is cleaved by the subject at the site of disease). In some embodiments, the subject is then administered a therapeutically effective amount of the AA and/or conjugated AA for which the subject tested positive. 
     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 AA 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 AA 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 AA comprises a first linker peptide (LP1) and a second linker peptide (LP2), 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 LP1 and LP2 is a peptide of about 1 to 20 amino acids in length, and wherein each of LP1 and LP2 need not be the same linker. In some embodiments of these methods and kits, one or both of LP1 and LP2 comprises a glycine-serine polymer. In some embodiments of these methods and kits, at least one of LP1 and LP2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GSGGS)n (SEQ ID NO: 22) and (GGGS)n (SEQ ID NO: 23), where n is an integer of at least one. In some embodiments of these methods and kits, at least one of LP1 and LP2 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 LP1 and LP2 comprises an amino acid sequence selected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO: 24), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 25), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 26), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 27), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 28), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 29). 
     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). 
     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 AA when the AA 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. 
     The antibodies, conjugated antibodies, AAs and conjugated AAs of the disclosure are used in diagnostic and prophylactic formulations. In one embodiment, an AA is administered to subjects that are at risk of developing one or more of the aforementioned inflammations, inflammatory disorders, cancer or other disorders. 
     A subject&#39;s or organ&#39;s predisposition to one or more of the aforementioned disorders can be determined using genotypic, serological or biochemical markers. 
     In some embodiments of the disclosure, an AA and/or a conjugated AA is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, an AA and/or a conjugated AA is administered to mitigate or reverse the effects of the clinical indication. 
     Antibodies, conjugated antibodies, AAs and conjugated AAs of the disclosure are also useful in the detection of the target in subject samples and accordingly are useful as diagnostics. For example, the antibodies, conjugated antibodies, the AAs and conjugated AAs of the disclosure are used in in vitro assays, e.g., ELISA, to detect target levels in a subject sample. 
     In one embodiment, an antibody and/or AA of the disclosure is immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized antibody and/or AA serves as a capture antibody for any target that may be present in a test sample. Prior to contacting the immobilized antibody and/or AA with a subject 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. 
     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. 
     It will be appreciated that based on the results obtained using the antibodies and/or AAs 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. 
     Antibodies, conjugated antibodies, AAs and conjugated AAs 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, AAs 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 AAs 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. 
     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, AAs 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. 
     In another example, AAs can be used to detect the presence or absence of a cleaving agent in a sample. For example, where the AAs contain a CM susceptible to cleavage by an enzyme, the AAs can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample. In another example, where the AAs contain a CM susceptible to cleavage by reducing agent, the AAs can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the AAs 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 AA 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 AAs 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 AAs prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the AA by the cleaving agent in the sample. 
     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 AAs 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 AAs 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. 
     AAs are also useful in in situ imaging for the validation of AA 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). 
     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. 
     In these techniques, an AA is labeled with a detectable label. The detectable label may 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). 
     Detection of the label in a sample that has been incubated with the labeled, AA 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 Nov. 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 AA 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. 
     In some embodiments, in situ imaging and/or in vivo imaging are useful in methods to identify which subjects to treat. For example, in in situ imaging, the AAs are used to screen subject samples to identify those subjects having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site. 
     In some embodiments in situ imaging is used to identify or otherwise refine a subject population suitable for treatment with an AA of the disclosure. For example, subjects that test positive for both the target and a protease that cleaves the substrate in the CM (CM) of the AA being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an AA comprising such a CM. Likewise, subjects that test negative for either or both of the target and the protease that cleaves the substrate in the CM in the AA being tested using these methods are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the AA being tested). In some embodiments, such subjects that test negative with respect to a first AA can be tested with other AAs comprising different CMs until a suitable AA for treatment is identified (e.g., an AA comprising a CM that is cleaved by the subject at the site of disease). 
     In some embodiments in vivo imaging is used to identify or otherwise refine a subject population suitable for treatment with an AA of the disclosure. For example, subjects that test positive for both the target and a protease that cleaves the substrate in the CM (CM) of the AA being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an AA comprising such a CM. Likewise, subjects that test negative are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the AA being tested). In some embodiments, such subjects that test negative with respect to a first AA can be tested with other AAs comprising different CMs until a suitable AA for treatment is identified (e.g., an AA comprising a CM that is cleaved by the subject at the site of disease). 
