Radiolabeled MET-binding proteins for immunoPET imaging

Abstract

Provision of radiolabeled MET-binding proteins for immunoPET imaging. [Solution] Radiolabeled anti-MET antibodies and MET×MET bispecific antibodies, as well as their use in immunoPET imaging, are provided herein. This includes methods for detecting the presence of MET protein in a subject or sample, and methods for monitoring the effectiveness of treatment for Met-expressing tumors. This disclosure includes radiolabeled anti-MET antibody conjugates and MET×MET bispecific antibody conjugates for use in immunoPET imaging.

Description

[Technical Field]

[0001] This disclosure relates to radiolabeled MET-binding proteins and their use in immunoPET imaging. Sequence List

[0002] An official copy of the sequence listing has been submitted electronically via EFS-Web at the same time as this Specified, as a sequence listing in ASCII format. The file name of this copy is "10649WO01_SEQ_LIST_ST25.txt", the creation date is September 15, 2020, and the size is approximately 136KB. The sequence listing contained in this ASCII format document is part of this Specified, and the entire document is incorporated herein by reference. [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 and is also known as MET. MET is a receptor tyrosine kinase consisting of a transmembrane beta chain linked to an extracellular alpha chain via disulfide crosslinks. When HGF binds to MET, the kinase catalytic activity of MET is activated, leading to phosphorylation of Tyr 1234 and Tyr 1235 of the beta chain and subsequent activation of downstream signaling pathways.

[0004] Overexpression, activation, or amplification of MET and / or HGF may be associated with non-small cell lung cancer (NSCLC), gastric, ovarian, pancreatic, thyroid, breast, head and neck, colon, and renal cancer (Sierra). and Tsao (Ther. Adv. Med. Oncol., 3(1 Suppl): S21-S35, 2011) have been shown to be involved. MET amplification is considered to be a major driver of oncogenesis in NSCLC and esophageal and gastric malignancies. Furthermore, mutations resulting in deletion of exon 14 of MET have been described as a driver of oncogenesis in a subset of NSCLC. Tumor cell lines with MET gene amplification are highly dependent on MET for proliferation and survival. Preclinical data encompass MET signaling in targeted therapy resistance in multiple tumor types, including 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 characteristics of the antibody with the sensitivity of a positron emission tomography camera. See, for example, The Oncologist, 12:1379 (2007); Journal of Nuclear Medicine, 52(8):1171 (2011). ImmunoPET enables the visualization and quantification of antigen and antibody accumulation in vivo, and can therefore function as an important tool for diagnosis and complementary therapy. For example, immunoPET can help in selecting promising target candidates for specific therapies and in monitoring treatment. Both preclinical and recent clinical outcomes suggest that tumors with MET gene modifications 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 those that enable 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 [Overview of the project] [Means for solving the problem]

[0007] This disclosure includes radiolabeled anti-MET antibody conjugates and MET×MET bispecific antibody conjugates for use in immunoPET imaging.

[0008] In one embodiment, the complex comprises an anti-MET antibody, a MET×MET bispecific antibody or its antigen-binding fragment, a chelate moiety, and a positron emitter.

[0009] This specification provides methods for synthesizing complexes and synthetic intermediates useful therefor.

[0010] This specification provides a method for imaging tissue expressing MET, the method comprising administering a radiolabeled anti-MET antibody conjugate or a MET×MET bispecific antibody conjugate described herein to the tissue and visualizing MET expression by positron emission tomography (PET) imaging.

[0011] This specification also provides a method for detecting MET in tissue, which includes administering the radiolabeled anti-MET antibody conjugate and MET×MET bispecific antibody conjugate described herein to the tissue and visualizing MET by PET imaging. In one embodiment, the tissue is present in a human subject. In a particular embodiment, the subject is a non-human mammal. In a particular embodiment, the subject has a disease or disorder such as cancer.

[0012] This specification also provides a method for determining the presence of MET-expressing cells in a subject. This method includes administering a radiolabeled anti-MET antibody conjugate or a MET×MET bispecific antibody conjugate as described herein to the subject and visualizing MET expression by PET imaging.

[0013] This specification also provides a method for identifying subjects having solid tumors suitable for antitumor therapy involving inhibitors of the HGF / MET signaling pathway, such as anti-MET antibodies, MET×MET bispecific antibodies, or antibody-drug conjugates (ADCs) thereof. The method comprises administering the radiolabeled antibody conjugate described herein to the subject and visualizing the administered radiolabeled antibody conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor determines that the subject is suitable for antitumor therapy involving inhibitors of the HGF / MET signaling pathway.

[0014] This specification also provides a method for treating a target solid tumor, which includes determining whether the solid tumor is MET-positive and administering an antitumor therapy to a subject requiring it. In certain embodiments, the antitumor therapy includes an anti-MET antibody or a MET×MET 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] This specification provides a method for monitoring the effectiveness of an antitumor therapy in a subject receiving antitumor therapy, the method comprising administering a radiolabeled complex described herein to the subject, and imaging the localization of the administered radiolabeled complex in the tumor by PET imaging, wherein a decrease from baseline in the uptake of the complex or radiolabeled signal indicates tumor reduction and the effectiveness of the antitumor therapy. In certain embodiments, the antitumor therapy comprises an inhibitor of the HGF / MET signaling pathway (e.g., an anti-MET antibody, a MET×MET bispecific antibody, or an ADC).

