Radiolabeled met binding proteins for immuno-pet imaging

Abstract

To provide radiolabeled MET binding proteins for immuno-PET imaging.SOLUTION: Radiolabeled anti-MET antibodies and MET×MET bispecific antibodies, and their use in immuno-PET imaging are provided herein. Included are methods of detecting the presence of MET proteins in a subject or sample and methods of monitoring the efficacy of treatment of a Met expressing tumor. Included in this disclosure are radiolabeled anti-MET antibody conjugates and MET×MET bispecific antibody conjugates for use in immuno-PET imaging.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to radiolabeled MET binding proteins and their use in immunoPET imaging. Sequence Listing

[0002] A public copy of the Sequence Listing has been submitted contemporaneously herewith electronically via EFS-Web as an ASCII formatted Sequence Listing, with a filename of "10649WO01_SEQ_LIST_ST25.txt", a creation date of September 15, 2020, and a size of approximately 136 KB. The Sequence Listing contained in this ASCII formatted document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a heterodimeric paracrine growth factor that exerts its activity by interacting with the HGF receptor (HGFR). HGFR is the product of the c-Met oncogene, also known as MET. MET is a receptor tyrosine kinase consisting of a transmembrane beta chain linked to an extracellular alpha chain via a disulfide bridge. Binding of HGF to MET activates the kinase catalytic activity of MET, leading to phosphorylation of the beta chain at Tyr 1234 and Tyr 1235 and subsequent activation of downstream signaling pathways.

[0004] Overexpression, activation, or amplification of MET and / or HGF is associated with a number of cancers, including non-small cell lung cancer (NSCLC), gastric, ovarian, pancreatic, thyroid, breast, head and neck, colon, and kidney cancers (Sierra and Tsao, Ther. Adv. Med. Oncol.,3(1 Suppl):S21-S35, 2011). MET amplification is thought to be a major driver of oncogenesis in NSCLC and esophagogastric malignancies. Furthermore, mutations resulting in deletion of exon 14 of MET have been described as an oncogenic driver in a subset of NSCLC. Tumor cell lines with MET gene amplification are highly dependent on MET for proliferation and survival. Preclinical data implicate MET signaling in resistance to targeted therapy in multiple tumor types, such as NSCLC, colorectal cancer, and head and neck squamous cell carcinoma (HNSCC).

[0005] Immunopositron emission tomography (PET) is an imaging diagnostic tool that utilizes monoclonal antibodies labeled with positron emitters, combining the targeting properties of antibodies with the sensitivity of positron emission tomography cameras. See, for example, The Oncologist, 12:1379 (2007); Journal of Nuclear Medicine, 52(8):1171 (2011). ImmunoPET allows the visualization and quantification of antigen and antibody accumulation in vivo, and therefore can serve as an important tool for diagnosis and complementary therapy. For example, immunoPET can help select potential subjects for a particular therapy, as well as monitor treatment. Both preclinical and recent clinical results suggest that tumors with MET gene alterations respond to MET inhibitors, validating MET as a driver of cancer. Thus, there is a need for diagnostic tools for anti-MET and / or anti-MET therapies, particularly diagnostic tools that allow for the detection of suitable candidates for therapy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sierra and Tsao, Ther.Adv.Med.Oncol.,3(1 Suppl):S21-S35, 2011 Summary of the Invention [Means for solving the problem]

[0007] The present disclosure includes radiolabeled anti-MET antibody conjugates and METxMET bispecific antibody conjugates for use in immunoPET imaging.

[0008] In one embodiment, the conjugate comprises an anti-MET antibody, a METxMET bispecific antibody or antigen-binding fragment thereof, a chelating moiety, and a positron emitter.

[0009] Provided herein are methods for synthesizing the conjugates and synthetic intermediates useful therefor.

[0010] Provided herein are methods of imaging a tissue expressing MET, the methods comprising administering to the tissue a radiolabeled anti-MET antibody conjugate or a METxMET bispecific antibody conjugate as described herein, and visualizing MET expression by positron emission tomography (PET) imaging.

[0011] Also provided herein are methods of detecting MET in a tissue, comprising administering to the tissue a radiolabeled anti-MET antibody conjugate and a METxMET bispecific antibody conjugate as described herein, and visualizing MET by PET imaging. In one embodiment, the tissue is in a human subject. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject has a disease or disorder, such as cancer.

[0012] Also provided herein is a method of determining the presence of MET-expressing cells in a subject, comprising administering to a subject a radiolabeled anti-MET antibody conjugate or a METxMET bispecific antibody conjugate described herein, and visualizing MET expression by PET imaging.

[0013] Also provided herein is a method for identifying a subject having a solid tumor suitable for anti-tumor therapy comprising an inhibitor of the HGF / MET signaling pathway, e.g., an anti-MET antibody, a METxMET bispecific antibody, or an antibody-drug conjugate (ADC) thereof. The method includes administering to the subject a radiolabeled antibody conjugate as described herein, visualizing the administered radiolabeled antibody conjugate in the tumor by PET imaging, and the presence of the radiolabeled antibody conjugate in the tumor identifies the subject as suitable for anti-tumor therapy comprising an inhibitor of the HGF / MET signaling pathway.

[0014] Also provided herein are methods of treating a solid tumor in a subject, comprising determining that the solid tumor is MET positive and administering an anti-tumor treatment to a subject in need thereof. In certain embodiments, the anti-tumor treatment comprises an anti-MET antibody or a METxMET bispecific antibody. In certain embodiments, the subject is administered a radiolabeled antibody conjugate as described herein, and the localization of the radiolabeled antibody conjugate is imaged by positron emission tomography (PET) imaging to determine whether the tumor is MET positive.

[0015] Provided herein is a method of monitoring the effectiveness of an anti-tumor treatment in a subject receiving an anti-tumor treatment, the method comprising administering to the subject a radiolabeled conjugate as described herein, imaging the localization of the administered radiolabeled conjugate in the tumor by PET imaging, wherein a decrease from baseline in uptake of the conjugate or radiolabeled signal indicates tumor shrinkage and the effectiveness of the anti-tumor treatment. In certain embodiments, the anti-tumor treatment comprises an inhibitor of the HGF / MET signaling pathway (e.g., either an anti-MET antibody or a METxMET bispecific antibody, or an ADC).

[0016] Also provided herein is a method of predicting a subject's response to an anti-tumor treatment comprising an inhibitor of the HGF / MET signaling pathway, the method comprising determining whether a tumor is MET positive, and if the tumor is MET positive, indicating a positive response of the subject to the anti-tumor treatment comprising an inhibitor of the HGF / MET signaling pathway. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present disclosure and localizing the radiolabeled antibody conjugate within the tumor by PET imaging, and the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is MET positive.

[0017] Provided herein are methods of diagnosing and treating a subject having a tumor, the methods comprising administering to the subject a radiolabeled conjugate as described herein, wherein localization of the radiolabeled antibody conjugate is imaged via PET imaging to determine if the tumor is MET positive, diagnosing the subject having a MET positive tumor, and administering to the subject an anti-tumor treatment comprising an inhibitor of the HGF / MET signaling pathway.

[0018] Provided herein are methods of diagnosing a subject having a MET-expressing tumor, the methods including administering to the subject a radiolabeled anti-MET antibody conjugate or a METxMET bispecific antibody conjugate described herein, visualizing MET expression by PET imaging, and diagnosing the subject as having a MET-expressing tumor when MET expression is visualized by PET imaging. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 shows a SE-HPLC chromatogram of a 5 μg injection of DFO-METxMET immunoconjugate complex on a Superdex 200 Increase column with UV 280 nm absorbance detection. Monomeric (99.6%) and high molecular weight (HMW) species (0.4%) are shown.

[0020] [Diagram 2]Figure 2 shows an image of SDS-PAGE of DFO-METxMET immune complex. The gel demonstrates that there is no change in the integrity of the antibody after DFO conjugation. Lanes are labeled as follows: 1) Standard ladder (BioRad, Cat. No.: 161-0374), 2) Non-reduced bispecific antibody, 3) Non-reduced DFO-Ab immune complex, 4) Blank, 5) Reduced bispecific antibody, 6) Non-reduced DFO-Ab immune complex. Each well was loaded with approximately 2 μg of protein. Note that non-reduced antibodies typically show less electrophoretic mobility than expected compared to ladder in a standard SDS-PAGE setup.

[0021] [Diagram 3] Figure 3 shows a representative SE-HPLC radiochromatogram of a 5 μg injection of radioimmunoconjugate (DFO-MET x MET bispecific antibody) with gamma emission detection. The RCP was greater than 95%, and unincorporated 89Zr accounted for less than 1% of the total integrated activity.

[0022] [Figure 4] Figure 4 shows a representative SE-HPLC UV absorption chromatogram of a 5 µg injection of radioimmunoconjugate (DFO-MET x MET bispecific antibody). The major species (97.9%) and HMW species (2.1%) are shown. The peak eluting from 25 min to 31 min is considered to be an event of formulation buffer / mobile phase mixing and not proteinaceous in origin.

[0023] [Diagram 5] Figure 5 shows PET / CT images of EBC-1 tumor xenografts in mice. Mice were administered radiolabeled METxMET bispecific antibody conjugates, and the conjugates specifically localized to the MET-expressing tumor xenografts over several days.

[0024] [Figure 6]Figure 6 shows PET / CT images of NCI-H441 tumor xenografts in mice. Mice were administered radiolabeled METxMET bispecific antibody conjugates, and the conjugates specifically localized to the MET-expressing tumor xenografts over several days.

[0025] [Figure 7] Figure 7 shows PET / CT images of NCI-H358 tumor xenografts in mice. Mice were administered radiolabeled METxMET bispecific antibody conjugates, and the conjugates specifically localized to the MET-expressing tumor xenografts over several days.

[0026] [Figure 8-1] Figures 8A, 8B, 8C, 8D, 8E, and 8F provide ex vivo biodistribution data of 89Zr-DFO-METxMET bispecific antibody conjugate in SCID mice bearing tumor xenografts. Mice were administered a single intravenous dose of 0.1 mg / kg, 0.5 mg / kg, or 5.0 mg / kg of 89Zr-DFO-METxMET bispecific antibody conjugate and sacrificed 6 days later. Blood was collected via cardiac puncture, and the indicated harvested tissues were weighed and radioactivity determined. Percent injected dose per gram (%ID / g) values ​​of individual samples collected on day 6 were calculated relative to the radioactivity of the dose standard from the injected material (89Zr-DFO-METxMET bispecific antibody conjugate) and the weight of the individual samples. Data are plotted as mean ± SD. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0027] [Figure 9] FIG. 9 shows the correlation between 89Zr-DFO-MET×MET bispecific antibody uptake and MET expression levels in tumor xenografts from three MET-expressing cell lines.

[0028] [Figure 10] Figures 10A and 10B show antibody saturation binding data for three MET-expressing cell lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] I. Definition Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described herein, as such methods and conditions may vary. It should also be understood that the terminology used herein is used for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the invention is limited only by the appended claims.

[0030] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.As used herein, the term "about" when used in relation to a specific recited numerical value means that the value can vary from the recited value by 1% or less.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0031] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, patent applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0032] MET protein The expressions "MET", "c-Met", and the like, as used herein, refer to: (1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 145, and / or the amino acid sequence set forth in NCBI Accession No. NM_001127500.2, which represents the unprocessed preproprotein of isoform "a"; (2) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 146, and / or the amino acid sequence set forth in NCBI Accession No. NM_000236.2, which represents the unprocessed preproprotein of isoform "b"; (3) a human transmembrane receptor tyrosine kinase having an amino acid sequence set forth in SEQ ID NO: 147, and / or an amino acid sequence set forth in NCBI Accession No. NM_001311330.1, which represents the unprocessed preproprotein of isoform "c", and / or (3) a mature protein comprising a cytoplasmic alpha subunit (SEQ ID NO: 148) common to all three isoforms, and a transmembrane beta subunit (SEQ ID NO: 149, 150 or 151 for isoforms a, b and c, respectively). The term "MET" includes both monomeric and multimeric MET molecules. As used herein, the term "monomeric human MET" refers to a MET protein or portion thereof that does not contain or possess any multimerization domain and exists under normal conditions as a single MET molecule without direct physical connection to another MET molecule. An exemplary monomeric MET molecule is a molecule designated herein as "hMET.mmh" comprising the amino acid sequence of SEQ ID NO: 152 (see, e.g., Example 3 of US-2018-0134794). As used herein, the phrase "dimeric human MET" refers to a construct comprising two MET molecules connected to each other via a linker, covalent bond, non-covalent bond, or via a multimerization domain, such as an antibody Fc domain. An exemplary dimeric MET molecule is a molecule designated herein as "hMET.mFc" comprising the amino acid sequence of SEQ ID NO: 153 (see, e.g., Example 3 of US-2018-0134794).

[0033] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless expressly specified as being from a non-human species. Thus, the term "MET" refers to human MET, unless specified as being from a non-human species, e.g., "mouse MET," "monkey MET," etc.

[0034] As used herein, the phrase "cell surface-expressed MET" refers to one or more MET proteins, or extracellular domains thereof, that are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the MET protein is exposed to the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody. "Cell surface-expressed MET" may include or consist of a MET protein expressed on the surface of a cell that normally expresses MET protein. Alternatively, "cell surface-expressed MET" may include or consist of a MET protein expressed on the surface of a cell that does not normally express human MET on its surface, but has been artificially engineered to express MET on its surface.

[0035] Other definitions The term "antibody" as used herein is intended to refer to an immunoglobulin molecule (i.e., an "intact antibody molecule") consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V H ") and the heavy chain constant region (C H 1 domain, C H 2 domain and C H Each light chain consists of a light chain variable region ("LCVR" or "V L ") and the light chain constant region (C L ) V H and V LThe regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs.

[0036] Substitution of one or more CDR residues or omission of one or more CDRs are also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be omitted for binding. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact areas between antibodies and their antigens based on published crystal structures and concluded that only about 1 / 5 to 1 / 3 of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have amino acids in contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0037] CDR residues that are not in contact with the antigen can be identified empirically and / or by molecular modeling from regions of the Kabat CDRs that are outside the Chothia CDRs, based on prior studies (e.g., residues H60-H65 in CDRH2 are often not necessary). When a CDR or its residue(s) is omitted, this CDR or its residue(s) is usually replaced with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. The positions for substitution within the CDR and the amino acids to be replaced can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0038] Human anti-MET antibodies or METxMET bispecific antibodies useful herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences of Table 1 to germline sequences available, for example, from public antibody sequence databases. Antibodies and antigen-binding fragments thereof useful in the present disclosure are derived from any of the amino acid sequences provided in Table 1, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences set forth in Table 1, one of skill in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll of the framework and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found in the first 8 amino acids of FR1, or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody is originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.