     Pharmaceutical Compositions 
     The AAs and conjugated AAs 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 AA and/or conjugated AA 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&#39;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&#39;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. 
     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. In an exemplary embodiment, the route of administration is intravenous. 
     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. 
     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 mannitol, 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. 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. 
     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. 
     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. 
     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. 
     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. 
     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. Pat. No. 4,522,811. 
     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. 
     Dosing 
     As provided herein, as subject is administered the AA or a conjugated AA at a dose of anywhere from about 1 ng/kg to 100 g/kg. In exemplary embodiments, the subject is administered the AA or the conjugated AA at a dose of greater than 6 mg/kg to about 10 mg/kg. In one embodiment, the subject is administered the AA or the conjugated AA at a dose of greater than 6 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 7 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 8 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 9 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 10 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of greater than 6 mg/kg to about 7 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 7 mg/kg to about 8 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 8 mg/kg to about 9 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 9 mg/kg to about 10 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of greater than 6 mg/kg to about 8 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 7 mg/kg to about 9 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a dose of about 8 mg/kg to about 10 mg/kg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of greater than 240 mg to about 1000 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of greater than 240 mg to about 400 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of greater than 600 mg to about 1000 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of greater than 240 mg to greater than 600 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of greater than 240 mg to about 280 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 280 mg to about 320 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 320 mg to about 360 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 360 mg to about 400 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of greater than 240 mg to about 320 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 280 mg to about 360 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 320 mg to about 400 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of greater than 600 mg to about 700 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 700 mg to about 800 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 800 mg to about 900 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 900 mg to about 1000 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of greater than 600 mg to about 800 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 700 mg to about 900 mg. In another embodiment, the subject is administered the AA or the conjugated AA at a fixed dose of about 800 mg to about 1000 mg. 
     In some embodiments the subject is administered a conjugated AA based on the weight of the subject. 
     In some embodiments the subject is administered a conjugated AA in which the dosage when measured in mg/kg is based on the actual body weight of the subject. 
     In some embodiments the subject is administered a conjugated AA in which the dosage when measured in mg/kg is based on the adjusted ideal body weight (AIBW) of the subject. In some embodiments, the adjusted ideal body weight is calculated based on a difference between the given subject&#39;s actual body weight and a predetermined ideal body weight (IBW) for male and female subjects as corresponding to the subject. In some embodiments, the ideal body weight of the given subject is based on the height of the subject. In some embodiments, the ideal body weight (IBW) for a given male subject in kilograms is determined as IBW=0.9×(height in cm)−88, and the IBW for a given female subject in kilograms is determined as IBW=0.9×(height in cm)−92. In some embodiments, the adjusted ideal body weight (AIBW) for a given subject in kilograms is determined by AIBW=IBW+0.4×(actual weight−IBW), where the IBW is based on their given height and gender. In some embodiments, the male and female subjects are human subjects. In some embodiments, the AIBW of the human subjects are from about 40 kg to about 100 kg. 
     In some embodiments, the subject is administered the AA or the conjugated AA intravenously every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 14 days, every 15 days, every 16 days, every 17 days, every 18 days, every 19 days, every 20 days, every 21 days, or even every 30 days. In some embodiments, the subject is administered the AA or the conjugated AA intravenously for as long as the AA and/or agent is effective. 
     In some embodiments, the subject is administered the AA or the conjugated AA once daily. In some embodiments, the subject is administered the AA or the conjugated AA multiple times a day, for example every 4 hours, every 6 hours, every 4-6 hours, every 8 hours, or every 12 hours. 
     In some embodiments of the present disclosure, in conjunction with administration of the AA of the present disclosure, the subject can be treated prophylactically with one or more treatment regimens and/or precautions intended to mitigate or prevent ocular toxicity. Without being bound by theory, these prophylactic measures are intended to mitigate and/or prevent ocular toxicity associated with maytansinoids, such as the DM4 associated with the conjugated AAs of the present disclosure. Exemplary prophylactic measures to mitigate and/or prevent ocular toxicity include use of UV AB eye protection (e.g., sunglasses), use of artificial tear eye drops, topical vasoconstrictor eye drops (e.g., brimonidine tartrate ophthalmic solution, tetrahydrozoline eye drops), and/or topical steroid eye drops (e.g., prednisolone acetate eye drops). In some embodiments, administration of ocular prophylactic measures to the treated subjects is optional. In some embodiments, administration of ocular prophylactic measures the treated subjects is mandatory. 
     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: CD166 Expression on Immune Cells and Activated T-Cells 