[0016] Furthermore, this specification provides a method for predicting a subject's response to an antitumor therapy comprising an inhibitor of the HGF / MET signaling pathway, the method comprising determining whether the tumor is MET-positive, and if the tumor is MET-positive, it indicates a positive response of the subject to an antitumor therapy comprising an inhibitor of the HGF / MET signaling pathway. In certain embodiments, the tumor is determined to be MET-positive by administering the radiolabeled antibody conjugate of this 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] In this specification, a method is provided for diagnosing and treating a subject having a tumor, the method comprising administering a radiolabeled conjugate described herein to the subject, the localization of the radiolabeled antibody conjugate being imaged via PET imaging to determine whether the tumor is MET-positive, diagnosing the subject having a MET-positive tumor, and administering an antitumor therapy to the subject, comprising an inhibitor of the HGF / MET signaling pathway.

[0018] This specification provides a method for diagnosing a subject having a MET-expressing tumor, the method comprising administering a radiolabeled anti-MET antibody conjugate or a MET×MET bispecific antibody conjugate described herein to the subject, visualizing MET expression by PET imaging, and diagnosing the subject having a MET-expressing tumor when MET expression is visualized by PET imaging. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 shows the SE-HPLC chromatogram after injection of 5 μg of DFO-MET×MET immunocomplex in a Superdex 200 Increase column with UV 280 nm absorption detection. Monomers (99.6%) and high molecular weight (HMW) species (0.4%) are shown.

[0020] [Figure 2]Figure 2 shows an SDS-PAGE image of DFO-MET×MET immunocomplexes. The gel demonstrates that the integrity of the antibodies remains unchanged after DFO conjugation. The lanes are labeled as follows: 1) Standard ladder (BioRad, catalog number: 161-0374), 2) Non-reducing bispecific antibody, 3) Non-reducing DFO-Ab immunocomplex, 4) Blank, 5) Reducing bispecific antibody, 6) Non-reducing DFO-Ab immunocomplex. Each well was packed with approximately 2 μg of protein. Note that non-reducing antibodies typically exhibit less electrophoretic motility than expected compared to ladders in a standard SDS-PAGE setup.

[0021] [Figure 3] Figure 3 shows a typical SE-HPLC radiochromatogram of a 5 μg injection of a radioimmune complex (DFO-MET×MET bispecific antibody) detected by gamma radiation. The RCP is over 95%, and the unincorporated 89Zr accounts for less than 1% of the total integrated activity.

[0022] [Figure 4] Figure 4 shows a typical SE-HPLC UV absorption chromatogram of a 5 μg injection of a radioimmune complex (DFO-MET×MET bispecific antibody). The main species (97.9%) and HMW species (2.1%) are shown. The elution peaks at 25–31 minutes are considered to be due to mixing of the formulation buffer / mobile phase and are not protein-derived in their origin.

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

[0026] [Figure 8-1] Figures 8A, 8B, 8C, 8D, 8E, and 8F provide ex vivo in vivo distribution data of the 89Zr-DFO-MET×MET bispecific antibody complex in SCID mice with tumor xenografts. Mice were administered a single intravenous dose of 0.1 mg / kg, 0.5 mg / kg, or 5.0 mg / kg of the 89Zr-DFO-MET×MET bispecific antibody complex and sacrificed after 6 days. Blood was collected via cardiac puncture, and the indicated collected tissues were weighed and their radioactivity determined. The percentage injection dose per gram (%ID / g) value (%ID / g) for individual samples collected on day 6 was calculated against the dose-standard radioactivity from the injected substance (89Zr-DFO-MET×MET bispecific antibody complex) and the weight of the individual sample. Data are plotted as mean ± SD. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0027] [Figure 9] Figure 9 shows the correlation between the uptake of the 89Zr-DFO-MET×MET bispecific antibody and the 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. [Modes for carrying out the invention]

[0029] I. Definition Before describing the present 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. Since the scope of the invention is limited only by the appended claims, it should also be understood that the terms used herein are used solely to describe specific embodiments and are not intended to be limiting.

[0030] Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which the present invention pertains. Where used herein, the term “about” means, when used in reference to a particular enumerated number, that value may vary by up to 1% or less from the enumerated value. For example, where used herein, the expression “about 100” includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0031] Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein. All patents, patent applications, and non-patent publications referenced herein are incorporated herein by reference in their entirety.