[0039] Useful herein are MET binding proteins, such as human anti-MET antibodies and METxMET bispecific antibodies, that comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences set forth in Table 1 herein, with one or more conservative substitutions. For example, the disclosure includes METxMET bispecific antibodies that have 10 or less, 8 or less, 6 or less, 4 or less conserved amino acid substitutions in the HCVR, LCVR, and / or CDR amino acid sequences, e.g., compared to any of the HCVR, LCVR, and / or CDR amino acid sequences in Table 1.

[0040] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies of the present disclosure may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly in CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antibody" as used herein is not intended to include monoclonal antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted onto human FR sequences.

[0041] As used herein, the term "multispecific antigen-binding molecule" refers to bispecific, trispecific, or multispecific antigen-binding molecules, and antigen-binding fragments thereof. A multispecific antigen-binding molecule may be specific for different epitopes of one target polypeptide, or may contain antigen-binding domains specific for one or more target polypeptides. A multispecific antigen-binding molecule may be a single multifunctional polypeptide, or may be a multimeric complex of two or more polypeptides covalently or non-covalently bound to each other. The term "multispecific antigen-binding molecule" includes an antibody of the present disclosure that may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as a protein or fragment thereof, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity. In accordance with the present disclosure, the term "multispecific antigen-binding molecule" also includes a bispecific, trispecific, or multispecific antibody or antigen-binding fragment thereof. In certain embodiments, an antibody of the present disclosure is operably linked to another antibody or antigen-binding fragment thereof to generate a bispecific antibody having a second binding specificity. Bispecific and multispecific antibodies of the present disclosure are described elsewhere herein.

[0042] The term "specifically binds" or "specifically binds to" or the like means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1x10 -8 M or less (e.g., a smaller K D indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by surface plasmon resonance, e.g., BIACORE™, that specifically bind to MET. Furthermore, multispecific antibodies that bind to one domain of MET and one or more additional antigens, or bispecific antibodies that bind to two different regions of MET, are nevertheless considered to be "specifically binding" antibodies as used herein.

[0043] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to MET.

[0044] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody or fragment thereof that specifically binds MET is substantially free of Abs that specifically bind antigens other than MET.

[0045] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows the analysis of biomolecular interactions in real time by detection of changes in protein concentration within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway Township, NJ).

[0046] "K D The term "as used herein" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0047] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. The term also refers to the region of an antigen to which an antibody binds. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that are directly involved in the affinity of the interaction. Epitopes may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0048] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP.

[0049] As applied to polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that share at least 90% sequence identity, and even more preferably at least 95%, 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using predefined gap weights. Preferably, residue positions that are not identical differ only by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix. Sequence similarity for polypeptides is typically measured using sequence analysis software.Protein analysis software uses similarity measures assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the region of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0050] By the phrase "therapeutically effective amount" is meant an amount that is administered to produce a desired effect. The exact amount will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0051] As used herein, the term "subject" refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as cancer.

[0052] II. Radiolabeled immunoconjugates of MET antibodies for immunoPET imaging Provided herein is a radiolabeled antigen binding protein that binds to a MET protein. In some embodiments, the radiolabeled antigen binding protein comprises an antigen binding protein covalently bound to one or more chelating moieties, where the chelating moiety is a chemical moiety capable of chelating a positron emitter.

[0053] In some embodiments, an antigen binding protein that binds MET, e.g., an anti-MET antibody or a MET x MET bispecific antibody, is provided, wherein the antigen binding protein that binds MET is covalently linked to one or more moieties having the structure: -LM Z wherein L is a chelating moiety, M is a positron emitter, and z is independently at each occurrence 0 or 1, with at least one occurrence of z being 1.

[0054] In some embodiments, the radiolabeled antigen binding protein is a compound of formula (I): MLA-[LM Z ] k (I) wherein A is a protein that binds to MET, L is a chelating moiety, M is a positive positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1.

[0055] In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (II): A-[LM] k (II) wherein A is a protein that binds to MET, L is a chelating moiety, M is a positive positron emitter, and k is an integer from 1 to 30.

[0056] In some embodiments, provided herein is a composition comprising a complex having the structure: AL k wherein A is a protein that binds to MET, L is a chelating moiety, and k is an integer from 1 to 30, and the conjugate is chelated with a sufficient amount of a positron emitter to provide a specific activity suitable for clinical PET imaging.

[0057] Suitable binding proteins, chelating moieties, and positron emitters are provided below.

[0058] A. MET-binding proteins Suitable MET binding proteins are proteins that specifically bind to MET, including those described in U.S. Patent Publication No. 2018-0134794, which is incorporated by reference in its entirety. Amino acid sequence identifiers for exemplary anti-MET antibodies useful herein are listed in Table 1 of U.S. Patent Publication No. 2018-0134794, and amino acid sequence identifiers for exemplary MET x MET bispecific antibodies useful herein are listed in Table 5 of U.S. Patent Publication No. 2018-0134794. Both tables are included below as Table 1 and Table 2, respectively. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0059] Table 1 provides amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-MET antibodies.

[0060] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that comprises an HCVR that comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0061] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising an LCVR amino acid sequence shown in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0062] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with the LCVR amino acid sequence shown in Table 1. According to certain embodiments, the disclosure provides an antibody or antigen-binding fragment thereof comprising an HCDR / LCDR amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 / 138, 10 / 138, 18 / 138, 26 / 138, 34 / 138, 42 / 138, 50 / 138, 58 / 138, 66 / 138, 74 / 138, 82 / 138, 90 / 138, 98 / 138, 106 / 138, 114 / 138, 122 / 138, and 130 / 138. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from SEQ ID NOs: 58 / 138 (e.g., H4H13306P2) and 82 / 138 (e.g., H4H13312P2).

[0063] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0064] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0065] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0066] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence set forth in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0067] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0068] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0069] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that comprises an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) that comprises any of the HCDR3 amino acid sequences listed in Table 1 that are paired with the LCDR3 amino acid sequence shown in Table 1. According to certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that comprises an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from SEQ ID NOs: 8 / 144 (e.g., H4H13290P2), 16 / 144 (e.g., H4H13291P2), 24 / 144 (H4H13295P2), 32 / 144 (H4H13299P2), 40 / 144 (H4H13300P2), 48 / 144 (H4H13301P2), 56 / 144 (H4H13302P2), 64 / 144 (H4H13306P2). , 72 / 144(H4H13309P2), 80 / 144(H4H13311P2), 88 / 144(H4H13312P2), 96 / 144(H4H13313P2), 104 / 144(H4H13316P2), 112 / 144(H4H13318P2), 120 / 144(H4H13319P2), 128 / 144(H4H13325P2), and 136 / 144(H4H13331P2).

[0070] In some embodiments, the binding protein is an antibody or antigen-binding fragment that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is set forth in SEQ ID NOs: 4-6-8-140-142-144 (e.g., H4H13290P2), 12-14-16-140-142-144 (e.g., H4H13291P2), 20-22-24-140-142-144 (H4H13295P2), 28-29-30-31-32-33-34 (H4H13296P2), 30-32-33-34-35-36 (H4H13297P2), 34-35-36-37-38-39 (H4H13298P2), 36-37-39-39-39 (H4H13299P2), 38-39-40 ... -30-32-140-142-144(H4H13299P2), 36-38-40-140-142-144(H4H13300P2), 44-44-48-140-142- 144(H4H13301P2), 52-54-56-140-142-144(H4H13302P2), 60-62-64-140-142-144(H4H13306P2), 68-70-72-140-142-144(H4H13309P2), 76-78-80-140-142-144(H4H13311P2), 84-86-88-140-14 2-144(H4H13312P2), 92-94-96-140-142-144(H4H13313P2), 100-102-104-140-142-144(H4H1331 6P2), 108-110-112-140-142-144 (H4H13318P2), 116-118-120-140-142-144 (H4H13319P2), 124-126-128-140-142-144 (H4H13325P2), and 132-134-136-140-142-144 (H4H13331P2).

[0071] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-MET antibodies listed in Table 1. For example, in some embodiments, the binding protein is selected from the group consisting of SEQ ID NOs: 2 / 138 (e.g., H4H13290P2), 10 / 138 (e.g., H4H13291P2), 18 / 138 (H4H13295P2), 26 / 138 (H4H13299P2), 34 / 138 (H4H13300P2), 42 / 138 (H4H13301P2), 50 / 138 (H4H13302P2), 58 / 138 (H4H13306P2), 66 / 138 (H4H13309P2), 74 / 138 (H4H13311P2), 82 / 138 (H4H13312P2), 90 / 138 (H4H13314P2), 100 / 138 (H4H13315P2), 110 / 138 (H4H13316P2), 120 / 138 (H4H13317P2), 132 / 138 (H4H13318P2), 140 / 138 (H4H13319P2), 150 / 138 (H4H13320P2), 160 / 138 (H4H13321P2), 170 / 138 (H4H13322P2), 180 / 138 (H4H13333P2), 190 / 138 (H4H13334P2), 200 / 138 (H4H13335P2), 210 / 138 (H4H13336P2), 220 / 138 (H4 The antibody or antigen-binding fragment thereof comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained in an HCVR / LCVR amino acid sequence pair selected from the group consisting of: 8 (H4H13312P2), 90 / 138 (H4H13313P2), 98 / 138 (H4H13316P2), 106 / 138 (H4H13318P2), 114 / 138 (H4H13319P2), 122 / 138 (H4H13325P2), and 130 / 138 (H4H13331P2).

[0072] Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are known in the art and can be used to identify CDRs in a particular HCVR and / or LCVR amino acid sequence useful herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, Kabat definition, Chothia definition, and AbM definition. In general terms, Kabat definition is based on sequence variability, Chothia definition is based on the position of structural loop regions, and AbM definition is a compromise between Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989). Public databases are also available to identify CDR sequences within antibodies.

[0073] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that competes for specific binding to MET with an antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1, respectively.

[0074] Table 2 lists amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of the first antigen binding region (D1) and second antigen binding region (D2) of several exemplary MET×MET bispecific antibodies.

[0075] The individual anti-MET antigen-binding domains used to construct the bispecific antibodies useful herein were derived from various bivalent, monospecific anti-MET antibodies described in Examples 1-3 of US Patent Publication No. 2018-0134794. All anti-MET antibodies described herein contain the same (common) light chain (comprising the amino acid sequence of the light chain variable region [LCVR] of SEQ ID NO: 138, and the amino acid sequences of the light chain CDRs [LCDR1, LCDR2, and LCDR3] of SEQ ID NOs: 140, 142, and 144). Additionally, all of the bispecific antibodies contain a "D2" arm derived from the exemplary anti-MET antibody, H4H13312P2. Thus, both antigen-binding domains (D1 and D2) of all bispecific antibodies described in this example contain this common light chain variable region, and all D2 binding arms contain the heavy chain variable region derived from H4H13312P2. However, the bispecific antibodies differ from each other with respect to their D1 heavy chain variable region (HCVR) and heavy chain CDRs (HCDRs). D1 and D2 are derived from different anti-MET antibodies and, as a result, bind distinct epitopes on the MET extracellular domain. That is, D1 can bind to a first epitope of human MET, e.g., an epitope comprising amino acids 192-204 of SEQ ID NO: 155, and D2 can bind to a second epitope of human MET comprising amino acids 305-315 and 421-455 of SEQ ID NO: 155.

[0076] As used herein, the phrase "antigen-binding domain" refers to any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide-containing construct that is capable of specifically binding to a particular antigen of interest (e.g., human MET). Terms such as "specifically bind," as used herein, refer to an antigen-binding region that binds to a polypeptide with a dissociation constant (K) of 500 pM or less. D) and does not bind other unrelated antigens under typical test conditions. An "unrelated antigen" is a protein, peptide, or polypeptide that shares less than 95% amino acid identity with one another.

[0077] Exemplary classes of antigen-binding domains that may be used in connection with the present disclosure include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising the ligand-binding portion of a receptor that specifically binds to a particular antigen, antigen-binding scaffolds (such as, for example, DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and aptamers or portions thereof.

[0078] Methods for determining whether two molecules specifically bind to one another are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, as used in the context of the present disclosure, an antigen-binding region has a K of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM as measured by surface plasmon resonance assay. D The present invention includes polypeptides that bind a specific antigen (e.g., a target molecule [T] or an internalization effector protein [E]) or a portion thereof, having the following structure:

[0079] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example using a BIAcore® system (Biacore Life Sciences Division, within GE Healthcare, Piscataway, NJ).

[0080] "K D The term "as used herein" refers to the equilibrium dissociation constant of a particular protein-protein interaction (e.g., an antibody-antigen interaction). Unless otherwise indicated, the K D Values ​​are K determined by surface plasmon resonance assay at 25 °C or 37 °C. D Points to a value.

[0081] As indicated above, the "antigen-binding domain" (D1 and / or D2) may comprise or consist of an antibody or an antigen-binding fragment of an antibody. The term "antibody" as used herein means any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., human MET). The term "antibody" encompasses immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (HCVR or VVR, as used herein). H The heavy chain constant region comprises a C H 1. C H 2, and C H Each light chain comprises three domains: a light chain variable region (herein referred to as LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H Area and V LThe regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs (or antigen-binding portions thereof) of the antibodies provided herein may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs.

[0082] The D1 and / or D2 components of the bispecific antigen-binding molecules useful herein may comprise or consist of antigen-binding fragments of full antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any natural, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from full antibody molecules using any suitable standard method, such as, for example, proteolytic or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding the antibody variable regions and, optionally, the constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or may be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, and the like.

[0083] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of an antibody, or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0084] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, H Domains and V L The domains may be arranged relative to one another in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0085] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L、 (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Additionally, antigen-binding fragments may be homodimers or heterodimers (or other multimers) of any of the above listed variable and constant domain configurations, non-covalently linked to each other and / or to one or more monomeric V H Or V L It may be included in a non-covalent bond with the domain (eg, via a disulfide bond).

[0086] In some embodiments, the binding protein is a bispecific antigen-binding molecule that comprises or consists of a human antibody and / or a recombinant human antibody, or a fragment thereof. The term "human antibody" as used herein includes antibodies with variable and constant regions derived from human germline immunoglobulin sequences. However, human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis, or by in vivo somatic mutation), for example in the CDRs and in particular in the CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0087] The term "recombinant human antibody", as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail below), antibodies isolated from a recombinant combinatorial human antibody library (described in more detail below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), thereby improving the V H Area and V L The amino acid sequence of the region is H Array and V L While the sequences are derived from and related to the sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0088] Methods for making bispecific antibodies are known in the art and may be used to construct bispecific antigen-binding molecules useful in the conjugates described herein. Exemplary bispecific formats that may be used in the context of the present disclosure include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab. 2 and bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0089] Examples of antigen-binding domains (D1 and D2) that may be included in a METxMET bispecific antigen-binding molecule useful herein include an antigen-binding domain derived from any of the anti-MET antibodies disclosed in Table 1. For example, the present disclosure includes METxMET bispecific antigen-binding molecules that comprise a D1 or D2 antigen-binding domain that comprises a HCVR that comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0090] The binding protein may be a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain comprising an LCVR comprising an amino acid sequence shown in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0091] Some binding proteins are anti-MET x MET bispecific antigen binding molecules that contain a D1 or D2 antigen binding domain that comprises an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) that includes any of the HCVR amino acid sequences listed in Table 1 paired with the LCVR amino acid sequence shown in Table 1.