     In this exemplary study, immune cells from human donors (peripheral blood mononuclear cells; PBMCs) were shown to express CD166. These exemplary results demonstrate that immune cells can be targeted selectively by antibodies that specifically bind CD166 and activatable antibodies that when activated specifically bind CD166. 
     To interrogate CD166 expression on the surface of immune cells, human PBMCs were isolated from the blood of four healthy donors (Leuko Pak, Stem Cell Technologies, Cambridge, Mass.). The PBMCs were isolated using Ficoll-Paque PLUS density gradient media (GE Healthcare Life Sciences Catalog #17 1440 02). The isolated PBMCs were analyzed by flow cytometry for membrane markers to detect CD166 expression of immune cells. For flow cytometry analysis in this and other studies described herein, cells were pre-incubated with Fc block reagents for 10 min on ice, then stained with conjugated antibody solution (anti-human CD166 antibody 3A6, BD Biosciences) in staining buffer (BioLegend #420201, or BD Biosciences #563794) for 30 minutes on ice (or one hour at room temperature for intracellular staining). Cells were washed 3 times in staining buffer (400 g, 5 minutes, 4 C). The live/dead fixable viability dye eFluor™ 780 (eBioScience #65-0865-14) was used to exclude dead cells. 
     Referring to  FIG. 1A , CD166 expression measured on monocytes, B cells, and blood myeloid dendritic cells (mDC) as compared to a fluorescence minus one (FMO) control. These exemplary results show the highest level of CD166 expression in mDCs. 
     Referring to  FIG. 1B , flow cytometry analysis was used to measure the percentage of the population of different immune cell sub-types that express CD166. These exemplary results show that blood myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDCs) showed the highest percentage of cells that expressed CD166, followed by monocytes and B cells. Natural killer (NK) cells, naïve CD4+ T cells, naïve CD8+ cells, and Treg cells showed essentially no CD166 expression. 
     Referring to  FIG. 1C , CD166 expression can be induced on naïve CD4+ T cells upon CD3/CD28 stimulation. In this exemplary study, CD4+ T cells isolated from PBMCs from healthy human donors were isolated by Ficoll and magnetic beads (#17952, Stem Cell Technology). CD4+ T cells were stimulated with aCD3/aCD28 beads for 4 days, and the cells were analyzed by flow cytometry on each day to detect CD166 expression. The results in  FIG. 1C  are from two independent donors, both showing CD166 expression in CD4+ T cells at days 3 and 4 following stimulation. Other exemplary studies showed that CD166 can also be induced in T cells and natural killer (NK) cells following phytohaemagglutinin (PHA) or staphylococcal enterotoxin B (SEB) treatment. 
     These results show that dendritic cells (mDC and pDC), as well as activated T cells, could be targeted by activatable antibodies that when activated bind CD166. 
     Example 2: Human CD166 Syngeneic Tumor Mice Model 
     In this exemplary study, a syngeneic mouse model was developed, as the anti-CD166 antibodies and activatable antibodies used in these studies do not bind mouse CD166. These results showed that a mouse cell line expressing human CD166 was sensitive in vitro to protease-activated activatable anti-CD166 antibody drug conjugate and could be used to establish a huCD166-expressing tumor model in immunocompetent mice. 
     CT26 cells were obtained from ATCC. Cells were cultured in cultured in RPMI-1640™ medium (Life Technologies, Inc, cat #72400120) supplemented with 10% (v/v) fetal bovine serum (FBS; Life Technologies, Inc, cat #16140-071). The cells were maintained in a humidified atmosphere of 5% CO2 at 37° C. CT26 murine colon carcinoma parental cells were transduced with a human CD166 lentiviral vector. CT26 cells that overexpressed human CD166 were selected with puromocyin. Referring to  FIG. 2A , these transgenic CT26 were analyzed by flow cytometry to confirm expression of human CD166. 
     An exemplary study was performed to demonstrate that CT26 huCD166 cells are sensitive to activatable anti-CD166 antibody conjugated to DM4 (Combination 55). Referring to  FIG. 2B , parental CT26 cells and transgenic CT26 huCD166 cells were cultured for four days in presence of an isotype (Synagis) conjugated to DM4 or Combination 55 activatable antibody drug conjugate (AADC) activated by matriptase. Recombinant human matriptase (R&amp;D systems Catalog #3946-SE) was active site titrated with MUGB (Sigma Aldrich Catalog #51010) and diluted in 50 mM Tris/HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.4. Cell viability was measured using Cell Titer Glo. These exemplary data demonstrate that transgenic CT26 huCD166 cells are sensitive to protease-activated anti-CD166 AADC. 
     An exemplary study was performed to show that CT26 huCD166 cells could be implanted in mice and form tumors. Referring to  FIG. 2C , BALB/C mice at 7 weeks of age were implanted with the indicated number of CT26 huCD166 cells, and the tumor volume was measured over time. 
     Example 3: In Vivo Efficacy in a Syngeneic Mouse Model of Combination of Anti-CD166 Conjugated Activatable Antibody and Anti-PD-1 Activatable Antibody 
     In this exemplary study, the in vivo efficacy of anti-CD166 DM4-conjugated activatable antibody (Combination 55 AADC) and activatable anti-PD-1 antibody (muPD-1 AA) were determined using a syngeneic mouse model. These results show that both of these drugs demonstrated in vivo efficacy against a huCD166-expressing tumor in a mouse model, and that a combined administration of both drugs showed higher level of in vivo efficacy. 
     For in vivo efficacy study, immunocompetent female (BALB/C) mice (7 weeks of age; Charles River Laboratories, Hollister, Calif.) were each inoculated with 10 6  CT26 huCD166 cells subcutaneously into the right hind flank in a volume of 0.1 mL serum free RPMI 1640 cell culture medium. Tumor volumes and body weights were recorded twice weekly after inoculation. Tumor dimensions were determined by caliper measurements and tumor volume was calculated using the formula (a×b 2 )/2 where a is the longest and b the shortest diameter. When tumor size reached 100-175 mm 3 , mice were randomized (day 0) and dosed according to the following regimen. Anti-CD166 AADC (Combination 55) was dosed intravenously 5 mg/kg once a week ×2 weeks (day 0 and day 7). Activatable anti-mouse PD-1 antibody (muPD-1 AA) was administered by intraperitoneal injection 10 mg/kg b.i.w. ×2.5 weeks (day 1, day 5, day 8, day 12, and day 15). In some experiments, mice that exhibited complete tumor regression were re-challenged with CT-26 huCD166 tumor cells 2 weeks after the last dose of drug. A control group was administered with vehicle control (PBS). 
     The activatable anti-CD166 antibody of Combination 55 includes a heavy chain of SEQ ID NO: 8 or SEQ ID NO: 9 and a light chain of SEQ ID NO: 14 or SEQ ID NO: 15. Conjugated activatable anti-CD166 antibodies can be conjugated to DM4 via a SPDB linker. 
     An exemplary amino acid sequence of human CD166 is: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 50) 
               