[0032] MET protein When used herein, expressions such as "MET" and "c-Met" refer to (1) an amino acid sequence having the amino acid sequence described in SEQ ID NO: 145 and / or the amino acid sequence described in NCBI accession number NM_001127500.2 representing the unprocessing preproprotein of isoform "a", or (2) an amino acid sequence having the amino acid sequence described in SEQ ID NO: 146 and / or the amino acid sequence described in NCBI accession number NM_000236.2 representing the unprocessing preproprotein of isoform "b". (3) refers to a human transmembrane receptor tyrosine kinase comprising an amino acid sequence having the amino acid sequence described in SEQ ID NO: 147 and / or the amino acid sequence described in NCBI accession number NM_001311330.1 representing the non-processing preproprotein of isoform "c", and / or (3) a mature protein comprising a cytoplasmic alpha subunit (SEQ ID NO: 148) and a transmembrane beta subunit (SEQ ID NOs: 149, 150, or 151 for isoforms a, b, and c, respectively), which are common to all three isoforms. The expression "MET" includes both monomeric and multimeric MET molecules. As used herein, the expression "monomeric human MET" means a MET protein or portion thereof that does not contain or possess any multimerizing domains and exists under normal conditions as a single MET molecule without direct physical connection to another MET molecule. An exemplary monomeric MET molecule is the molecule referred to herein as "hMET.mmh" containing the amino acid sequence of SEQ ID NO: 152 (see, for example, Example 3 of US-2018-0134794). As used herein, the expression "dimeric human MET" means a construct comprising two MET molecules linked to each other via a linker, covalent bond, non-covalent bond, or via a multimerizing domain such as an antibody Fc domain. An exemplary dimeric MET molecule is the molecule referred to herein as "hMET.mFc" containing the amino acid sequence of SEQ ID NO: 153 (see, for example, Example 3 of US-2018-0134794).

[0033] In this specification, all references to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment unless explicitly specified as originating from a non-human species. Therefore, the expression "MET" means human MET unless otherwise specified as originating from a non-human species, such as "mouse MET" or "monkey MET."

[0034] As used herein, the expression “cell surface expressed MET” means one or more MET proteins or their extracellular domains 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 is accessible to the antigen-binding portion of an antibody. “Cell surface expressed MET” may include, or consist of, MET proteins expressed on the surface of a cell that normally expresses MET proteins. Alternatively, “cell surface expressed MET” may include, or consist of MET proteins 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 As used herein, the term “antibody” is intended to refer to an immunoglobulin molecule (i.e., a “complete antibody molecule”) consisting of four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, as well as their polymers (e.g., IgM) or their antigen-binding fragments. Each heavy chain has a heavy chain variable region ("HCVR" or "V"). H ) and heavy chain constant region (C H 1 domain, C H 2 domains and C H It consists of 3 domains. Each light chain has a light chain variable region ("LCVR" or "V"). L ) and light chain steady region (C L ) consists of V H and V LThe region can be further subdivided into a hypervariable region called a complementarity determining region (CDR) in which more conserved regions, called framework regions (FR), are interspersed. Each V H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FR of an antibody (or antigen-binding fragment thereof) may be identical to a human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence can be defined based on the alignment of two or more CDRs.

[0036] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. The scientific literature has described that one or two CDRs can be omitted for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions 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 that do not have amino acids in one or two CDRs that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0037] CDR residues that do not contact the antigen can be identified empirically, by molecular modeling, and / or based on prior studies, from the region of Kabat CDRs outside of Chothia CDRs (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 is usually replaced with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within a CDR and the amino acids to be substituted can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[0038] Human anti-MET antibodies or MET×MET bispecific antibodies useful in this specification may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy chain variable domain and light chain variable domain compared to the corresponding germline sequence. Such mutations can be readily identified by comparing the amino acid sequences in Table 1 with germline sequences available, for example, from publicly available antibody sequence databases. Antibodies and their antigen-binding fragments useful in this disclosure are derived from any of the amino acid sequences provided in Table 1, and one or more amino acids in one or more frameworks and / or CDR regions are mutated to corresponding residues in the germline sequence from which the antibody is derived, or to corresponding residues in another human germline sequence, or to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy chain variable domain sequences and light chain variable domain sequences described in Table 1. In certain embodiments, V H and / or V LIn other embodiments, all of the framework and / or CDR residues within the domain are mutated again to residues found in the original germline sequence from which the antibody originates. In other embodiments, only certain residues, for example, only mutant residues found in the first eight amino acids of FR1, or only mutant residues found in the last eight amino acids of FR4, or only mutant residues found in CDR1, CDR2, or CDR3 are mutated again to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originates). Furthermore, the antibodies of this disclosure may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues, different from the original germline sequence, are maintained or mutated to corresponding residues 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 (optionally obtained) the biological properties of the antagonist or agonist, or reduced immunogenicity. Antibodies and antigen-binding fragments obtained in this general manner are included in this disclosure.

[0039] This specification provides for the usefulness of MET-binding proteins, such as human anti-MET antibodies and MET×MET bispecific antibodies, which include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences shown in Table 1 of this specification, having one or more conservative substitutions. For example, this disclosure includes MET×MET bispecific antibodies that have 10 or fewer, 8 or fewer, 6 or fewer, and 4 or fewer protective amino acid substitutions compared to, for example, any of the HCVR, LCVR, and / or CDR amino acid sequences in Table 1.