[0092] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0093] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0094] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain comprising a heavy chain CDR3 (HCDR3) comprising any HCDR3 amino acid sequence listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0095] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from the LCDR1 amino acid sequences shown in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0096] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain comprising a light chain CDR2 (LCDR2) comprising the LCDR2 amino acid sequence shown in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0097] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain comprising a light chain CDR3 (LCDR3) comprising an LCDR3 amino acid sequence shown in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0098] Some binding proteins are anti-MET x MET bispecific antigen binding molecules that contain a D1 or D2 antigen binding domain that contains an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) that includes any HCDR3 amino acid sequence listed in Table 1 paired with the LCDR3 amino acid sequence shown in Table 1.

[0099] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-MET antibodies listed in Table 1.

[0100] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in the HCVR / LCVR amino acid sequence pair defined in any of the exemplary anti-MET antibodies listed in Table 1.

[0101] A METxMET bispecific antigen binding molecule useful herein may comprise a D1 antigen binding domain derived from any of the anti-MET antibodies in Table 1, and a D2 antigen binding domain derived from any other anti-MET antibody in Table 1. Non-limiting examples of METxMET bispecific antibodies of the present disclosure are depicted in FIG. 1 of U.S. Patent Publication No. 2018-0134794, illustrating 272 exemplary METxMET bispecific antibody components. Each numbered cell of the matrix (numbered 1 through 272) identifies a unique bispecific antibody comprising a "D1" antigen binding domain and a "D2" antigen binding domain, where the D1 antigen binding domain comprises the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDRs of the corresponding anti-MET antibody listed along the Y-axis, and the D2 antigen binding domain comprises the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDRs of the corresponding anti-MET antibody listed along the X-axis. Thus, for example, the METxMET bispecific antigen binding molecule "number 10" shown in the matrix comprises a D1 antigen binding domain that comprises an HCVR / LCVR pair or six-CDR set derived from the exemplary anti-MET antibody H4H13290P2, and a D2 antigen binding domain that comprises an HCVR / LCVR pair or six-CDR set derived from the exemplary anti-MET antibody H4H13321P2. Additional examples of METxMET bispecific antibodies provided herein are described in Example 4 of U.S. Patent Publication No. 2018-0134794.

[0102] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 antigen binding domain and a D2 antigen binding domain, wherein the D1 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:58 / 138, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity, or the set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs:60-62-64-140-142-144, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. and the D2 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 82 / 138, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, or the set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO: 84-86-88-140-142-144, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0103] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 antigen binding domain and a D2 antigen binding domain, wherein the D1 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 18 / 138 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity, or the heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) set comprising SEQ ID NO: 20-22-24-140-142-144, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 82 / 138 or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, or the set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO: 84-86-88-140-142-144 or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0104] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 antigen binding domain and a D2 antigen binding domain, wherein the D1 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:58 / 138 or a set of heavy or light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO:60-62-64-140-142-144, and the D2 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:82 / 138 or a set of heavy or light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO:84-86-88-140-142-144. An exemplary MET×MET bispecific antibody having these sequence characteristics is the bispecific antibody designated H4H14639D, also referred to as bispecific antibody number 122, which comprises D1 derived from H4H13306P2 and D2 derived from H4H13312P2 (see Table 2 herein).

[0105] In some embodiments, the binding protein is a METxMET bispecific antigen binding molecule comprising a D1 antigen binding domain and a D2 antigen binding domain, wherein the D1 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 18 / 138 or a set of heavy or light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO: 20-22-24-140-142-144, and the D2 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 82 / 138 or a set of heavy or light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO: 84-86-88-140-142-144. An exemplary MET×MET bispecific antibody having these sequence characteristics is the bispecific antibody designated H4H14635D, also referred to as bispecific antibody number 42, which comprises D1 derived from H4H13295P2 and D2 derived from H4H13312P2 (see Table 2 herein).

[0106] The bispecific antigen-binding molecule useful in the present invention may also include one or more multimerizing components. The multimerizing components can function to maintain the association between the antigen-binding domains (D1 and D2). As used herein, a "multimerizing component" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing component of the same or similar structure or composition. For example, the multimerizing component may be an immunoglobulin C. H The multimerizing component may be a polypeptide comprising three domains. Non-limiting examples of multimerizing components include the Fc portion of an immunoglobulin (e.g., an Fc domain of an isotype selected from the following isotypes: IgG1, IgG2, IgG3, and IgG4, and all allotypes within each isotype group). In certain embodiments, the multimerizing component is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerizing component is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides that include or consist of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0107] In certain embodiments, a bispecific antigen-binding molecule useful herein comprises two multimerization domains, M1 and M2, where D1 is bound to M1 and D2 is bound to M2, and the association of M1 and M2 promotes the physical linkage of D1 and D2 to each other in one bispecific antigen-binding molecule. In certain embodiments, M1 and M2 are identical to each other. For example, M1 may be an Fc domain having a specific amino acid sequence, and M2 is an Fc domain having the same amino acid sequence as M1. Alternatively, M1 and M2 may differ from each other at one or more amino acid positions. For example, M1 may be an Fc domain having a specific amino acid sequence, and M2 may be an Fc domain having the same amino acid sequence as M1. H 3 domain, M2 being a second Ig C H3 domains, in which case the first and second Ig C H The three domains differ from each other by at least one amino acid, where the at least one amino acid difference reduces binding of the targeting construct to Protein A compared to a reference construct having identical M1 and M2 sequences. In one embodiment, the IgC domain of M1 H The 3 domain binds protein A and is involved in IgC in M2. H The C3 domain contains mutations that reduce or abolish Protein A binding, such as the H95R modification (according to the IMGT exon numbering; H435R in the EU numbering). H 3 may further contain a Y96F modification (according to IMGT; Y436F according to EU). H Further modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; in the case of an IgG1 Fc domain, D356E, L358M, N384S, K392N, V397M, and V422I are according to EU); in the case of an IgG2 Fc domain, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I are according to EU); and in the case of an IgG4 Fc domain, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I are according to EU).

[0108] In some embodiments, a binding protein may be "isolated". As used herein, an "isolated bispecific antigen-binding molecule" refers to a bispecific antigen-binding molecule that has been identified and separated and / or recovered from at least one component of its natural environment. For example, a bispecific antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it was produced, is an "isolated bispecific antibody" for purposes of this disclosure. An isolated bispecific antigen-binding molecule also includes a molecule in situ within a recombinant cell. An isolated bispecific antigen-binding molecule is a molecule that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated bispecific antigen-binding molecule may be substantially free of other cellular material and / or chemicals.

[0109] A bispecific antigen binding molecule useful herein, or an antigen binding domain thereof (D1 and / or D2), may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and / or light chain variable domains, compared to the corresponding germline sequence from which the antigen binding protein or antigen binding domain is derived. Such mutations can be readily ascertained by comparing the amino acid sequences in Table 1 to germline sequences available, for example, from public antibody sequence databases. A bispecific antigen binding molecule useful herein, or an antigen binding domain thereof (D1 and / or D2), may be derived from any of the amino acid sequences shown in Tables 1 and 2, where one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations").

[0110] Starting with the heavy and light chain variable region sequences of Tables 1 and 2, one of skill in the art can readily generate many bispecific antigen binding molecules, or antigen binding domains thereof (D1 and / or D2), that contain one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V L All of the framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a different germline sequence than the germline sequence from which the antibody was originally derived).

[0111] In some embodiments, the binding protein may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residues of a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. For example, a bispecific antigen-binding molecule or its antigen-binding domain (D1 and / or D2) containing one or more germline mutations can be easily verified for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules or their antigen-binding domains (D1 and / or D2) obtained in this general manner are included in the present disclosure.

[0112] In some embodiments, the binding protein is an anti-MET antibody or bispecific antigen binding molecule that comprises a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences provided in Tables 1 and 2. Exemplary variants included in this aspect include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences in Tables 1 and 2 that have one or more conservative substitutions. For example, the disclosure provides that the binding proteins useful herein include anti-MET antibodies and METxMET bispecific antigen binding molecules that have HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences that have, e.g., 10 or less, 8 or less, 6 or less, or 4 or less conservative amino acid substitutions relative to any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences set forth in Table 1 herein.

[0113] Exemplary variants also include variants that have substantial sequence identity with any of the HCVR, LCVR, and / or CDR amino acid sequences provided in Table 1. As used herein in the context of amino acid sequences, the term "substantial identity" or "substantially identical" means that two amino acid sequences share at least 95%, 98% or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weighting. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445., which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0114] The sequence identity between two different amino acid sequences is typically measured using sequence analysis software. Sequence analysis software matches similar sequences using a measure of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutants. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences provided herein to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0115] In some embodiments, the binding protein is an anti-MET antibody or a METxMET bispecific antigen binding protein that comprises an Fc domain that contains one or more mutations that enhance or decrease antibody binding to the FcRn receptor at, e.g., acidic pH compared to neutral pH. For example, anti-MET antibodies and METxMET bispecific antigen binding proteins include those that include an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor at, e.g., acidic pH compared to neutral pH. H 2 or C H The antibody can contain mutations in the 3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations can result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P).

[0116] For example, the binding protein may be a METxMET bispecific antigen binding protein comprising an anti-MET antibody and an Fc domain that comprises one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations in the antibody variable domains useful herein, are contemplated to be within the scope of the present disclosure.

[0117] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that cross-competes with binding to MET with a reference antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1, respectively.

[0118] Binding protein characteristics In some embodiments, the binding protein is an isolated antibody or antigen-binding fragment that binds to monomeric human MET with high affinity. For example, a binding protein useful herein has a K of less than about 230 nM as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format set forth in Example 3 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay. DAccording to certain embodiments, anti-MET antibodies useful herein have a K of less than about 230 nM, less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, or less than about 3 nM, as measured by surface plasmon resonance, such as using the assay format set forth in Example 3 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay. D and binds to monomeric human MET at 37°C.

[0119] In some embodiments, the binding protein has a dissociation half-life (t) of greater than about 1 minute as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format set forth in Example 3 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay. 1 / 2 ) that binds to monomeric human MET (e.g., hMET.mmh). In certain embodiments, such anti-MET antibodies have a t of greater than about 1 minute, greater than about 2 minutes, greater than about 4 minutes, greater than about 6 minutes, greater than about 8 minutes, greater than about 10 minutes, greater than about 12 minutes, greater than about 14 minutes, greater than about 16 minutes, greater than about 18 minutes, or greater than about 20 minutes as measured by surface plasmon resonance, e.g., using the assay format set forth in Example 3 of U.S. Patent Publication No. 2018-0134794 or a substantially similar assay. 1 / 2 and binds to monomeric human MET at 37°C.

[0120] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that binds to dimeric human MET (e.g., hMET.mFc) with high affinity. For example, such an anti-MET antibody has a K of less than about 3 nM as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format set forth in Example 3 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay.D Anti-MET antibodies useful herein have a K of less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, or less than about 0.25 nM, as measured by surface plasmon resonance, e.g., using the assay format set forth in Example 3 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay. D and binds to dimeric human MET at 37°C.

[0121] In some embodiments, the binding protein has a dissociation half-life (t) of greater than about 4 minutes as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format set forth in Example 3 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay. 1 / 2 ) that binds to dimeric human MET (e.g., hMET.mmh). According to certain embodiments, anti-MET antibodies useful herein have a t of greater than about 4 minutes, greater than about 5 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, or greater than about 105 minutes, as measured by surface plasmon resonance, e.g., using the assay format set forth in Example 3 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay. 1 / 2 and binds to dimeric human MET at 37°C.

[0122] In some embodiments, the binding protein has a dissociation half-life (t) of greater than about 10 minutes as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format set forth in Example 5 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay. 1 / 2) that binds to dimeric human MET (e.g., hMET.mmh). According to certain embodiments, a METxMET bispecific antigen-binding protein useful herein has a t of greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1100 minutes, as measured by surface plasmon resonance, e.g., using the assay format set forth in Example 5 of U.S. Patent Publication No. 2018-0134794, or a closely similar assay. 1 / 2 and binds to dimeric human MET at 37°C.

[0123] In some embodiments, the binding protein is an antibody or an antigen-binding fragment thereof, such as, for example, a METxMET bispecific antigen-binding protein, which blocks the interaction between HGF and MET, for example, in an in vitro ligand binding assay.The METxMET bispecific antigen-binding protein useful herein can block HGF binding to cells expressing human MET, and induces minimal or no MET activation in the absence of HGF signaling.For example, the METxMET bispecific antigen-binding protein useful herein exhibits a degree of MET agonist activity in a cell-based MET activity reporter assay that is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1% of that observed in a comparable activity reporter assay using a monospecific antibody containing D1 or D2 alone.

[0124] In one embodiment, the antibody or fragment thereof is a human monoclonal antibody or antigen-binding fragment thereof that binds to MET, and the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 130, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; ii) an LCVR having the amino acid sequence of SEQ ID NO: 138 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and (iii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128 and 136 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. (iv) an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124 and 132, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto; and an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126 and 134, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and an HCDR2 domain having an amino acid sequence of SEQ ID NO: 140 or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.(v) a multispecific antigen-binding molecule comprising a first binding specificity for MET and a second binding specificity for a tumor-specific antigen; (vi) a multispecific antigen-binding molecule comprising a first binding specificity for an epitope of MET and a second binding specificity for a second epitope of MET; (vii) a K of less than about 230 nM as measured by surface plasmon resonance at 25° C. or 37° C.; D and (viii) binds to monomeric human MET (e.g., hMET.mmh) with a K of less than about 3 nM as measured by surface plasmon resonance at 25° C. or 37° C. D (ix) binds to dimeric human MET, (ix) blocks the binding of HGF to MET, and (x) inhibits tumor growth and increases survival in a subject with cancer.