               
                   
                 MESKGASSCRLLFCLLISATVFRPGLGWYTVNSAYG 
               
               
                   
               
               
                   
                 DTIIIPCRLDVPQNLMFGKWKYEKPDGSPVFIAFR 
               
               
                   
               
               
                   
                 SSTKKSVQYDDVPEYKDRLNLSENYTLSISNARIS 
               
               
                   
               
               
                   
                 DEKRFVCMLVTEDNVFEAPTIVKVFKQPSKPEIVS 
               
               
                   
               
               
                   
                 KALFLETEQLKKLGDCISEDSYPDGNITWYRNGKV 
               
               
                   
               
               
                   
                 LHPLEGAVVIIFKKEMDPVTQLYTMTSTLEYKTTK 
               
               
                   
               
               
                   
                 ADIQMPFTCSVTYYGPSGQKTIHSEQAVFDIYYPT 
               
               
                   
               
               
                   
                 EQVTIQVLPPKNAIKEGDNITLKCLGNGNPPPEEF 
               
               
                   
               
               
                   
                 LFYLPGQPEGIRSSNTYTLMDVRRNATGDYKCSLI 
               
               
                   
               
               
                   
                 DKKSMIASTAITVHYLDLSLNPSGEVTRQIGDALP 
               
               
                   
               
               
                   
                 VSCTISASRNATVVWMKDNIRLRSSPSFSSLHYQD 
               
               
                   
               
               
                   
                 AGNYVCETALQEVEGLKKRESLTLIVEGKPQIKMT 
               
               
                   
               
               
                   
                 KKTDPSGLSKTIICHVEGFPKPAIQWTITGSGSVI 
               
               
                   
               
               
                   
                 NQTEESPYINGRYYSKIIISPEENVTLTCTAENQL 
               
               
                   
               
               
                   
                 ERTVNSLNVSAISIPEHDEADEISDENREKVNDQA 
               
               
                   
               
               
                   
                 KLIVGIVVGLLLAALVAGVVYWLYMKKSKTASKHV 
               
               
                   
               
               
                   
                 NKDLGNMEENKKLEENNHKTEA. 
               