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

[0041] As used herein, the term “multispecific antigen-binding molecule” refers to a bispecific, triplicate, or multispecific antigen-binding molecule and its antigen-binding fragment. A multispecific antigen-binding molecule may be specific to different epitopes of one target polypeptide, or may contain antigen-binding domains specific to one or more target polypeptides. A multispecific antigen-binding molecule may be a single multifunctional polypeptide, or a polymeric complex of two or more polypeptides covalently or noncovalently linked to each other. The term “multispecific antigen-binding molecule” includes antibodies of this disclosure that can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, gene fusion, noncovalent association, or otherwise) to one or more other molecular entities, e.g., a protein or fragment thereof, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity. According to this disclosure, the term “multispecific antigen-binding molecule” also includes bispecific, triplicate, or multispecific antibodies or their antigen-binding fragments. In certain embodiments, the antibodies of this disclosure are functionally linked to another antibody or its antigen-binding fragment to produce a bispecific antibody having a second binding specificity. The bispecific and multispecific antibodies of this disclosure are described elsewhere in this specification.

[0042] The terms "specifically bind to" or "specifically bind to," or similar phrases, mean that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least approximately 1 x 10⁻¹⁶. -8 It can be characterized by an equilibrium dissociation constant less than or equal to M (for example, a smaller K D (This indicates a tighter binding.) Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis and surface plasmon resonance. Where used herein, antibodies are identified by surface plasmon resonance that specifically binds to MET, for example, BIACORE®. 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 “specifically binding” antibodies where used herein.

[0043] As used herein, the terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms “antigen-binding fragment” or “antibody fragment” of an antibody refer to one or more fragments of an antibody that retain the ability to bind to MET.

[0044] As used herein, “isolated antibody” is intended to mean an antibody that substantially does not contain other antibodies (Abs) with different antigen specificities (for example, an isolated antibody or fragment thereof that specifically binds to MET substantially does not contain Abs that specifically bind to antigens other than MET).

[0045] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE® system (Pharmacia Biosensor AB, Uppsala, Sweden and Piskataway, New Jersey).

[0046] "K D When used herein, the term "equilibrium dissociation constant" 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 a paratope. A single antigen may have two or more epitopes. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. The term “epitope” also refers to the site on an antigen to which B cells and / or T cells respond. This term also refers to the region of the antigen to which an antibody binds. Epitopes may be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and have residues that are directly involved in the affinity of the interaction. Epitopes can also be conformational, i.e., they may consist 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, they may have specific three-dimensional structural features and / or specific charge-to-mass features.

[0048] The terms “substantially identical” or “substantially identical” refer to nucleic acids or fragments thereof, and indicate that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), they have nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP.

[0049] When applied to polypeptides, the term “substantial similarity” or “substantially identical” means that two peptide sequences share at least 90% sequence identity, and more preferably at least 95%, 98%, or 99%, when optimally aligned using predefined gap weights, such as by programmed GAP or BESTFIT. Preferably, non-identical residue positions differ only by conserved amino acid substitutions. A “conservative amino acid substitution” is when 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). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ only by conservative substitutions, the percentage or degree of homology may be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331 (incorporated herein by reference). Examples of amino acid groups with side chains possessing 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 conserved amino acid substituents 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, as disclosed by 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 matches similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes 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 proteins and their mutant proteins. 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 of best overlap regions and sequence identity percentage between the query sequence and the search sequence (Pearson (2000) above). Another preferred algorithm for comparing the sequences of this disclosure with databases containing numerous sequences from different organisms is the computer program BLAST, in particular BLASTP or TBLASTN, with default parameters. For example, see Altschul et al. (1990) J.Mol.Biol.215:403-410 and (1997) Nucleic Acids Res.25:3389-3402, which are incorporated herein by reference, respectively.

[0050] The phrase "therapeutic dose" refers to the amount administered to produce the desired effect. The exact amount depends on the therapeutic purpose and can be determined by those 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, that requires remission, prevention, and / or treatment for a disease or disorder such as cancer.

[0052] II. Radiolabeled immunoconjugates of MET antibodies for immunoPET imaging This specification provides radiolabeled antigen-binding proteins that bind to MET proteins. In some embodiments, the radiolabeled antigen-binding proteins include antigen-binding proteins covalently bound to one or more chelate moieties, the chelate moieties being chemical moieties capable of chelating positron emitters.

[0053] In some embodiments, antigen-binding proteins that bind to MET, such as anti-MET antibodies or MET×MET bispecific antibodies, are provided, and the antigen-binding proteins that bind to MET are covalently bound to one or more portions having the following structure: -LM Z In the formula, L is the chelate portion, M is the positron emitter, and z is independently 0 or 1 in each occurrence, with at least one of the z values ​​being 1.

[0054] In some embodiments, the radiolabeled antigen-binding protein is a compound of formula (I), MLA-[LM Z ] k (I) In the formula, A is a protein that binds to MET, L is a chelate, M is a cation 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) In the formula, A is a protein that binds to MET, L is the chelate portion, M is a cation emitter, and k is an integer between 1 and 30.

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

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

[0058] A. MET-binding protein Suitable MET-binding proteins are proteins that specifically bind to MET, including those described in U.S. Patent Publication 2018-0134794, which is incorporated herein 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 2018-0134794, and amino acid sequence identifiers for exemplary MET×MET bispecific antibodies useful herein are listed in Table 5 of U.S. Patent Publication 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 lists the 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 containing HCVR or an antigen-binding fragment thereof, which contains an amino acid sequence selected from any of the HCVR 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 thereto.

[0061] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising the LCVR 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 thereto.