[0125] In one embodiment, the antibody or fragment thereof is a human monoclonal antibody or antigen-binding fragment thereof that blocks HGF binding to MET, and the antibody or fragment thereof exhibits one or more of the following characteristics: (i) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 130, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. (ii) an LCVR having the amino acid sequence of SEQ ID NO: 138 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and (iii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128 and 136 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. (iv) an HCDR3 domain having an amino acid sequence of SEQ ID NO: 144 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto; and (v) an HCDR3 domain having an amino acid sequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124 and 132 or ... having at least 90%, at least 95%, at least 98% or less sequence identity thereto. and a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126 and 134, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a HCDR2 domain having an amino acid sequence of SEQ ID NO: 140 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.(v) a multispecific antigen-binding molecule comprising a first binding specificity for MET and a second binding specificity for a tumor-specific antigen; (vi) a multispecific antigen-binding molecule comprising a first binding specificity for an epitope of MET and a second binding specificity for a second epitope of MET; (vii) a K of less than about 230 nM as measured by surface plasmon resonance at 25° C. or 37° C.; D and (viii) binds to monomeric human MET (e.g., hMET.mmh) with a K of less than about 3 nM as measured by surface plasmon resonance at 25° C. or 37° C. D and (ix) inhibiting tumor growth and increasing survival in subjects with cancer.

[0126] In certain embodiments, the binding protein is a METxMET bispecific antibody or antigen-binding fragment thereof, in which the first antigen-binding domain (D1) binds to a first epitope of human MET and the second antigen-binding domain (D2) binds to a second epitope of human MET or a fragment thereof, either naturally occurring or recombinantly produced. In some aspects, D1 and D2 do not compete with each other for binding to human MET. In some embodiments, the binding protein exhibits minimal agonist activity in a cell-based MET activity reporter assay. In some embodiments, the bispecific antigen-binding molecule exhibits a degree of MET agonist activity in a cell-based MET activity reporter assay that is less than 10% of the MET agonist activity of a monovalent antigen-binding molecule comprising D1 or D2 alone. In some embodiments, the bispecific antigen-binding molecule promotes degradation of cell surface-expressed MET. In some embodiments, the bispecific antigen-binding molecule inhibits the growth or promotes tumor regression of tumors with MET gene alterations. In some embodiments, the bispecific antigen binding molecule inhibits growth or promotes tumor regression of tumors whose growth is driven by autocrine HGF signaling.

[0127] In some embodiments, an anti-MET antibody or METxMET bispecific antibody useful herein binds to the same epitope, or a portion of an epitope, as any of the specific exemplary antibodies described in Table 1 or Table 2, or an antibody having the CDR sequences of any of the exemplary antibodies described in Table 1 or Table 2. Similarly, suitable binding proteins also include anti-MET antibodies or METxMET bispecific antibodies that compete for binding to MET or a MET fragment with any of the specific exemplary antibodies set forth in Table 1 or Table 2, or an antibody having the CDR sequences of any of the exemplary antibodies set forth in Table 1 or Table 2. For example, suitable binding proteins include anti-MET antibodies and METxMET bispecific antibodies that cross-compete for binding to MET with one or more antibodies defined in U.S. Patent Publication No. 2018-0134794, or that cross-compete for binding to MET with one or more antibodies defined in U.S. Patent Publication No. 2018-0134794.

[0128] The antibodies and antigen-binding fragments described herein specifically bind to MET and modulate the interaction between MET and HGF. METxMET bispecific antibodies can bind to MET with high or low affinity. In certain embodiments, the antibodies are blocking antibodies, where the antibodies bind to MET and block the interaction between MET and HGF. In some embodiments, the blocking antibodies of the present disclosure block the binding of HGF to MET. In some embodiments, blocking antibodies are useful for treating subjects suffering from cancer. They can be used to inhibit the growth of tumor cells in a subject. They can also be used as adjunctive therapy alone or with other therapeutic moieties or modalities known in the art for treating cancer. In certain embodiments, METxMET bispecific antibodies that bind to MET with low affinity are used as multispecific antigen-binding molecules, where a first binding specificity binds to MET with low affinity and a second binding specificity binds to a different epitope of MET or a tumor-specific antigen.

[0129] Certain anti-MET antibodies and METxMET bispecific antibodies of the present disclosure can bind to and neutralize MET activity as measured by in vitro or in vivo assays. The ability of the disclosed antibodies to bind to and neutralize MET activity may be measured using any standard method known to those of skill in the art, including the binding or activity assays described herein.

[0130] Non-limiting exemplary in vitro assays for measuring binding activity are shown in Examples 3 and 6 of US-2018-0134794A1. In Example 6, the binding affinity and kinetic constants of human MET x MET bispecific antibodies for human MET were determined by surface plasmon resonance, and measurements were performed on a T200 Biacore instrument. In Example 7 of US-2018-0134794, a blocking assay was used to determine the ability of anti-MET antibodies and MET x MET bispecific antibodies to block the MET binding ability of HGF. In Example 4 of US-2018-0134794, a blocking assay was used to determine cross-competition between different anti-MET antibodies. Example 8 of US-2018-0134794 describes the growth inhibition of cells overexpressing MET by anti-MET antibodies and MET x MET bispecific antibodies. In Example 10 of US-2018-0134794, a METxMET bispecific antibody is shown to induce MET degradation and inhibit phosphorylation of both MET and ERK. US-2018-0134794 also provides several examples showing tumor growth inhibition or tumor shrinkage induced by a METxMET bispecific antibody, both in vivo and in vitro.

[0131] Unless specifically stated otherwise, the term "antibody" as used herein is understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule") as well as antigen-binding fragments thereof. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody or "antibody fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to MET. Antibody fragments include Fab fragments, F(ab') ... 2 The term "antigen-binding fragment" may include a fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide of a multispecific antigen-binding molecule or a fragment thereof. In such embodiments, the term "antigen-binding fragment" includes, for example, an extracellular domain of HGF that specifically binds to MET. Antigen-binding fragments of antibodies can be obtained from complete antibody molecules using any suitable standard technique, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable domains and (optionally) the constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0132] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0133] Antigen-binding fragments of antibodies typically contain at least one variable domain. A variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, H Domains and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0134] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a VH -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H Or V LIt may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domains (eg, via disulfide bonds).

[0135] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the examples of bispecific antibody formats disclosed herein, may be adapted for use in the context of antigen-binding fragments of the disclosed antibodies using routine techniques available in the art.

[0136] Anti-MET antibodies and METxMET bispecific antibodies and antibody fragments useful herein include proteins having amino acid sequences that differ from those of the described antibodies but which retain the ability to bind to MET. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Similarly, antibody-encoding DNA sequences of the present disclosure include sequences that encode antibodies or antibody fragments that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but are essentially biologically equivalent to the antibodies or antibody fragments of the present disclosure.

[0137] Two antigen-binding proteins or antibodies are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes and do not show significant differences in their rate and extent of absorption when administered in the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies are considered equivalents, or pharmaceutical substitutes, if the extent of absorption is comparable but the rate of absorption is not, but the antibodies can be considered bioequivalent because the difference in absorption rate is intentional, reflected in the labeling, is not essential, for example, to achieving effective body drug concentrations in chronic use, and is not considered medically significant for the particular pharmaceutical product being tested.

[0138] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically relevant differences in their safety, purity, or efficacy.

[0139] In one embodiment, two antigen binding proteins are bioequivalent if a subject is able to make one or more switches between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or decreased efficacy, as compared to continuous therapy without such switches.

[0140] In one embodiment, two antigen binding proteins are biologically equivalent if they both act by a common mechanism of action to the extent that mechanism is known for the conditions of use.

[0141] Bioequivalence may be demonstrated by in vivo and in vitro methods, including, for example, (a) in vivo tests in humans or other mammals in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum or other biological fluids as a function of time, (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo tests in humans or other mammals in which the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time, and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0142] Biologically equivalent variants of the antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other situations, biologically equivalent antibodies can include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that abolish or eliminate glycosylation.

[0143] Anti-MET antibodies and METxMET bispecific antibodies useful herein can include, for example, an Fc domain that contains one or more mutations that increase or decrease antibody binding to the FcRn receptor at acidic compared to neutral pH. For example, the present disclosure provides an Fc domain that contains one or more mutations that increase or decrease antibody binding to the FcRn receptor at acidic compared to neutral pH. H 2 or C HThe present invention includes a METxMET bispecific antibody that contains mutations in three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0144] For example, anti-MET antibodies and METxMET bispecific antibodies are useful that contain an Fc domain that contains one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T and 256E (e.g., M252Y, S254T and T256E), 428L and 434S (e.g., M428L and N428L), 434S and 434S (e.g., M434S ... 34S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A and 434A (e.g., T307A, E380A and N434A), and 433K and 434F (e.g., H433K and N434F). In one embodiment, the present disclosure includes a METxMET bispecific antibody comprising an Fc domain that comprises an S108P mutation in the hinge region of IgG4 that promotes dimer stabilization. All possible combinations of the aforementioned Fc domain mutations, as well as other mutations in the antibody variable domains provided herein in Table 1, are contemplated to be within the scope of the present disclosure.

[0145] The anti-MET antibodies and MET×MET bispecific antibodies useful herein comprise a chimeric heavy chain constant (C H ) region, H The region is composed of one or more C H For example, the antibody may include a segment derived from a C region derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule. H C from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule in combination with some or all of the three domains H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibody may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric hinges described herein may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1, IgG2 or IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. H Antibodies comprising the region, in certain embodiments, exhibit modified Fc effector functions without negatively affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., USSN. 14 / 170,166, filed January 31, 2014, the disclosure of which is incorporated herein by reference in its entirety).

[0146] B. Positron Emitters and Chelating Moieties Suitable positron emitters include, but are not limited to, those that form stable complexes with chelating moieties and have suitable physical half-lives for purposes of immunoPET imaging. Exemplary positron emitters include: 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Suitable positron emitters also include, but are not limited to, Y. 76 Br and 124 I, and prosthetic groups, e.g., 18 These include, but are not limited to, those induced by F that directly bind to the MET binding protein.

[0147] Chelating moieties as described herein are chemical moieties covalently attached to a MET binding protein, e.g., a METxMET bispecific antibody, and include moieties that can chelate with a positron emitter, i.e., can react with a positron emitter to form a coordinate chelate complex. Suitable moieties include those that allow efficient loading of a particular metal and form a metal chelator complex that is sufficiently stable for in vivo diagnostic applications, e.g., immunoPET imaging. Exemplary chelating moieties include those that minimize dissociation of the positron emitter and accumulation in bone minerals, plasma proteins, and / or bone marrow deposits to an extent suitable for diagnostic applications.

[0148] Examples of chelating moieties include positron emitters. 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Exemplary chelating moieties include, but are not limited to, those that form stable complexes with Y. Exemplary chelating moieties include those described in Nature Protocols, 5(4):739, 2010; Bioconjugate Chem., 26(12):2579 (2015); Chem Commun (Camb), 51(12):2301 (2015); Mol. Pharmaceutics, 12:2142 (2015); Mol. Imaging Biol., 18:344 (2015); Eur. J. Nucl. Med. Mol. Imaging, 37:250 (2010); Eur. J. Nucl. Med. Mol. Imaging (2016). doi:10.1007 / s00259-016-3499-x; Bioconjugate Chem., 26(12):2579(2015); WO2015 / 140212A1; and U.S. Pat. No. 5,639,879.

[0149] Exemplary chelating moieties include desferrioxamine (DFO) (also known as deferoxamine), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic) acid (DOTP), 1R,4R,7R,10R)-α′α″α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H 4 Octape, H 6 Hospa, H 2 Dedopa, H 5 Dekapa, H 2 Azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazacyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (Cy clen), 1,4,8,11-tetraazacyclododecane (Cyclam), octadentate chelators, hexadentate chelators, phosphonate based chelators, macrocyclic chelators, chelators containing macrocyclic terephthalamide ligands, bifunctional chelators, fusarinine C and fusarinine C derivative chelators, triacetylfusarinine C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), ferrichrome A (FCHA), and the like.

[0150] In some embodiments, the chelating moiety is covalently attached to a MET binding protein, e.g., an antibody or antigen-binding fragment thereof, via a linker moiety that covalently attaches the chelating portion of the chelating moiety to the binding protein. In some embodiments, these linker moieties are formed from the reaction between a reactive moiety of the MET binding protein, e.g., a cysteine ​​or lysine of the antibody, and a reactive moiety attached to a chelator, e.g., a p-isothiocyanatophenyl group and a reactive moiety provided in the conjugation methods below. In addition, such linker moieties optionally contain chemical groups that are used to adjust the polarity, solubility properties, steric interactions, rigidity, and / or length between the chelating moiety and the MET binding protein.

[0151] C. Preparation of Radiolabeled MET-binding Protein Complex A radiolabeled anti-MET antibody or METxMET bispecific antibody complex can be prepared by (1) reacting a MET binding protein, e.g., a METxMET bispecific antibody, with a molecule containing a positron emitter chelator and a moiety reactive to the desired conjugation site on the MET binding protein, and (2) loading it with the desired positron emitter.

[0152] Suitable conjugation sites include, but are not limited to, lysine and cysteine, both of which can be, for example, natural or engineered, and can be present, for example, on the heavy or light chain of an antibody. Cysteine ​​conjugation sites include, but are not limited to, those obtained from mutation, insertion, or reduction of antibody disulfide bonds. Methods for making cysteine ​​engineered antibodies include, but are not limited to, those disclosed in WO2011 / 056983. Site-specific conjugation methods can also be used to direct the conjugation reaction to a specific site on an antibody, to achieve a desired stoichiometry, and / or to achieve a desired drug-to-antibody ratio. Such conjugation methods are known to those skilled in the art and include, but are not limited to, glutamine conjugation, Q295 conjugation, and transglutaminase-mediated conjugation, and those described in J.Clin.Immunol.,36:100(2016), the entirety of which is incorporated herein by reference. The appropriate moiety reactive to the desired conjugation site generally allows efficient and easy conjugation of the MET binding protein, e.g., an antibody, with the positron emitter chelator. The moiety reactive to lysine and cysteine ​​sites includes electrophilic groups known to those skilled in the art. In certain embodiments, when the desired conjugation site is lysine, the reactive moiety is an isothiocyanate, e.g., a p-isothiocyanatophenyl group or a reactive ester. In certain embodiments, when the desired conjugation site is cysteine, the reactive moiety is a maleimide.

[0153] When the chelating agent is desferrioxamine (DFO) (also known as deferoxamine), suitable reactive moieties include isothiocyanatobenzyl groups, n-hydroxysucrinimide esters, 2,3,5,6 tetrafluorophenol esters, n-sucrinimidyl-S-acetylthioacetate, and 1,2-diamino-1,3-diphenylmethane, ... 2014, Article ID 203601. In certain embodiments, the MET binding protein is an antibody and the molecule comprising a positron emitter chelator and a moiety reactive to a conjugation site is p-isothiocyanatobenzyl-desferrioxamine (p-SCN-Bn-DFO). [ka]

[0154] Loading of the positron emitter is accomplished by incubating the MET binding protein chelator complex with the positron emitter for a period of time sufficient to allow attachment of the positron emitter to the chelator, e.g., by carrying out the methods described in the Examples provided herein, or substantially similar methods.