            
           
         
       
     
     Referring to  FIGS. 3A-3D , the tumor growth curves of three (3) independent studies are shown. As single agents, both huCD166 AADC ( FIG. 3C ) and muPD-1 AA ( FIG. 3B ) only slow down tumor growth as compare to vehicle control ( FIG. 3A ), but they do not induce complete tumor regression. In contrast, when both drugs are combined ( FIG. 3D ), there is a significant increase of antitumor activity as compare to either of the single drugs (huCD166 AADC alone versus combination: Log 10 difference: −0.7699, p&lt;0.0001), with multiple complete responses (tumor volume at 20 days post-initial dose lower than the measurement at day 0) observed only with the combinations of the two drugs. Analysis of 3 independent studies showed that the combination of huCD166 AADC plus muPD-1 AA led to a 51% overall response rate (ORR). 
     These results show that in vivo efficacy of the combination of the anti-PD-1 activatable antibody and the anti-CD166 activatable antibody drug conjugate showed a significantly higher efficacy than either drug alone. 
     Example 4: Combination of Anti-CD166 Conjugated Activatable Antibody and Anti-PD-1 Activatable Antibody Induce Memory T Cell Response 
     In this exemplary study, the results demonstrated that mice studied in Example 3 that remained tumor-free after treatment were protected from a tumor rechallenge, indicating that an immunological memory response had been established in the mice. 
     Treated mice from Example 3 that remained tumor-free 15 days after the last dose of the combination treatment were re-challenged with CT-26 huCD166 tumor cells. As shown in  FIG. 4B , 50% (3 of 6) of the tumor-free mice were protected from tumor rechallenge, whereas all naïve mice (0 of 10) rapidly developed tumors ( FIG. 4A ). 
     Two weeks after tumor rechallenge, splenocytes derived from these protected mice were stimulated with the AH1, a CT26-specific peptide (Eurogentec #AS-64798) in presence of brefeldin and were evaluated by flow cytometry to determine the production of IFN-gamma by peripheral CD8+ T cells. Referring to  FIG. 4C , CD8+ T cells isolated from mice protected in the tumor rechallenge assay produce IFN-gamma ( FIG. 4C , bottom panels), whereas CD8+ T cells isolated from unprotected mice do not produce IFN-gamma in response to AH1 peptide ( FIG. 4C , top panels). These results indicate that the combination of huCD166 AADC plus mouse PD-1 AA induce a T cell-centered, immunological memory response in the mice treated with the combination. 
     To provide further evidence of the role of CD8+ cells in these results, the effect of CD8+ T cell depletion on the in vivo efficacy of huCD166 AADC, a combination treatment of huCD166 AADC and muPD-1 AA, or a vehicle control (PBS) in the immunocompetent mouse tumor model was determined. In this exemplary study, tumors were formed by implanting CT26 huCD166 in immunocompetent BALB/C mice as described in Example 3. For the mice that underwent CD8+ T cell depletion, anti-CD8 depleting antibodies (53-6.72; rat IgG2a, Bio X Cell) were administered to the mice at 10 mg/kg at day −2, day 0, day 7, and day 8. Referring to  FIG. 6 , CD8+ T cell depletion was confirmed by flow cytometry analysis of blood samples obtained at day 10. CD8+ T-cell depleted and non-depleted tumor-bearing mice were treated with huCD166 AADC, both huCD166 AADC and muPD-1 AA, or vehicle control (PBS). Anti-huCD166 AADC (Combination 55) was administered intravenously 5 mg/kg once a week ×2 weeks (day 0 and day 7). Activatable anti-mouse PD-1 antibody (muPD-1 AA) was administered by intraperitoneal injection 10 mg/kg b.i.w. ×2.5 weeks (day 1, day 5, day 8, day 12, and day 15). 
     Referring to  FIGS. 5A-5C , in CD8+ T-cell undepleted tumor-bearing mice the combination treatment of huCD166 AADC and muPD-1 AA showed a greater efficacy ( FIG. 5C ) than either huCD166 AADC monotherapy ( FIG. 5B ) or vehicle control ( FIG. 5A ). Referring to  FIGS. 5D-5F , the efficacy of huCD166 AADC monotherapy ( FIG. 5E ) or the combination of huCD166 AADC and muPD-1 AA ( FIG. 5F ) showed significant reduction of anti-tumor efficacy in CD8+ T cell depleted mice. 