[0062] In some embodiments, the binding protein is an antibody or antigen-binding fragment containing an HCVR / LCVR amino acid sequence pair (HCVR / LCVR) which includes one of the HCVR amino acid sequences listed in Table 1 that is paired with the LCVR amino acid sequence shown in Table 1. According to a particular embodiment, the present disclosure provides an antibody or antigen-binding fragment containing an HCVR / LCVR amino acid sequence pair contained in one of the exemplary anti-MET antibodies listed in Table 1. In a particular embodiment, 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 a particular embodiment, 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 containing a heavy chain CDR1 (HCDR1) that includes 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.

[0064] In some embodiments, the binding protein is a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or an antibody or antigen-binding fragment containing a substantially similar sequence 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 containing a heavy chain CDR3 (HCDR3) that includes an amino acid sequence selected from any of the HCDR3 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 thereto.

[0066] In some embodiments, the binding protein is an antibody or an antigen-binding fragment thereof, comprising a light chain CDR1 (LCDR1) having the 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 thereto.

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

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

[0069] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising an HCDR3-LCDR3 amino acid sequence pair (HCDR3 / LCDR3) containing one of the HCDR3 amino acid sequences listed in Table 1, which is paired with the LCDR3 amino acid sequence shown in Table 1. According to one embodiment, the present disclosure provides an antibody or antigen-binding fragment comprising an HCDR3 / LCDR3 amino acid sequence pair contained in one of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pairs are: 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) The group is selected from the following: 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 containing a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) found in one of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences is 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 -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 The group is selected from the following: 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 its antigen-binding fragment containing a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) included in an HCVR / LCVR amino acid sequence pair defined by one of the exemplary anti-MET antibodies listed in Table 1. For example, in some embodiments, the binding protein is sequence numbers 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 / 13 An antibody or its antigen-binding fragment containing the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set included 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 within HCVR and LCVR amino acid sequences are known in the art and can be used herein to identify CDRs within specific HCVR and / or LCVR amino acid sequences useful to this specification. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Under general conditions, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. 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 for identifying CDR sequences within antibodies.

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

[0074] Table 2 lists the 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 bispecific antibodies useful herein were derived from various bivalent, monospecific anti-MET antibodies described in Examples 1-3 of U.S. Patent Publication No. 2018-0134794. All anti-MET antibodies described herein contain the same (common) light chain (including 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). Furthermore, all 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 these examples contain this common light chain variable region, and all D2 binding arms contain the heavy chain variable region derived from H4H13312P2. However, these bispecific antibodies differ from each other with respect to their D1 heavy chain variable region (HCVR) and heavy chain CDR (HCDR). D1 and D2 originate from different anti-MET antibodies and consequently bind to distinct epitopes on the extracellular domain of MET. That is, D1 can bind to the first epitope of human MET, e.g., the epitope containing amino acids 192-204 of SEQ ID NO: 155, while D2 can bind to the second epitope of human MET, containing amino acids 305-315 and 421-455 of SEQ ID NO: 155.

[0076] As used herein, the term "antigen-binding domain" means any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide-containing construct that can specifically bind to a specific antigen of interest (e.g., human MET). Terms such as "specifically bind" as used herein mean that the antigen-binding region has a dissociation constant (K) of 500 pM or less. DThis means that it forms a complex with a specific antigen characterized by ) and does not bind to other unrelated antigens under general test conditions. An "unrelated antigen" is a protein, peptide, or polypeptide that has less than 95% amino acid homology to one another.

[0077] Exemplary classifications of antigen-binding domains that may be used in connection with this disclosure include antibodies, antigen-binding moieties of antibodies, peptides that specifically interact with a particular antigen (e.g., peptide bodies), receptor molecules that specifically interact with a particular antigen, proteins containing ligand-binding moieties of receptors that specifically bind to a particular antigen, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on native repeat proteins [see, for example, Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and the references cited in that literature]), as well as aptamers or moieties thereof.

[0078] Methods for determining whether two molecules specifically bind to each other are known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, and similar methods. For example, when used in connection with the present disclosure, the antigen-binding region has a K content of less than approximately 500 pM, less than approximately 400 pM, less than approximately 300 pM, less than approximately 200 pM, less than approximately 100 pM, less than approximately 90 pM, less than approximately 80 pM, less than approximately 70 pM, less than approximately 60 pM, less than approximately 50 pM, less than approximately 40 pM, less than approximately 30 pM, less than approximately 20 pM, less than approximately 10 pM, less than approximately 5 pM, less than approximately 4 pM, less than approximately 2 pM, less than approximately 1 pM, less than approximately 0.5 pM, less than approximately 0.2 pM, less than approximately 0.1 pM, or less than approximately 0.05 pM as measured by surface plasmon resonance assay. D The polypeptide contains a specific antigen (e.g., a target molecule [T] or an internally distributed effector protein [E]) or a portion thereof, which has the properties of a polypeptide that binds to that antigen.

[0079] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time interaction analysis by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore® system (GE Healthcare's Biacore Life Sciences division, Piscataway, New Jersey).

[0080] "K D The term "K" as used herein means the equilibrium dissociation constant of a particular protein-protein interaction (e.g., antibody-antigen interaction). Unless otherwise specified, K is represented by antibodies useful herein. D The value was determined by surface plasmon resonance assay at 25°C or 37°C. D It refers to a value.