[0155] D. Exemplary Embodiments of the Conjugate Included in the disclosure is a radiolabeled antibody conjugate comprising an antibody or antigen-binding fragment that binds to human MET, e.g., an anti-MET antibody or a METxMET bispecific antibody, and a positron emitter. Additionally included in the disclosure is a radiolabeled antibody conjugate comprising an anti-MET antibody or a METxMET bispecific antibody, a chelating moiety, and a positron emitter.

[0156] In some embodiments, the chelating moiety is 89 The chelating agent can form a complex with Zr. In certain embodiments, the chelating moiety comprises desferrioxamine. In certain embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine.

[0157] In some embodiments, the positron emitter comprises: 89In some embodiments, less than 1.0% of the MET binding protein is bound to a positron emitter, less than 0.9% of the MET binding protein is bound to a positron emitter, less than 0.8% of the MET binding protein is bound to a positron emitter, less than 0.7% of the MET binding protein is bound to a positron emitter, less than 0.6% of the MET binding protein is bound to a positron emitter, less than 0.5% of the MET binding protein is bound to a positron emitter, less than 0.4% of the MET binding protein is bound to a positron emitter, less than 0.3% of the MET binding protein is bound to a positron emitter, less than 0.2% of the MET binding protein is bound to a positron emitter, and less than 0.1% of the MET binding protein is bound to a positron emitter.

[0158] In some embodiments, the chelating moiety to antibody ratio of the conjugate is 1-2. As used herein, the "chelating moiety to antibody ratio" is the average chelating moiety to antibody ratio, which is a measure of the chelating loading per antibody. This ratio is similar to the drug-antibody ratio used by those skilled in the art to measure the "DAR", i.e., drug loading per antibody for antibody drug conjugates (ADCs), and in the case of the conjugates described herein for iPET imaging, the chelating moiety to antibody ratio can be determined by the methods described herein and other methods known in the art for determining the DAR, e.g., Wang et al., Antibody-Drug Conjugates, The 21st Symposium on Imaging of Antibodies, vol. 1, no. 1, pp. 217-219, 2002. st The antibody to chelating moiety ratio can be ascertained using those described in Century Magic Bullets for Cancer (2015). In some embodiments, the chelating moiety to antibody ratio is 1.0 to 4.0, or about 1.0 to 3.0, or about 1.0 to 2.0. In some embodiments, the chelating moiety to antibody ratio is about 1.26, such as about 1.3.

[0159] In certain embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine and the positron emitter is 89 In another particular embodiment, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine and the positron emitter is89 Zr, and the chelating moiety to antibody ratio of the conjugate is 1-2.

[0160] In some embodiments, provided herein is an antigen binding protein that binds MET, wherein the antigen binding protein that binds MET is covalently linked to one or more moieties having the structure: -LM Z wherein L is a chelating moiety, M is a positron emitter, and z is independently at each occurrence 0 or 1, and at least one of the z's is 1. In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (I): MLA-[LM Z ] k (I) A is a protein that binds to MET, L is a chelating moiety, M is a positive positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1. In some embodiments, k is 2.

[0161] In some embodiments, L is: [ka]

[0162] In some embodiments, M is 89 Zr.

[0163] In some embodiments, k is an integer from 1 to 2. In some embodiments, k is 1. In some embodiments, k is 2.

[0164] In some embodiments, -LM is: [ka]

[0165] Also, the compound of formula (III) [ka] 89 Also included in the disclosure is a method of synthesizing a radiolabeled antibody conjugate comprising contacting A with Zr, where A is an antibody or antigen-binding fragment thereof that binds to MET. In certain embodiments, the compound of formula (III) is synthesized by contacting an antibody or antigen-binding fragment thereof that binds to MET with p-SCN-Bn-DFO.

[0166] With a compound of formula (III) 89 Also provided herein are products of the reaction between Zr.

[0167] Provided herein is a compound of formula (III): [ka] wherein A is an antibody or antigen-binding fragment thereof that binds to MET, and k is an integer from 1 to 30. In some embodiments, k is 1 or 2.

[0168] Provided herein is an antibody conjugate comprising: (i) an antibody, or antigen-binding fragment thereof, that binds to MET; and (ii) one or more chelating moieties.

[0169] In some embodiments, the chelating moiety comprises: [ka] [ka] is a covalent bond to the antibody or antigen-binding fragment thereof.

[0170] In some embodiments, the antibody conjugate has a chelating moiety to antibody ratio of about 1.0 to about 2.0, hi some embodiments, the antibody conjugate has a chelating moiety to antibody ratio of about 1.3.

[0171] In some embodiments, provided herein is a composition comprising a complex having the following structure: AL k where A is a protein that binds to MET, L is a chelating moiety, k is an integer from 1 to 30, and the conjugate is chelated with a positron emitter in an amount sufficient to provide a specific activity suitable for clinical PET imaging. In some embodiments, the amount of chelated positron emitter is sufficient to provide a specific activity of about 1 to about 50 mCi per 1 to 50 mg of protein that binds MET.

[0172] In some embodiments, the amount of chelated positron emitter is sufficient to provide a specific activity in the range of up to 25 mCi, up to 20 mCi, up to 15 mCi, up to 12 mCi, up to 10 mCi, e.g., about 3 to about 25 mCi, about 10 to about 25 mCi, about 1 to about 15 mCi, about 3 to about 15 mCi, about 5 to about 25 mCi, or about 15 to about 25 mCi, or about 3 to about 10 mCi, or about 12 mCi, or about 21 mCi, per 1 to 50 mg of protein that binds MET.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof has a binding dissociation equilibrium constant (K) of less than about 230 nM as measured by surface plasmon resonance assay at 25° C. or 37° C. D ) binds to monomeric human MET.

[0174] In some embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 3 nM in a surface plasmon resonance assay at 25° C. or 37° C. D It binds to dimeric human MET at

[0175] In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to human MET with a reference antibody that comprises a complementarity determining region (CDR) of a HCVR, where the HCVR has an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1, and a CDR of a LCVR, where the LCVR has an amino acid sequence as set forth in Table 1. In some embodiments, the reference antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair set forth in Table 1. In some embodiments, the reference antibody comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 138, 10 / 138, 18 / 138, 26 / 138, 34 / 138, 42 / 138, 50 / 138, 58 / 138, 66 / 138, 74 / 138, 82 / 138, 90 / 138, 98 / 138, 106 / 138, 114 / 138, 122 / 138 and 130 / 138.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof blocks HGF binding to MET, hi some embodiments, the antibody or antigen-binding fragment thereof does not increase or decrease MET binding to its ligand.

[0177] In some embodiments, the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) of an HCVR, wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 130, and a CDR of an LCVR, wherein the LCVR has the amino acid sequence of SEQ ID NO: 138. In certain embodiments, the isolated antibody comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 138, 10 / 138, 18 / 138, 26 / 138, 34 / 138, 42 / 138, 50 / 138, 58 / 138, 66 / 138, 74 / 138, 82 / 138, 90 / 138, 98 / 138, 106 / 138, 114 / 138, 122 / 138 and 130 / 138.

[0178] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MET, and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the HCVR sequences listed in Table 1.

[0179] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MET, and the antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) having the amino acid sequence shown in Table 1.

[0180] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MET, and the antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the HCVR sequences listed in Table 1, and (b) a light chain variable region (LCVR) having an amino acid sequence shown in Table 1.

[0181] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in any one of the heavy chain variable region (HCVR) sequences set forth in Table 1, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light chain variable region (LCVR) sequence shown in Table 1.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124 and 132; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, and 134; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128 and 136; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 140; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 142, and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 144.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 138, 10 / 138, 18 / 138, 26 / 138, 34 / 138, 42 / 138, 50 / 138, 58 / 138, 66 / 138, 74 / 138, 82 / 138, 90 / 138, 98 / 138, 106 / 138, 114 / 138, 122 / 138 and 130 / 138.

[0184] In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDRs of an HCVR, wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 130, and the CDRs of an LCVR, wherein the LCVR has the amino acid sequence of SEQ ID NO: 138.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-MET antibody comprising CDRs in the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 82 / 138. In some aspects, the antibody or antigen-binding fragment thereof is an anti-MET antibody comprising the HCVR amino acid sequence of SEQ ID NO: 82 and the LCVR amino acid sequence of SEQ ID NO: 138.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-MET antibody comprising CDRs in the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 18 / 138. In some aspects, the antibody or antigen-binding fragment thereof is an anti-MET antibody comprising the HCVR amino acid sequence of SEQ ID NO: 18 and the LCVR amino acid sequence of SEQ ID NO: 138.

[0187] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-MET antibody comprising CDRs in the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 58 / 138. In some aspects, the antibody or antigen-binding fragment thereof is an anti-MET antibody comprising the HCVR amino acid sequence of SEQ ID NO: 58 and the LCVR amino acid sequence of SEQ ID NO: 138.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof is a METxMET bispecific antigen-binding protein comprising a CDR at the D1-HCVR amino acid sequence of SEQ ID NO: 58, and a CDR at the D2-HCVR amino acid sequence of SEQ ID NO: 82. In some aspects, the METxMET bispecific antigen-binding protein further comprises a CDR at the LCVR amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody or antigen-binding fragment thereof is a METxMET bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 58, and the D2-HCVR amino acid sequence of SEQ ID NO: 82. In some aspects, the METxMET bispecific antigen-binding protein further comprises the LCVR amino acid sequence of SEQ ID NO: 138.

[0189] In some embodiments, the antibody or antigen-binding fragment thereof is a METxMET bispecific antigen-binding protein comprising a CDR at the D1-HCVR amino acid sequence of SEQ ID NO: 18, and a CDR at the D2-HCVR amino acid sequence of SEQ ID NO: 82. In some aspects, the METxMET bispecific antigen-binding protein further comprises a CDR at the LCVR amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody or antigen-binding fragment thereof is a METxMET bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 18, and the D2-HCVR amino acid sequence of SEQ ID NO: 82.

[0190] In some embodiments, the radiolabeled antibody conjugate comprises an antibody or antigen-binding fragment thereof that binds to MET, a chelating moiety, and a positron emitter, wherein the antibody or antigen-binding fragment thereof that binds to MET comprises a CDR to the D1-HCVR amino acid sequence of SEQ ID NO:58, a CDR to the D2-HCVR amino acid sequence of SEQ ID NO:82, and a CDR to the LCVR amino acid sequence of SEQ ID NO:138, wherein the chelating moiety is desferrioxamine, and the positron emitter is 89 Zr.

[0191] In some embodiments, the radiolabeled antibody conjugate comprises an antibody or antigen-binding fragment thereof that binds to MET, a chelating moiety, and a positron emitter, wherein the antibody or antigen-binding fragment thereof that binds to MET comprises the D1-HCVR amino acid sequence of SEQ ID NO:58, the D2-HCVR amino acid sequence of SEQ ID NO:82, and the LCVR amino acid sequence of SEQ ID NO:138, the chelating moiety is desferrioxamine, and the positron emitter is 89 Zr.

[0192] III. Methods Using Radiolabeled Immunoconjugates In certain aspects, the present disclosure provides diagnostic and therapeutic methods using the radiolabeled antibody conjugates of the present disclosure.

[0193] According to one aspect, the present disclosure provides a method for detecting MET in a tissue, the method comprising administering to the tissue a radiolabeled antibody conjugate provided herein and visualizing MET expression by positron emission tomography (PET) imaging. In certain embodiments, the tissue comprises a cell or a cell line. In certain embodiments, the tissue is present in the body of a subject, and the subject is a mammal. In certain embodiments, the subject is a human subject. In certain embodiments, the subject has cancer.

[0194] According to one aspect, the present disclosure provides a method of imaging a tissue expressing MET, comprising administering to the tissue a radiolabeled antibody conjugate of the present disclosure and visualizing MET expression by positron emission tomography (PET) imaging. In one embodiment, the tissue is contained within a tumor. In one embodiment, the tissue is contained within a tumor cell culture or tumor cell line. In one embodiment, the tissue is contained within a tumor lesion in a subject.

[0195] According to one aspect, the present disclosure provides a method of measuring response to anti-MET therapy in a subject having cancer, where the response to therapy is measured by a change in MET expression relative to pre-therapy MET expression. The method according to this aspect includes administering to a subject in need thereof a radiolabeled antibody conjugate provided herein and visualizing MET expression by positron emission tomography (PET) imaging. A decrease in MET expression relative to pre-treatment MET expression correlates with a positive response to the anti-MET therapy.

[0196] According to one aspect, the present disclosure provides a method for determining whether a subject having a solid tumor is suitable for anti-tumor treatment comprising an inhibitor of the HGF / MET signaling pathway, the method comprising administering to the subject a radiolabeled antibody conjugate of the present disclosure and localizing the administered radiolabeled antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate within the tumor determines that the subject is suitable for anti-tumor treatment comprising an inhibitor of the HGF / MET signaling pathway.

[0197] Anti-tumor therapy useful according to the methods disclosed herein can be any therapeutically useful inhibitor of the HGF / MET signaling pathway, i.e., an inhibitor of HGF, an inhibitor of MET, or an inhibitor of EKR or any other downstream protein of the HGF / MET signaling pathway. In some embodiments, the anti-tumor therapy comprises an anti-MET antibody or antigen-binding fragment thereof, e.g., any one or more of the antibodies listed in Table 1. In some embodiments, the anti-tumor therapy comprises any one or more of the METxMET bispecific antibodies, e.g., any one of the bispecific antibodies listed in Table 2. In some embodiments, the antibody, bispecific antibody, or antigen-binding fragment thereof is conjugated to an agent (i.e., ADC) useful for treating cancer. Exemplary anti-MET ADCs are disclosed in US-2018-0134794A1.

[0198] According to one aspect, the present disclosure identifies candidate subjects for anti-tumor treatment comprising an inhibitor of the HGF / MET signaling pathway, the method comprising administering a radiolabeled antibody conjugate of the present disclosure to a subject having a tumor, localizing the administered radiolabeled antibody conjugate to the tumor by PET imaging, and the presence of the radiolabeled antibody conjugate in the tumor determines that the subject is suitable for anti-tumor treatment comprising an inhibitor of the HGF / MET signaling pathway.

[0199] According to one aspect, the present disclosure provides a method of predicting a subject's response to an anti-tumor treatment, the method comprising determining whether a tumor is MET positive, where if the tumor is MET positive, a positive response to the anti-tumor treatment is predicted for the subject. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present disclosure and localizing the radiolabeled antibody conjugate in the tumor by PET imaging, where the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is MET positive.

[0200] According to one aspect, the present disclosure provides a method for predicting a response of a subject having a solid tumor to an anti-tumor treatment, the method comprising determining whether the tumor is MET positive, and if the tumor is MET positive, a positive response of the subject is predicted. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present disclosure and localizing the radiolabeled antibody conjugate in the tumor by PET imaging, and the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is MET positive.