     These results show that the anti-tumor efficacy of huCD166 AADC and the anti-tumor efficacy of the combination of huCD166 AADC and muPD-1 AA is partially dependent on CD8+ T cells in the treated subject. 
     Example 5: Cytotoxic Activity of huCD166 AADC Towards Mature Dendritic Cells and Activated T Cells 
     In this exemplary study, the results demonstrated that while anti-CD166 antibody drug conjugate (huCD166 ADC, VH of SEQ ID NO: 12 and VL of SEQ ID NO: 13) demonstrated moderate cytotoxic activity against human dendritic cells and activated human T cells, both of which express CD166, this cytotoxicity was lower than its efficacy against tumor cells, e.g. transgenic CT26 expressing huCD166. 
     The cytotoxicity of a DM4-conjugated anti-CD166 antibody (huCD166 ADC) towards immune cells was tested in this exemplary study. CD4+ T cells and monocytes were isolated from PBMCs using Stem Cell Technology magnetic beads (#17952 for CD4+ T cells, and #119359 for Monocytes) according to manufacturer instructions. Isolated monocytes were differentiated into dendritic cells (MoDC) using Stem Cell Technology dendritic cell differentiation kit (#10988) according to manufacturer instructions. In some assays, dendritic cells were then maturated to fully activated MoDC using Stem Cell Technology DC maturation kit (#10989). MoDC were cultured using ImmunoCult dendritic cell medium (StemCell Technologies #10987). In some assay, purified CD4+ T cells were activated with Dynabeads covalently coupled to anti-CD3 and anti-CD28 antibodies (Gibco Catalog #11132D) at a ratio of one T cell to one bead. 
     The T cells were pre-activated for 24 hours to allow the expression of CD166 before treatment with the test article. Referring to  FIG. 7A , the activated T cells were treated for 72 hours with the indicated test article (Synagis isotype antibody conjugated to DM4 (Iso-DM4 ADC), anti-human CD166 antibody conjugated to DM4 (CD166-DM4 ADC), or anti-human CD166 antibody (CD166 mAb) at the indicated concentrations. Cell viability was measured for each treatment using CellTiter-Glo Luminescent viability assay (Promega #G7570). Referring to  FIG. 7B , fully activated MoDC were incubated with the indicated treatments at the indicated concentrations for 48 hours. Cell viability was measured using CytoTox-Glo cytoxicity assay (Promega #G92901). These exemplary results demonstrate that the anti-CD166 ADC has a minimal cytotoxic activity towards both MoDCs and activated T cells. Cytotoxicity was observed only at doses above 10 nM, similar to the activity of an isotype control conjugated to DM4. 
     These exemplary results suggest that anti-CD166 ADC has a moderate and off-target cytotoxic activity towards these immune cells, rather than a target-mediated toxicity, despite the levels of CD166 expression on these cells. 
     Example 6: Effect of huCD166 ADC on Dendritic Cell Maturation and T Cell Stimulation 
     In this exemplary study, the results demonstrated that anti-CD166 antibody drug conjugate (huCD166 ADC, VH of SEQ ID NO: 12 and VL of SEQ ID NO: 13) promoted maturation of dendritic cells in vitro. 
     In this study, dendritic cells were cultured with 10 mM anti-CD166 antibody conjugated to DM4 (CD166-DM4 ADC) or a dendritic cell maturation cocktail (Stem Cell Technology DC maturation kit (#10989)) for 48 hours. Dendritic cells were also treated with 10 mM of either free DM4, a Synagis isotype-DM4 ADC or were untreated as controls. The MoDCs were then assessed for maturation by flow cytometric measurement of dendritic cell maturation markers CD80 (2D10 antibody, BioLegend), CD83 (HB15e antibody, BioLegend), HLA-DR (L243 antibody, BioLegend), and CD86 (IT2.2 antibody, BioLegend). 
     Referring to  FIG. 8A , expression of CD83 and CD86 was slightly increased in anti-CD166-DM4 ADC or free DM4 treated cells as compared to untreated cells. These exemplary results indicate that DM4 may promote dendritic cell activation, but to a lesser extent than the maturation cocktail of cytokines, which are known to fully induce dendritic cell maturation. 