[0081] As shown above, the “antigen-binding domain” (D1 and / or D2) may include 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 comprising 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 comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and their polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (HCVR or V) as used herein. H It includes the heavy chain steady region (often abbreviated as C). The heavy chain steady region is C H 1, C H 2, and C H It contains three domains. Each light chain has a light chain variable region (LCVR or V in this specification). L It includes a domain (abbreviated as C) and a light chain steady region. The light chain steady region is one domain (C L Includes 1). V H Region and V LThe region can be further subdivided into a hypervariable region called the complementarity-determining region (CDR), which contains more conserved regions called the framework region (FR). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs of the antibodies provided herein (or their antigen-binding moieties) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0082] The D1 and / or D2 components of bispecific antigen-binding molecules useful herein may include, or consist of, antigen-binding fragments of full antibody molecules. Terms such as “antigen-binding portion” and “antigen-binding fragment” of an antibody, 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 protein digestion or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding an antibody variable region and optionally a constant domain. 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. DNA can be sequenced and manipulated, for example, by chemical or molecular biological techniques, to position one or more variable and / or constant domains in a suitable configuration, introduce codons, create cysteine ​​residues, modify, add, or delete amino acids.

[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 that mimic the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide) or the restricted FR3-CDR3-FR4 peptide. Other manipulated molecules, such as domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included in the expression “antigen-binding fragment” as used herein.

[0084] The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain may be of any size or have any amino acid composition, and may generally contain at least one CDR adjacent to or within one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, V H Domain and V L The domains can be arranged relative to each other in any preferred configuration. For example, the variable region may be a dimer, V H -V H , V H -V L or V L -V L It may contain a dimer. Alternatively, the antigen-binding fragment of the antibody may be a monomer V H or V L It may contain a domain.

[0085] In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen-binding fragment of the antibody of this disclosure include (i)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 LExamples include: In any configuration of the variable domain and constant domain, including any of the exemplary configurations listed above, the variable domain and constant domain may be directly linked to each other or 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 result in a mobile or semi-mobile chain between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment may consist of homodimers or heterodimers (or other polymers) of any of the variable and constant domain configurations listed above, non-covalently linked to each other and / or one or more monomers V H Or V L It may be included by a non-covalent bond with the domain (for example, by a disulfide bond).

[0086] In some embodiments, the binding protein is a bispecific antigen-binding molecule comprising or consisting of human antibodies and / or recombinant human antibodies, or fragments thereof. The term "human antibody," as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. However, human antibodies may also include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or mutations introduced by somatic mutations in vivo), for example, in the CDR and in certain CDR3 sequences. However, the term "human antibody," as used herein, is not intended to include antibodies in which germline CDR sequences from another mammalian species, such as mouse, are transplanted onto the human framework sequence.

[0087] When used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (detailed below), antibodies isolated from recombinant combinatorial human antibody libraries (detailed below), antibodies isolated from animals transgenic to human immunoglobulin genes (e.g., mice) (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means, including splicing human immunoglobulin gene sequences to 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, if transgenic animals are used for the human Ig sequence, in vivo mutagenesis of somatic cells), so the V of the recombinant antibody H Region and V L The amino acid sequence of the region is human germline V H Array and V L While it is derived from and related to the sequence, it cannot naturally exist in the in vivo human antibody germline repertoire.

[0088] Methods for making bispecific antibodies are known in the art, and using such methods, bispecific antigen-binding molecules useful for the complexes described herein may be constructed. Exemplary bispecific formats that may be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chains (e.g., common light chains having knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein for a review of the formats described above) are included.

[0089] Examples of antigen-binding domains (D1 and D2) that may be included in the MET×MET bispecific antigen-binding molecules useful herein include antigen-binding domains derived from any of the anti-MET antibodies disclosed in Table 1. For example, the present disclosure includes 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, and includes a MET×MET bispecific antigen-binding molecule comprising a D1 or D2 antigen-binding domain.

[0090] The binding protein may be a MET×MET bispecific antigen-binding molecule comprising a D1 or D2 antigen-binding domain comprising an LCVR having the 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×MET bispecific antigen-binding molecules that contain a D1 or D2 antigen-binding domain containing an HCVR-LCVR amino acid sequence pair (HCVR / LCVR) which includes one of the HCVR amino acid sequences listed in Table 1 that is paired with the LCVR amino acid sequence shown in Table 1.

[0092] In some embodiments, the binding protein is a MET×MET bispecific antigen-binding molecule containing a D1 or D2 antigen-binding domain that includes a heavy chain CDR1 (HCDR1) containing 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 MET×MET bispecific antigen-binding molecule containing a D1 or D2 antigen-binding domain that includes a heavy chain CDR2 (HCDR2) containing 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 MET×MET bispecific antigen-binding molecule containing a D1 or D2 antigen-binding domain containing a heavy chain CDR3 (HCDR3) containing 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 MET×MET bispecific antigen-binding molecule containing a D1 or D2 antigen-binding domain that includes a light chain CDR1 (LCDR1) containing 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 MET×MET bispecific antigen-binding molecule containing a D1 or D2 antigen-binding domain that includes a light chain CDR2 (LCDR2) containing 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 MET×MET bispecific antigen-binding molecule containing a D1 or D2 antigen-binding domain that includes a light chain CDR3 (LCDR3) containing the 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×MET bispecific antigen-binding molecules that contain an HCDR3 / LCDR3 amino acid sequence pair (HCDR3 / LCDR3) which includes any HCDR3 amino acid sequence listed in Table 1 that is paired with the LCDR3 amino acid sequence shown in Table 1, and include a D1 or D2 antigen-binding domain.