[0201] According to one embodiment, the present disclosure provides a method for detecting a MET-positive tumor in a subject. The method according to this embodiment includes administering to a subject a radiolabeled antibody conjugate of the present disclosure and determining the localization of the radiolabeled antibody conjugate by PET imaging, where the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is MET-positive. In some embodiments, the detection method identifies the location of the tumor. In some embodiments, the detection method allows for monitoring the progress of an anti-tumor treatment, for example, whether the tumor shrinks or stops growing.

[0202] According to one aspect, the present disclosure provides a method of determining the size of a MET-positive tumor in a subject, the method according to this aspect includes administering to a subject a radiolabeled antibody conjugate of the present disclosure and visualizing the radiolabeled antibody conjugate by PET imaging, whereby the size of the tumor can be determined.

[0203] Also provided herein is a method of determining the presence of MET-expressing cells in a subject, comprising administering to a subject a radiolabeled anti-MET antibody conjugate or a METxMET bispecific antibody conjugate described herein, and visualizing MET expression by PET imaging.

[0204] Provided herein are methods of diagnosing and treating a subject having a tumor, the methods comprising administering to the subject a radiolabeled conjugate as described herein, wherein localization of the radiolabeled antibody conjugate is imaged via PET imaging to determine if the tumor is MET positive, diagnosing the subject having a MET positive tumor, and administering to the subject an anti-tumor treatment comprising an inhibitor of the HGF / MET signaling pathway.

[0205] Provided herein are methods of diagnosing a subject having a MET-expressing tumor, the methods including administering to the subject a radiolabeled anti-MET antibody conjugate or a METxMET bispecific antibody conjugate described herein, visualizing MET expression by PET imaging, and diagnosing the subject as having a MET-expressing tumor when MET expression is visualized by PET imaging.

[0206] As used herein, the term "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or signs of cancer, and / or a human or non-human mammal diagnosed with cancer, including solid tumors, and in need of treatment for cancer. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may be diagnosed with a primary or metastatic tumor and / or with one or more symptoms or signs, including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, abdominal distension, chest pain / pressure, enlarged spleen, and elevated levels of cancer-related biomarkers (e.g., CA125). This term includes subjects with primary or established tumors. In certain embodiments, this term includes human subjects with and / or in need of treatment for solid tumors, such as colon cancer, breast cancer, lung cancer, prostate cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, cervical cancer, pancreatic cancer, head and neck cancer, and brain cancer. The term includes subjects with primary or metastatic tumors (advanced malignancies). In certain embodiments, the term "subject in need thereof" includes patients with solid tumors that are resistant or refractory to previous therapies (e.g., treatment with anticancer drugs) or that are not adequately controlled by previous therapies. For example, the term includes subjects that have been treated with one or more previous line of therapies, such as chemotherapy treatment (e.g., carboplatin or docetaxel). In certain embodiments, the term "subject in need thereof" includes patients with solid tumors that have been treated with one or more previous line of therapies but have subsequently recurred or metastasized.

[0207] In certain embodiments, the method of the present disclosure is used in subjects with solid tumors. The terms "tumor", "cancer" and "malignant tumor" are used interchangeably herein. As used herein, the term "solid tumor" refers to a mass of abnormal tissue that does not usually contain cysts or liquid areas. Solid tumors can be benign (not cancerous) or malignant (cancerous). For the purposes of this disclosure, the term "solid tumor" refers to malignant solid tumors. This term includes different types of solid tumors named for the cell type that forms them, i.e., sarcoma, carcinoma and lymphoma. In certain embodiments, the term "solid tumor" includes cancers including, but not limited to, colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer, and myeloma.

[0208] In some embodiments, the tumor is selected from the group consisting of acute myeloid leukemia, adult T-cell leukemia, astrocytoma, cholangiocarcinoma, chronic myeloid leukemia, gastric cancer (e.g., gastric cancer with MET amplification), glioblastoma, head and neck cancer (e.g., head and neck squamous cell carcinoma [HNSCC]), Kaposi's sarcoma, leiomyosarcoma, lung cancer (e.g., non-small cell lung cancer [NSCLC]), lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal carcinoma, osteosarcoma, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung carcinoma, synovial sarcoma, thyroid cancer, and Wilms' tumor.

[0209] According to one aspect, the disclosure provides a method of treating a tumor in a subject. The method according to this aspect includes determining that the tumor is MET positive and administering one or more doses of an inhibitor of the HGF / MET signaling pathway. In some aspects, the inhibitor is an anti-MET antibody, a METxMET bispecific antibody, or a drug conjugate thereof. In certain embodiments, the tumor is determined to be MET positive by administering to the subject a radiolabeled antibody conjugate of the disclosure and visualizing the radiolabeled antibody conjugate in the tumor by PET imaging. The presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is MET positive. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is gastric cancer.

[0210] As used herein, the terms "treat", "treating" and the like mean alleviating symptoms, eliminating the cause of symptoms either temporarily or permanently, slowing or inhibiting tumor growth, reducing tumor cell mass or burden, causing tumor shrinkage, necrosis and / or elimination, suppressing or inhibiting metastasis, inhibiting the growth of metastatic tumors, and / or prolonging the survival of a subject.

[0211] According to one aspect, the present disclosure provides a method for monitoring the effectiveness of an anti-tumor treatment in a subject undergoing cancer treatment, the method comprising administering to the subject a radiolabeled antibody conjugate of the present disclosure, imaging the localization of the administered labeled conjugate in the tumor by PET imaging, determining tumor growth, wherein a decrease in the radiolabeled signal from baseline indicates tumor regression and the effectiveness of the anti-tumor treatment. In certain embodiments, the anti-tumor treatment comprises an inhibitor of the HGF / MET signaling pathway (e.g., a METxMET bispecific antibody).

[0212] As used herein, the term "baseline" with respect to MET expression in tumors refers to the numerical value of radiolabeled conjugate uptake for a subject before or at the time of administration of a dose of antitumor treatment. Radiolabeled conjugate uptake is determined using methods known in the art (see, for example, Oosting et al 2015, J.Nucl.Med.56:63-69). In certain embodiments, the antitumor treatment comprises an inhibitor of the HGF / MET signaling axis.

[0213] To determine the presence or absence of tumor regression, uptake of the radiolabeled conjugate is quantified at baseline and at one or more time points following administration of an inhibitor of the HGF / MET signaling pathway (e.g., a METxMET bispecific antibody). For example, uptake of the administered radiolabeled antibody conjugate (e.g., a radiolabeled METxMET bispecific antibody) is quantified at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 22, 25, 29, 36 days after initial treatment with an inhibitor of the HGF / MET signaling pathway (e.g., a METxMET bispecific antibody). The uptake may be measured at the end of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-twoth, twenty-third, twenty-fourth, or more weeks. The difference between the uptake value at a particular time point after the start of treatment and the uptake value at baseline is used to establish whether there is a difference in the amount of tumor tissue (tumor shrinkage or progression). For example, a decrease in uptake from baseline upon treatment with at least one dose of an inhibitor of the HGF / MET signaling pathway signifies tumor shrinkage and indicates the effectiveness of the anti-tumor treatment.

[0214] In certain embodiments, the radiolabeled antibody conjugate is administered intravenously or subcutaneously to the subject. In certain embodiments, the radiolabeled antibody conjugate is administered intratumorally. Upon administration, the radiolabeled antibody conjugate is localized in the tumor. The localized radiolabeled antibody conjugate is imaged by PET imaging, and the uptake of the radiolabeled antibody conjugate by the tumor is measured by methods known in the art. In certain embodiments, imaging is performed 1, 2, 3, 4, 5, 6, or 7 days after administration of the radiolabeled conjugate. In certain embodiments, imaging is performed on the same day as administration of the radiolabeled antibody conjugate.

[0215] In certain embodiments, the radiolabeled anti-MET conjugate can be administered at a dose of from about 0.1 mg / kg to about 100 mg / kg of the subject's body weight, e.g., from about 0.1 mg / kg to about 50 mg / kg of body weight, or from about 0.5 mg / kg to about 25 mg / kg, or from about 0.1 mg / kg to about 1.0 mg / kg of body weight. EXAMPLES

[0216] IV. Working Examples Certain embodiments of the present disclosure are illustrated by the following non-limiting examples.

[0217] Example 1: Generation of human antibodies against MET Human anti-MET antibodies, including those listed in Table 1, were prepared and characterized as described in US-2018-0134794, the entirety of which is incorporated herein by reference. Briefly, human antibodies against MET were generated using an immunogen comprising recombinant human MET extracellular domain fused to human Fc (R&D Systems, Catalog No. 358-MT, Minneapolis, MN). The mice used for immunization express a "universal light chain"; that is, the antibodies generated in the mice have essentially identical light chain variable domains, but different heavy chain variable regions.

[0218] The antibody immune response was monitored by MET-specific immunoassay. When the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines producing MET-specific antibodies. Using this technique and the aforementioned immunogens, several anti-MET chimeric antibodies (i.e., antibodies bearing human variable domains and mouse constant domains) were obtained. Furthermore, several fully human anti-MET antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in US2007 / 0280945. Exemplary antibodies so generated are designated H4H13290P2, H4H13291P2, H4H13295P2, H4H13299P2, H4H13300P2, H4H13301P2, H4H13302P2, H4H13306P2, H4H13309P2, H4H13311P2, H4H13312P2, H4H13313P2, H4H13316P2, H4H13318P2, H4H13319P2, H4H13325P2, and H4H13331P2, and the sequences of these antibodies are set forth in Table 1 above.

[0219] METxMET bispecific antibodies were constructed from the anti-MET antibodies of Table 1. All anti-MET antibodies described herein contain the same (common) light chain (comprising the amino acid sequence of the light chain variable region [LCVR] of SEQ ID NO: 138, and the amino acid sequences of the light chain CDRs [LCDR1, LCDR2, and LCDR3] of SEQ ID NOs: 140, 142, and 144). In addition, all bispecific antibodies exemplified in this example contain a "D2" arm derived from the exemplary anti-MET antibody, H4H13312P2. Thus, both antigen-binding domains (D1 and D2) of all bispecific antibodies described in this example contain this common light chain variable region, and all D2 binding arms contain the heavy chain variable region derived from H4H13312P2. However, the bispecific antibodies differ from each other with respect to their D1 heavy chain variable region (HCVR) and heavy chain CDRs (HCDRs). Examples of these antibodies so generated are designated H4H14634D, H4H14635D, H4H14636D, H4H14637D, H4H14638D, H4H14639D, H4H14640D, H4H14641D, H4H16445D, H4H16446D, H4H16447D, H4H16448D, and H4H16449D, and the sequences of these antibodies are shown above in Table 2. For example, the METxMET bispecific antibody H4H14639D contains the D1-HCVR (SEQ ID NO:58) from the anti-MET antibody H4H13306 and the D2-HCVR (SEQ ID NO:82) from the anti-MET antibody H4H13312P2.

[0220] Example 2: Binding of MET x MET bispecific antibody H4H14639D to p-SCN-Bn-DFO The parental MET x MET bispecific antibody, H4H14639D, and an isotype control antibody were modified with a chelator, p-SCN-bn-deferoxamine (DFO, also known as desferrioxamine, Macrocylics, catalog number: B-705), to make them suitable for ImmunoPET studies involving radiolabeling, and conjugated to the antibodies.

[0221] For modification, 100 mg of 26.1 mg / mL H4H14639D was divided into four aliquots and buffer exchanged into binding buffer (150 mM NaCl, 50 mM sodium carbonate, pH 9.0; Sigma-Aldrich, Catalog No. S6297-1KG and Gibco, Catalog No. 24740-011, respectively) via four pre-equilibrated PD-10 desalting columns (GE Healthcare, Catalog No. 17-0851-01) according to the manufacturer's instructions. The elution products were combined and analyzed by UV absorption spectroscopy (Thermo Scientific) measured at 280 nm and calculated from the primary sequence-based extinction coefficient. The concentration was determined by a Scientific NanoDrop 2000c, catalog number ND-2000c-US-CAN). The elution product was further diluted to 10.4 mg / mL with binding buffer. In a separate vial, p-SCN-Bn-DFO was prepared in pure anhydrous dimethyl sulfoxide (DMSO; Sigma-Aldrich, catalog number 276855-100ML) at a concentration of 13.8 mM. The p-SCN-Bn-DFO solution was added in 1 / 4 increments to the diluted elution product and mixed by gentle pipetting, with the final reaction solution consisting of 10 mg / mL bispecific antibody in binding buffer, 2% DMSO, and a 4-fold molar-to-molar excess of p-SCN-Bn-DFO relative to bispecific antibody. The solution was allowed to incubate in a 37° C. water bath without further agitation. After 30 min at 37° C., the reaction solution was divided into four aliquots and quickly passed through four PD-10 desalting columns pre-equilibrated with a buffer containing 50 mM sodium acetate at pH 5.0 (Formulation Buffer, Sigma-Aldrich, Cat. No. 32319-1KG-R). The final eluted solutions were combined, sterile filtered through syringe filters (Acrodisc 13 mm Syringe Filter, Pall Corporation, Cat. No. 4602) and designated DFO-Ab immunoconjugate, DFO-H4H14639D immunoconjugate.

[0222] The concentrations and DFO to antibody ratios (DAR, chelator to antibody ratio) were then measured by UV absorption spectroscopy. For absorbance measurements, DFO-conjugated antibodies were measured against formulation buffer at 252 nm (A252), 280 nm (A280), and 600 nm (A600), see Tables 3 and 4. For calculations, the background was corrected for each absorbance using the following equation:

number

[0223] The antibody concentration, conjugate concentration, and DAR were calculated using the following equation: MW=144950 g mol -1 ,ε 280 =207729M -1 cm -1 ,ε 252 =79048M -1 cm -1 Calculating antibody concentration

number

number

number

[0224] The yield of the final DFO-Ab immunoconjugate was 61 mg.

[0225] DFO-Ab immune complexes were analyzed for monomeric purity using steric exclusion high performance liquid chromatography (SE-HPLC) using a Superdex 200 Increase 10 / 300 GL column (GE Healthcare, Cat. No. 28990944) with an in-line UV absorbance detector monitored at 280 nm, and a PBS mobile phase at 0.75 mL / min (see Figure 1). The major elution peak at approximately 15 min corresponds to the monomeric species. DFO-Ab immune complexes were also evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Invitrogen, Novex 4-20% Tris-Glycine Mini Gels, Cat. No. XP04200) against the unmodified bispecific antibody H4H14639D, performed according to the manufacturer's instructions (see Figure 2). The target binding equilibrium constant, i.e., K of DFO-Ab immune complexes, was determined by the following steps: Dを , and analyzed by SPR (GE Healthcare, Biacore 8k), and the bispecific antibody K D It was determined that the difference was within 10% of the [Table 3] [Table 4]

[0226] The bispecific antibody was successfully conjugated with p-SCN-Bn-DFO via primary amine chemistry as shown by UV absorption spectroscopy, SE-HPLC and SDS-PAGE. The calculated DAR of 1.26 was within the expected range of 1.0-2.0. SE-HPLC chromatograms showed a highly monomeric product (99.6%) with no detectable low molecular weight species. SDS-PAGE showed that the bispecific antibody remained unchanged after DFO-conjugation.