     The effect of these treated dendritic cells on their ability to activate T cells was determined. In this exemplary study, MoDCs treated as indicated above with the test articles were then co-cultured for 2 days with allogenic CD4+ T cells at a ratio of 1:25 dendritic cells to T cells. T cell activation was assessed after 48 hours by measuring IL-2 production as determined by ELISA. Referring to  FIG. 8B , MoDCs pre-treated with CD166-DM4 ADC, an isotype conjugated to DM4, or free DM4, only slightly increase the production of IL-2 by allogeneic T cells. However, when MoDCs were pretreated with the previous drugs in combination with a dendritic cell maturation cocktail, IL-2 production was significantly higher as compared to T cells co-cultured with dendritic cells treated with maturation cocktail only observed with the maturation cocktail only. 
     Altogether, these exemplary data suggest that in contrast to its cytotoxic activity towards CD166+tumor cells, CD166 ADC spares T cells and dendritic cells, and may enhance T cell priming. Similarly, these results suggest that activated activatable anti-CD166 drug conjugate (anti-CD166 AADC) could potentiate T cell priming in vivo through maturation of dendritic cells, and without being bound by theory, would provide a rationale for the observed synergistic effect of combined therapies of anti-CD166 AADC and anti-PD-1 AA. 
     Example 7: huCD166 AADC Induces Immunogenic Cell Death in Cell Lines 
     In this exemplary study, the results demonstrated that anti-CD166 antibody drug conjugate (huCD166 ADC conjugated to DM4, VH of SEQ ID NO: 12 and VL of SEQ ID NO: 13) induced immunogenic cell death (ICD) in vitro. These exemplary results show that anti-huCD166 ADC can increase signals relating to ICD in cancer cells and CD166-expressing cells. 
     Immunogenic cell death (ICD) is a process by which certain cytotoxic drugs can induce apoptosis of tumor cells in a manner that stimulates the immune system. Cells, such as tumor cells, when treated with drug conjugates, can increase their immunogenic potential through the release of cellular danger signals such as damage-associated molecular patterns (DAMPSs). DAMPs can in turn activate antigen-presenting cells, such as dendritic cells, which can elicit a tumor-targeting immune response and immunological memory. 
     ICD can be measured by markers such as the expression of calreticulin on the surface of cancer cells and secretion of HMGB1. In this exemplary study, A375 cells (ATCC) derived from a human malignant melanoma were cultured in RPMI-1640™ medium with 10% (v/v) fetal bovine serum for 48 hours in the presence of the indicated amount of drug. HCC1806 cells (ATCC) derived from human breast carcinoma were cultured in DME medium for 48 hours in the presence of the indicated amount of drug. CT26 cells and CT26 huCD166 cells (CT26 cells expressing human CD166 polypeptide as discussed herein) were cultured in RPMI-1640™ medium with 10% (v/v) fetal bovine serum for 72 hours in the presence of the indicated amount of drug. Following incubation, calreticulin expression was detected by flow cytometry using a FITC conjugated calreticulin antibody (Novus Bio, clone 1G6A7; #NBP1-47518F). HMGB1 protein was detected by ELISA (Tecan HMGB1 ELISA kit, #NC9959947). 
     Referring to  FIGS. 9A and 9B , in this exemplary study the indicated amount of free DM4 ( FIG. 9A ) or anti-CD166 ADC ( FIG. 9B ) showed that melanoma cells A375 and breast cancer cells HCC1806 showed increased expression of calreticulin (CRT) and increased amounts of secreted HMGB1. Referring to  FIG. 9C , in this exemplary study the indicated amount of free DM4 or CD166 ADC also induced increased surface expression of calreticulin (CRT) on CT26 or CT26 huCD166 cells. 
     These exemplary data suggest that anti-CD166 ADC induce ICD-related signals in cancer cells and cells expressing CD166. These exemplary results show that anti-tumor efficacy may occur by ICD. 
     OTHER EMBODIMENTS 
     While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following.