[0099] In some embodiments, the binding protein is a MET×MET bispecific antigen-binding molecule containing a D1 or D2 antigen-binding domain, comprising 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 MET×MET bispecific antigen-binding molecule containing a D1 or D2 antigen-binding domain, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in an HCVR / LCVR amino acid sequence pair as defined in any of the exemplary anti-MET antibodies listed in Table 1.

[0101] MET×MET bispecific antigen-binding molecules useful herein may include 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 MET×MET bispecific antibodies in this disclosure are illustrated in Figure 1 of U.S. Patent Publication No. 2018-0134794, illustrating 272 exemplary MET×MET bispecific antibody components. Each numbered cell in the matrix (numbered from 1 to 272) identifies a unique bispecific antibody containing a “D1” antigen-binding domain and a “D2” antigen-binding domain, where the D1 antigen-binding domain includes the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDR of the corresponding anti-MET antibody listed along the Y axis, and the D2 antigen-binding domain includes the immunoglobulin variable domain (HCVR / LCVR amino acid sequence) or CDR of the corresponding anti-MET antibody listed along the X axis. Therefore, for example, “number 10” of the MET×MET bispecific antigen-binding molecule shown in the matrix comprises a D1 antigen-binding domain containing an HCVR / LCVR pair or 6-CDR set derived from H4H13290P2 of the exemplary anti-MET antibody, and a D2 antigen-binding domain containing an HCVR / LCVR pair or 6-CDR set derived from H4H13321P2 of the exemplary anti-MET antibody. Additional examples of MET×MET 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 MET×MET bispecific antigen-binding molecule comprising a D1 antigen-binding domain and a D2 antigen-binding domain, wherein the D1 antigen-binding domain is the HCVR / LCVR amino acid sequence pair of SEQ ID NOs. 58 / 138, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) including SEQ ID NOs. 60-62-64-140-142-144, or at least 90%, at least 95%, at least 98%, or The D2 antigen-binding domain includes substantially similar sequences having at least 99% sequence identity, the HCVR / LCVR amino acid sequence pair of SEQ ID NOs. 82 / 138, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) including SEQ ID NOs. 84-86-88-140-142-144, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0103] In some embodiments, the binding protein is a MET×MET bispecific antigen-binding molecule comprising a D1 antigen-binding domain and a D2 antigen-binding domain, wherein the D1 antigen-binding domain is the HCVR / LCVR amino acid sequence pair of SEQ ID NOs. 18 / 138 or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) including SEQ ID NOs. 20-22-24-140-142-144, or at least 90%, at least 95%, at least 98%, or The D2 antigen-binding domain includes a substantially similar sequence having at least 99% sequence identity, the HCVR / LCVR amino acid sequence pair of SEQ ID NOs. 82 / 138 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) including SEQ ID NOs. 84-86-88-140-142-144, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

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

[0105] In some embodiments, the binding protein is a MET×MET bispecific antigen-binding molecule comprising a D1 antigen-binding domain and a D2 antigen-binding domain, wherein the D1 antigen-binding domain comprises a set of heavy or light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs. 18 / 138 or SEQ ID NOs. 20-22-24-140-142-144, and the D2 antigen-binding domain comprises a set of heavy or light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs. 82 / 138 or SEQ ID NOs. 84-86-88-140-142-144. The exemplary MET×MET bispecific antibody having these sequence characteristics is a bispecific antibody designated as H4H14635D, also referred to as bispecific antibody number 42, and includes D1 derived from H4H13295P2 and D2 derived from H4H13312P2 (see Table 2 herein).

[0106] A bispecific antigen-binding molecule useful in the present invention may also comprise one or more multimer-forming components. These multimer-forming components may function to maintain association between antigen-binding domains (D1 and D2). As used herein, “multimer-forming component” is any polymer, protein, polypeptide, peptide, or amino acid having the ability to associate with a second multimer-forming component of the same or similar structure or composition. For example, a multimer-forming component may be immunoglobulin C H The polypeptide may also contain three domains. Non-limiting examples of multimerizing components include the Fc portion of an immunoglobulin (e.g., the Fc domains of isotypes selected from the following isotypes: IgG1, IgG2, IgG3, and IgG4, as well as any allotype 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 containing or comprising a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0107] In one embodiment, a bispecific antigen-binding molecule useful herein comprises two multimer-forming 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 a single 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 may be 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 a first immunoglobulin (Ig) C H It may contain 3 domains, and M2 is the second Ig C HIt may include 3 domains, and in this case, the first and second Ig C H The 3 domains have at least one amino acid different from each other, and in this case, the difference in at least one amino acid reduces the binding of the targeting construct to Protein A compared to a reference construct having the same M1 and M2 sequences. In one embodiment, the Ig C of M1 H The 3 domain binds to Protein A, and the Ig C of M2 H The 3 domain contains a mutation that reduces or abolishes Protein A binding, such as, for example, the H95R modification (according to IMGT exon numbering; H435R in EU numbering). The C of M2 H The 3 of M2 may further include the Y96F modification (according to IMGT; Y436F according to EU). The C of M2 H Additional modifications that can be found in the 3 of M2 include the following: D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; in the case of the IgG1 Fc domain, D356E, L358M, N384S, K392N, V397M, and V422I are according to EU); in the case of the IgG2 Fc domain, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I are according to EU); and in the case of the 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, the binding protein may be “isolated.” As used herein, “isolated bispecific antigen-binding molecule” means a bispecific antigen-binding molecule identified, isolated, and / or recovered from at least one component of its natural environment. For example, a bispecific antibody isolated or removed from at least one component of a living organism, or from the tissue or cell from which the antibody was produced, is an “isolated bispecific antibody” for the purposes of this disclosure. An isolated bispecific antigen-binding molecule also includes the molecule in situ within a recombinant cell. An isolated bispecific antigen-binding molecule is a molecule that has undergone at least one purification or isolation step. According to some embodiments, an isolated bispecific antigen-binding molecule may not substantially contain other cellular material and / or chemicals.