[0227] Example 3: Synthesis of DFO-conjugated bispecific antibodies 89 Zr chelation To use ImmunoPET in in vivo studies, the DFO-conjugated MET × MET bispecific antibody, H4H14639D, and the DFO-conjugated isotype control antibody were 89 It was radiolabeled with Zr.

[0228] DFO-conjugated antibody (250ug) was first diluted to a total of 200μL with 1M HEPES, pH 7.4 (Teknova, Cat#: H1030). Separately, approximately 5mCi (<150uL) of 89 Zr-oxalate solutions (3D Imaging, Little Rock AR) were neutralized and buffered to pH 6.8-7.4 to a total volume of up to 1000 uL of 1 M HEPES, pH 7.4. DFO-Ab immune complexes and buffered 89 The Zr solutions were combined, mixed gently by pipette, and allowed to rest for 45 minutes at room temperature. Upon completion, the reaction mixture was immediately buffer exchanged through a PD-10 column (GE Healthcare, Catalog No. 17-0851-01) preconditioned with 250 mM sodium acetate, pH 5.5 (Sigma-Aldrich, Catalog No. 32319-1KG-R) according to the manufacturer's instructions. The concentration of the eluted product, termed DFO-Ab radioimmunoconjugate, was determined by UV absorption spectroscopy (Thermo Scientific NanoDrop 2000c, Catalog No. ND-2000c-US-CAN), measured at 280 nm and calculated from the DFO contribution-adjusted primary sequence-based extinction coefficient using the following equation: Concentration in mg / mL = Absorbance at 280 nm in AU ÷ 1.86 mL / mg 1 / cm

[0229] The DFO-Ab radioimmunoconjugates were sterile filtered and analyzed for protein yield, specific activity (SA), radiochemical purity (RCP), protein purity, and target specific binding, i.e., immunoreactivity (IR). The data are reported in Table 5. Activity yield in mCi was measured using a dose calibrator (Capintec CRC-25R; Catalog No.: 5130-3215). Protein yield and specific activity of the DFO-Ab radioimmunoconjugates were determined using the following equations: a. Protein yield in mg = concentration in mg / mL x mass of solution in grams b. SA in mCi / mg = activity yield in mCi ÷ mass of complex in mg

[0230] RCP, not captured 89 Zr, and protein purity were assayed using a Superdex 200 with in-line UV280 absorbance and gamma emission detectors (Agilent Technologies, Model 1260 configured with a Lablogic SCAN-RAM radioactivity detector) connected in series using a PBS mobile phase at a flow rate of 0.75 mL / min. Analysis was performed by steric exclusion high performance liquid chromatography (SE-HPLC) using an Increase 10 / 300 GL column (GE Healthcare, Cat. 28990944). Percent protein purity was determined by comparing the relative integrals in the UV280 chromatogram of the high molecular weight (HMW) species peak (~10-~15 min) with the main peak (~15-~18 min). No low molecular weight species (~18-~25 min) were observed. Unincorporated chromatographs were analyzed using radiation chromatography (gamma radiation). 89 Radiochemical purity was determined by relative comparison of the integrals of the main peak to the Zr peak (approximately 25 min) and % HMW species.

[0231] The IR of DFO-Ab radioimmunoconjugate was administered at 2.0 × 107 cells / mL A and 0.5 × 10 7 The binding was measured by a cell binding assay involving two 500 uL aliquots, A and B, of EBC-1 cells (JCRB no. JCRB0820) at cells / mL B. DFO-Ab radioimmunoconjugate (20 ng) was added to aliquot A and incubated at 37° C., 5% CO 2The cells were incubated at 40° C. for 45 min. Both aliquots A and B were centrifuged at 1500 rpm for 5 min (Eppendorf; model #5504R). The supernatant from cell pellet B was discarded. The supernatant from cell pellet A was transferred to cell pellet B followed by incubation and separation as above. Each cell pellet (A and B) was washed twice with 1 mL of fresh cell culture medium and centrifuged at 1500 rpm for 5 min between each wash. The supernatant from the washes was collected. The final activity for all components (each cell pellet resuspended in 500 uL of cell culture medium, supernatant, and the four wash supernatants) was measured using a gamma counter (Perkin Elmer Wizard2; model #2470-0020). The IR was determined as the sum of the activities of both pellets divided by the sum of the activities of all components, x 100%. This process was tested against a non-specific DFO-Ab radioimmunoconjugate (n=1) and the IR was determined to be 2.8%.

[0232] The assay results (n=5) of the DFO-Ab radioimmunoconjugates produced above are recorded in Table 5. In particular, the average RCP was 94.6±1.2%, indicating that no 89 Zr was 2.7 ± 1.9% and protein purity was 98.3 ± 1.5%. Representative chromatograms are shown in Figures 3 and 4, respectively. SA ranged from 11.9 to 21.3 mCi / mg, appropriate for in vivo administration. The mean IR was 84 ± 7%, compared to 2.8% for the nonspecific control. [Table 5]

[0233] Example 4: Binding affinity and kinetic constants of METxMET bispecific and DFO-bound METxMET bispecific The equilibrium dissociation constants (K ) for hMET.mmh binding to purified anti-METxMET bispecific mAb (H4H14639D) or anti-METxMET bispecific mAb bound to DFO (H4H14639D-DFO) were DValues) were determined using a real-time surface plasmon resonance biosensor using a Biacore T-200 instrument. A CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody to capture purified H4H14639D or H4H14639D-DFO. The Biacore binding study was performed in a buffer consisting of 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 0.05% v / v surfactant P20 (HBS-P running buffer). Different concentrations of hMET with a C-terminal myc.myc hexahistidine tag prepared in HBS-ET running buffer were injected over the captured antibody at a flow rate of 50 μL / min. Binding of hMET.mmh to the captured monoclonal antibody was monitored for 5 min, and dissociation of hMET.mmh in HBS-ET running buffer was monitored for 10 min. Binding kinetic experiments were carried out at 25° C. Kinetic association (K) was determined by fitting the real-time sensorgrams to a 1:1 binding model using 2.0c curve fitting software. a ) and dissociation (K d The binding / dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) was calculated from the following kinetic rate constants: K D (M)=k d / k a , and t 1 / 2 (min)=0.693 / k d / 60

[0234] The binding kinetic parameters for human MET binding to purified antibodies at 25° C. are shown in Table 6 below. [Table 6]

[0235] Example 5: In vivo biodistribution of radiolabeled MET x MET bispecific antibodies Different tumor xenografts based on MET expression levels, EBC1 (MET high), NCI-H441 (MET moderate), and NCI-H358 (MET low), were selected for imaging in immunodeficient mice. 6 Tumor cells were implanted and allowed to grow for 10-14 days. Mice were then treated with 0.1 mg / kg 89 Zr-DFO-H4H14639D was administered with increasing amounts of unlabeled, unconjugated antibody to achieve final protein doses of 0.1, 0.5 and 5 mg / kg. Control animals received 0.1 mg / kg 89 Zr-DFO-isotype control antibody and unlabeled non-conjugated antibody were administered at a final protein dose of 0.5 mg / kg. PET imaging was performed on days 0, 1, 4, and 6. Biodistribution was performed on day 6.

[0236] PET / CT images were acquired using a Sofie Biosciences G8 PET / CT (Sofie Biosciences and Perkin Elmer). The instrument was 89 The system was pre-calibrated for the detection of Zr. The energy window ranged from 150 to 650 keV with a reconstructed resolution of 1.4 mm in the center of the field of view. Mice were induced anesthetized using isoflurane and kept under a continuous flow of isoflurane during imaging. Static 10 min images were collected using G8 acquisition software and subsequently reconstructed using pre-configured settings. Image data were corrected for attenuation and other parameters. CT images were collected after PET acquisition and subsequently co-registered with the PET images. Images were analyzed using VivoQuant post-processing software (invicro The images were created using 3D Imaging Services.

[0237] For biodistribution studies, mice were cultured at the final time point ( 89 Six days after administration of Zr-DFO-H4H14639D, the mice were euthanized and blood was collected via cardiac puncture. Tumor and normal tissues were excised, placed in counting tubes, and weighed. The mice were then cultured in CPM. 89Zr counting data was collected by measuring samples on an automated gamma counter (Wizard 2470, Perkin Elmer). The percent injected dose per gram (%ID / g) was calculated for each sample using standards prepared from the injected material.

[0238] The imaging results are 89 We show that Zr-DFO-H4H14639D specifically localizes to MET-expressing tumor xenografts (Figures 5-7), which is further substantiated by biodistribution data. 89 The blocking doses of Zr-DFO-H4H14639D were 0.1 and 0.5 mg / kg. 89 Compared to the low dose of Zr-DFO-H4H14639D, the NCI-H441 (MET medium) and NCI-H358 (MET low) tumors showed increased blood uptake (% ID / g) and decreased tumor uptake (% ID / g) (Figure 8). 89 Overall tumor uptake of Zr-DFO-H4H14639D correlates well with relative MET expression (Figure 9).

[0239] Example 6: MET x MET Bispecific Antibody Binding Ability (ABC) by Saturation Radioligand Binding Assay 89 Zr-DFO-anti-Met single-arm comparator antibody (Comp1; Onartuzumab, described in US2016 / 0222115 and Martens et al., Clin Cancer Res 2006,12(20):6144-6152) and 89 Antibody binding ability (ABC) assays were performed in EBC-1, NCI-H441, and NCI-H358 cell lines using Zr-DFO-H4H14639D as the radioligand. Radioligand preparation (DFO-antibody binding and subsequent Zr 89An example of 10-HT-radiolabeled IgG1 (-radiolabeled IgG1) has been described above. All six experiments were performed in a similar manner with the aid of a Hamilton-Starlett liquid handling system. Briefly, cells were first harvested, analyzed for viability (>90% confirmed by trypan blue exclusion staining), and cultured at 2–5 x 10 6 Next, 100 μL (200,000 to 500,000 cells, N) was plated along rows arranged in duplicate or quadruplicate in a V-bottom 96-well plate ("cell plate") c ) was aliquoted. The cell plate was kept at 4°C until the radiolabeled antibody was dosed in a later step. In a second V-bottom 96-well plate ("mAb plate"), 150uL of radiolabeled antibody (0.10-0.12mg / mL) was aliquoted across a row in column 1 as well as a single well in column 12 as an internalization control. Columns 2-9 were then serially diluted by a factor of 2.8 with cold medium. 50uL of the mAb plate was then bled onto the cell plate. The cell plate was incubated at 4°C for 45 minutes with gentle agitation. After incubation, the wells in columns 1-9 were first gently mixed by pipette to create an instant cell suspension, then 30uL (or 20% of the total) was collected from each well into a flip-cap tube representing the total antibody dosed. The remaining cell plate was centrifuged at 150g for 5 minutes, after which the supernatant was removed and discarded. The cell plate was then washed with 200 uL of cold medium, the plate was spun again with five aspirate / dispense cycles, and the supernatant was then removed and discarded. This washing process was repeated two more times before the cells were resuspended in 200 uL of cold buffer (10% FBS in PBS, v / v). Of the 200 uL, 180 uL was collected from each well and dispensed into flip-cap tubes representing cell-bound antibody. The activity of the total antibody samples was assayed using a gamma counter (Hidex Automatic The antibody concentrations were measured using a Gamma Counter, model 425-601. A standard curve was generated from the total antibody dose samples using the number detected vs. total antibody administered per well, T, based on a serial dilution (divided 5 times for sampling) starting from the initial antibody concentration. The standard curve was used to convert the fraction bound (multiplied by 1.38 for sampling) to the mass (or concentration) of antibody B bound.

[0240] Internalization control Internalization of the radioligand was also analyzed at the highest dose concentration for 45 min at 4°C. If significant, the fraction of internalized antibody was used to scale the ABC accordingly. For the internalization control, the well contents (200 μL) of the end column 12 were transferred to a 1.5 mL Eppendorf tube containing 1 mL of low pH stripping buffer (50 mM glycine, 150 mM NaCl, pH 2.4) and left at room temperature for 10 min. After 10 min incubation, the Eppendorf tube was centrifuged at 150 g for 5 min. The supernatant was removed without disturbing the cell pellet and saved for counting. The cell pellet was washed with 1 mL of cold buffer (10% FBS in PBS, v / v) and between washes the supernatant was aspirated, spun and removed 10 times. Activity of the cell pellet removed supernatant and washed supernatant was measured using a gamma counter. The fraction internalized was calculated as I, the ratio of activity in the cell pellet to the sum of the total activity of the cell pellet, the removed supernatant, and the washed supernatant.

[0241] ABC of Comp1: The binding saturation data were fitted using Equation 1, assuming the law of mass action under single-site conditions. Boundary values ​​were determined and converted to ABC via Equation 2. In our binding / wash protocol, nonspecific binding was determined to be a negligible component for all runs and was not considered as part of the analysis. Internalization was determined to have a minor contribution to bound radioactivity and was also not considered as part of the analysis.

[0242]

number

[0243] Formula 2:

number

[0244] Since H4H14639D has multiple binding modes, the data set was fitted by the Hill-Langmuir equation (Equation 3). ABC was then calculated using Equation 2. Nonspecific binding was determined to be a negligible component for all runs and was not considered as part of the analysis. Radioligand internalization was determined to have a non-negligible contribution to the determination of bound radioactive dose and was therefore compensated for accordingly.

[0245] Formula 3 (for H4H14639D):

number

[0246] The ABC results are summarized in Tables 7 and 8 and shown in Figures 10A and 10B. [Table 7] [Table 8]

[0247] ABC values ​​using the anti-Met, single arm / monovalent format antibody Comp1 are reasonable estimates of Met receptor copy number (i.e., one-to-one antibody to the receptor). However, ABC values ​​using antibody H4H14639D are not expected to a priori indicate one-to-one Met receptor copy number.

[0248] The above-described embodiments and examples are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific compounds, materials and procedures. All such equivalents are considered to be within the scope and encompassed by the appended claims. The present invention provides, for example, the following items. (Item 1) A radiolabeled antibody conjugate comprising an antibody or antigen-binding fragment thereof that binds to MET, a chelating moiety, and a positron emitter. (Item 2) the conjugate comprises an antibody or antigen-binding fragment thereof that binds to MET, the antibody or antigen-binding fragment thereof being covalently bound to one or more moieties of formula (A): -LM Z (A) 2. The conjugate of claim 1, wherein L is a chelating moiety, M is a positron emitter, and z is independently 0 or 1 in each occurrence, and at least one of the z's is 1. (Item 3) 3. The conjugate of claim 1 or 2, wherein the chelating moiety comprises desferrioxamine. (Item 4) The positron emitter 89 4. The complex according to any one of items 1 to 3, wherein Zr is (Item 5) -LM, [ka] wherein Zr is the positron emitter 89 5. The complex according to any one of items 1 to 4, wherein Zr is (Item 6) 6. The conjugate of any one of items 1 to 5, wherein the antibody or antigen-binding fragment thereof is covalently bound to one, two, or three moieties of formula (A). (Item 7) The antibody is selected from the group consisting of: (i) comprising a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 130, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) comprises an LCVR having an amino acid sequence of SEQ ID NO: 138, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128 and 136, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR3 domain having an amino acid sequence of SEQ ID NO: 144, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, (iv) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, and 132, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, and 134, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an HCDR2 domain having an amino acid sequence of SEQ ID NO: 140 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; an LCDR1 domain having an amino acid sequence of SEQ ID NO: 140 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and an LCDR2 domain having an amino acid sequence of SEQ ID NO: 142 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; (v) a multispecific antigen-binding molecule comprising a first binding specificity for MET and a second binding specificity for a tumor-specific antigen; (vi) a multispecific antigen-binding molecule that comprises a first binding specificity for one epitope of MET and a second binding specificity for a second epitope of MET; (vii) a K of less than about 230 nM as measured by surface plasmon resonance at 25° C. or 37° C. D binds to monomeric human MET (e.g., hMET.mmh) at (viii) a K of less than about 3 nM as measured by surface plasmon resonance at 25° C. or 37° C. D binds to dimeric human MET at (ix) blocking the binding of HGF to MET; and (x) inhibiting tumor growth and increasing survival in a subject with cancer. (Item 8) 8. The conjugate according to any one of items 1 to 7, wherein the antibody comprises in its heavy chain variable region (HCVR) three heavy chain complementarity determining regions (HCDRs), the HCVRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 130, and in its light chain variable region (LCVR) three light chain complementarity determining regions (LCDRs), the LCVRs having the LCVR amino acid sequence of SEQ ID NO: 138. (Item 9) 9. The conjugate of any one of items 1 to 8, wherein the antibody comprises three CDRs in the HCVR of SEQ ID NO: 18. (Item 10) 9. The conjugate of any one of items 1 to 8, wherein the antibody comprises three CDRs in the HCVR of SEQ ID NO: 58. (Item 11) 9. The conjugate of any one of items 1 to 8, wherein the antibody comprises three CDRs in the HCVR of SEQ ID NO: 82. (Item 12) 12. The conjugate of any one of items 1 to 11, wherein the antibody comprises three CDRs in the LCVR of SEQ ID NO: 138. (Item 13) The antibody, (i) a first antigen-binding domain (D1), and (ii) a second antigen-binding domain (D2); D1 specifically binds to the first epitope of human MET, 13. The conjugate according to any one of items 1 to 12, wherein D2 specifically binds to a second epitope of human MET. (Item 14) (i) D1 comprises the CDRs in the HCVR amino acid sequence of SEQ ID NO:58; (ii) The conjugate of item 13, wherein D2 comprises the CDR in the HCVR amino acid sequence of SEQ ID NO: 82. (Item 15) (i) D1 comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of amino acid sequences of SEQ ID NOs: 60-62-64-140-142-144; (ii) The complex according to item 13, wherein D2 comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 having the amino acid sequences of SEQ ID NOs: 84-86-88-140-142-144. (Item 16) 16. A method for imaging a tissue expressing MET, comprising administering to said tissue a radiolabeled antibody conjugate according to any one of items 1 to 15, and visualizing MET expression by positron emission tomography (PET) imaging. (Item 17) 16. A method for identifying a MET-expressing tumor in a subject, comprising administering to the subject a radiolabeled antibody conjugate according to any one of items 1 to 15, imaging the radiolabeled antibody conjugate via positron emission tomography (PET), wherein localization of the radiolabeled antibody conjugate in the subject is indicative of a MET-expressing tumor. (Item 18) 1. A method of treating a subject having a solid tumor, comprising: (a) determining that the solid tumor is MET positive; (b) administering to said subject in need thereof one or more doses of said inhibitor of the HGF / MET signaling pathway. (Item 19) Step (a) comprises the steps of: (i) administering to a subject in need thereof the radiolabeled antibody conjugate according to any one of items 1 to 15; (ii) imaging localization of the radiolabeled antibody conjugate in the tumor by positron emission tomography (PET) imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is MET positive. (Item 20) 20. The method of claim 19, wherein the subject is administered 0.1 to 10 mg / kg of the radiolabeled antibody conjugate. (Item 21) 21. The method of claim 19 or 20, wherein the radiolabeled antibody conjugate is administered to the subject subcutaneously or intravenously. (Item 22) 22. The method of any one of items 19 to 21, wherein PET imaging is performed 2 to 7 days after administration of the radiolabeled antibody conjugate. (Item 23) 23. The method according to any one of items 18 to 22, wherein step (a) is performed prior to treating the subject with the inhibitor of the HGF / MET signaling pathway. (Item 24) below: (a) treating the subject with at least one dose of an inhibitor of the HGF / MET signaling pathway followed by administering the radiolabeled antibody conjugate; (b) imaging the localization of the radiolabeled antibody conjugate in the tumor by PET imaging, wherein a decrease from baseline in the area of ​​localization of the radiolabeled antibody conjugate in the tumor indicates tumor shrinkage. (Item 25) 25. The method of any one of items 19 to 24, wherein the radiolabeled antibody conjugate is administered to the subject 1 to 20 weeks after administration of the inhibitor of the HGF / MET signaling pathway. (Item 26) 26. The method of any one of items 18 to 25, wherein the tumor is selected from the group consisting of acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer (e.g., gastric cancer with MET amplification), glioblastoma, head and neck cancer (e.g., head and neck squamous cell carcinoma [HNSCC]), Kaposi's sarcoma, renal cancer, leiomyosarcoma, hepatic cancer, lung cancer (e.g., non-small cell lung cancer [NSCLC]), lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung carcinoma, synovial sarcoma, thyroid cancer, and Wilms' tumor. (Item 27) 27. The method according to any one of items 18 to 26, wherein the tumor is selected from the group consisting of gastric cancer or non-small cell lung cancer. (Item 28) 28. The method of any one of items 18 to 27, wherein the inhibitor of the HGF / MET signaling pathway is an antibody or an antigen-binding fragment thereof. (Item 29) 29. The method of any one of items 18 to 28, wherein the inhibitor of the HGF / MET signaling pathway is a METxMET bispecific antibody or an antigen-binding fragment thereof. (Item 30) The METxMET bispecific antibody or antigen-binding fragment thereof is (i) a first antigen-binding domain (D1) comprising the CDRs in the HCVR amino acid sequence of SEQ ID NO: 58; and (ii) a second antigen-binding domain (D2) comprising the CDR in the HCVR amino acid sequence of SEQ ID NO: 82. (Item 31) 31. The method of claim 30, wherein the METxMET bispecific antibody or antigen-binding fragment thereof comprises the CDRs in the LCVR amino acid sequence of SEQ ID NO: 138. (Item 32) 31. The method of item 30, wherein D1 comprises the heavy chain variable region (HCVR) of SEQ ID NO: 58 and D2 comprises the HCVR of amino acid SEQ ID NO: 82. (Item 33) A compound of formula (III), [ka] wherein A is an antibody or antigen-binding fragment thereof that binds to MET, and k is an integer from 1 to 30. (Item 34) 34. The compound according to item 33, wherein k is 1 or 2. (Item 35) An antibody conjugate comprising: (i) an antibody or antigen-binding fragment thereof that binds to MET; and (ii) one or more chelating moieties. (Item 36) The chelating moiety is [ka] During the ceremony, [ka] is a covalent bond to the antibody or antigen-binding fragment thereof. (Item 37) 37. The antibody conjugate of claim 35, wherein the conjugate has a chelating moiety for the antibody of 1.0 to 3.0. (Item 38) 38. The antibody conjugate of any one of items 35 to 37, wherein the chelating moiety to antibody ratio is about 1.3.

Claims

1. 1. A method for making a radiolabeled AntiMET binding protein, comprising: loading a positron emitter onto a complex comprising a chelator-modified antiMET binding protein, thereby forming said radiolabeled antiMET binding protein. A method comprising:

2. The method further comprises: forming the conjugate by reacting the antiMET binding protein with the chelating agent, wherein the chelating agent comprises a chelating moiety and a reactive moiety for conjugation to the antiMET binding protein.

2. The method of claim 1, comprising: forming the complex prior to loading the positron emitter.

3. 3. The method of claim 2, wherein the reactive moiety is selected from the group consisting of an isothiocyanatobenzyl group, an n-hydroxysuccinimide ester, a 2,3,5,6 tetrafluorophenol ester, and an n-succinimidyl-S-acetylthioacetate moiety.

4. The chelating agent has the following formula: 【Chemistry 1】 The method according to any one of claims 1 to 3, comprising:

5. The method of any one of claims 1 to 3, wherein the chelating moiety of the chelating agent comprises desferrioxamine.

6. The method of any one of claims 1 to 3, wherein the chelating agent is p-isothiocyanatobenzyl-desferrioxamine.

7. The positron emitter ８９ The method according to any one of claims 1 to 6, wherein Zr is used.

8. The positron emitter ８９ The method according to any one of claims 1 to 6, wherein the Zr is an oxalic acid or other salt thereof.

9. The method of any one of claims 1 to 8, wherein the chelator to MET binding protein ratio is 1 to 4.

10. The method of any one of claims 1 to 8, wherein the chelator to MET binding protein ratio is 1-2.

11. 9. The method of any one of claims 1 to 8, wherein the chelator to MET binding protein ratio is 1.3±0.

013.

12. The complex has the following formula: 【Chemistry 2】 (III) or a salt thereof, wherein -A is the AntiMET binding protein.

13. the radiolabeled antiMET binding protein is 【Transformation 3】 wherein A is the antiMET binding protein and Zr is the positron emitter. ８９ The method according to any one of claims 1 to 12, wherein Zr is used.

14. 4. The method of claim 1, wherein the step of loading the positron emitter comprises incubating the complex with the positron emitter to promote the formation of a stable complex between the positron emitter and the complex.

15. the antiMET binding protein is (i) three heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 130; (ii) three light chain complementarity-determining regions (LCDRs) of a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 138; and The method according to any one of claims 1 to 14, comprising:

16. The antiMET binding protein is selected from the group consisting of: (i) comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 130; (ii) comprises an LCVR having the amino acid sequence of SEQ ID NO: 138; (iii) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, and 136, and an LCDR3 domain having the amino acid sequence of SEQ ID NO: 144; (iv) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, and 132, an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, and 134, an LCDR1 domain having the amino acid sequence of SEQ ID NO: 140, and an LCDR2 domain having the amino acid sequence of SEQ ID NO: 142; (v) a multispecific antigen-binding molecule comprising a first binding specificity for MET and a second binding specificity for a tumor-specific antigen; (vi) a multispecific antigen-binding molecule that has a first binding specificity for one epitope of MET and a second binding specificity for a second epitope of MET; (vii) a K of less than 230±2.3 nM as measured by surface plasmon resonance at 25° C. or 37° C. Ｄ binds to monomeric human MET at (viii) a K of less than 3±0.03 nM as measured by surface plasmon resonance at 25° C. or 37° C. Ｄ binds to dimeric human MET at (ix) blocking the binding of HGF to MET; and (x) inhibiting tumor growth and increasing survival in subjects with cancer; The method of any one of claims 1 to 15, having one or more properties selected from the group consisting of:

17. 17. The method of any one of claims 1 to 16, wherein the antiMET binding protein is an antiMET antibody or antigen-binding fragment thereof, or a METxMET bispecific antibody or antigen-binding fragment thereof.

18. 18. The method of any one of claims 1 to 17, wherein the AntiMET binding protein comprises the three CDRs of the HCVR of SEQ ID NO:

18.

19. 18. The method of any one of claims 1 to 17, wherein the AntiMET binding protein comprises the three CDRs of the HCVR of SEQ ID NO:

58.

20. 18. The method of any one of claims 1 to 17, wherein the AntiMET binding protein comprises the three CDRs of the HCVR of SEQ ID NO:

82.

21. 18. The method of any one of claims 1 to 17, wherein the AntiMET binding protein comprises the three CDRs of the LCVR of SEQ ID NO:

138.

22. 18. The method of any one of claims 1 to 17, wherein the AntiMET binding protein comprises an HCDR1 set forth in SEQ ID NO:20, an HCDR2 set forth in SEQ ID NO:22, and an HCDR3 set forth in SEQ ID NO:

24.

23. 18. The method of any one of claims 1-17, wherein the AntiMET binding protein comprises an HCDR1 set forth in SEQ ID NO:60, an HCDR2 set forth in SEQ ID NO:62, and an HCDR3 set forth in SEQ ID NO:

64.

24. 18. The method of any one of claims 1 to 17, wherein the AntiMET binding protein comprises an HCDR1 set forth in SEQ ID NO:84, an HCDR2 set forth in SEQ ID NO:86, and an HCDR3 set forth in SEQ ID NO:

88.

25. 18. The method of any one of claims 1 to 17, wherein the AntiMET binding protein comprises an LCDR1 set forth in SEQ ID NO: 140, an LCDR2 set forth in SEQ ID NO: 142, and an LCDR3 set forth in SEQ ID NO:

144.

26. the antiMET binding protein is a METxMET bispecific antibody or antigen-binding fragment thereof, and the bispecific antibody or antigen-binding fragment thereof is (i) a first antigen-binding domain (D1); and (ii) a second antigen-binding domain (D2) Including, D1 specifically binds to a first epitope of human MET; and D2 specifically binds to a second epitope on human MET; The method according to any one of claims 1 to 17.

27. (i) D1 comprises a CDR within the HCVR amino acid sequence of SEQ ID NO: 58; and (ii) D2 comprises a CDR within the HCVR amino acid sequence of SEQ ID NO: 82; 27. The method of claim 26.

28. (i) D1 comprises a CDR within the LCVR amino acid sequence of SEQ ID NO: 138; and (ii) D2 comprises a CDR within the LCVR amino acid sequence of SEQ ID NO: 138; 27. The method of claim 26.

29. (i) D1 comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of the amino acid sequences of SEQ ID NOs: 60-62-64-140-142-144; and (ii) D2 comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of the amino acid sequences of SEQ ID NOs: 84-86-88-140-142-144; 27. The method of claim 26.

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