[0109] The bispecific antigen-binding molecules or their antigen-binding domains (D1 and / or D2) useful herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the variable domains of the heavy and / or light chains, compared to the corresponding germline sequence from which the antigen-binding protein or antigen-binding domain originates. Such mutations can be readily identified by comparing the amino acid sequences in Table 1 with germline sequences available, for example, from publicly available antibody sequence databases. The bispecific antigen-binding molecules or their antigen-binding domains (D1 and / or D2) useful herein originate from any of the amino acid sequences shown in Tables 1 and 2, in which case one or more amino acids in one or more framework regions and / or CDR regions are mutated to the corresponding residues in the germline sequence from which the antibody originates, or to the corresponding residues in another human germline sequence, or to conserved amino acid substitutions in the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”).

[0110] Those skilled in the art can easily construct many bispecific antigen-binding molecules, or their antigen-binding domains (D1 and / or D2), starting from the heavy chain and light chain variable region sequences of Tables 1 and 2, including one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V L In other embodiments, all of the framework and / or CDR residues within the domain are mutated back to residues found in the original germline sequence from which the antibody originates. In other embodiments, only certain residues, for example, only mutant residues found in the first eight amino acids of FR1, or only mutant residues found in the last eight amino acids of FR4, or only mutant residues found in CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originates).

[0111] In some embodiments, the binding protein may contain any combination of two or more germline mutations within its framework and / or CDR region, for example, in which certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues, different from the original germline sequence, are maintained or mutated to corresponding residues in 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 readily validated for one or more desired properties. Desired properties include, for example, improved binding specificity, increased binding affinity, improved or enhanced (in some cases) antagonistic or agonist biological properties, or decreased immunogenicity. A bispecific antigen-binding molecule or its antigen-binding domain (D1 and / or D2) obtained in this general manner is included in this disclosure.

[0112] In some embodiments, the binding protein is an anti-MET antibody or a bispecific antigen-binding molecule comprising 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 embodiment include any of the HCVR, LCVR, and / or CDR amino acid sequences from Tables 1 and 2 having one or more conserved substitutions. For example, this disclosure includes anti-MET antibodies and MET×MET bispecific antigen-binding molecules having an HCVR amino acid sequence, LCVR amino acid sequence, and / or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conserved amino acid substitutions relative to any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences listed in Table 1 herein.

[0113] Exemplary variants include variants having 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 terms “substantial identity” or “substantially identical” mean that two amino acid sequences share at least 95%, 98%, or 99% sequence identity when optimally aligned, for example, by GAP or BESTFIT in a program using default gap weighting. In certain embodiments, non-identical residue positions are due to differing conservative amino acid substitutions. A “conservative amino acid substitution” is a substitution 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). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. If two or more amino acid sequences differ from each other only by conservative substitutions, the percentage of sequence identity or degree of homology may be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331 (incorporated herein by reference). Examples of amino acid groups 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 substituents 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 by Gonnet et al., (1992) Science 256:1443-1445.The relevant literature 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] Sequence identity between two different amino acid sequences is typically measured using sequence analysis software. Sequence analysis software matches similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between very closely related polypeptides, such as homologous polypeptides from different species, or homologous polypeptides between wild-type proteins and their variants. 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 of best overlap regions and sequence identity percentage between the query sequence and the search sequence (Pearson (2000), above). Another preferred algorithm for comparing the sequences provided herein with databases containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN using default parameters. For example, see Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402, which are incorporated herein by reference, respectively.

[0115] In some embodiments, the binding protein is, for example, an anti-MET antibody or MET×MET bispecific antigen-binding protein containing an Fc domain with one or more mutations that enhance or reduce the antibody's binding to the FcRn receptor at acidic pH compared to neutral pH. For example, anti-MET antibodies and MET×MET bispecific antigen-binding proteins contain the C of the Fc domain. H 2 or C H The antibody can contain mutations in three regions, which increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to animals. Non-restrictive examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 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 position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F and / or 308P).

[0116] For example, the binding protein may be a MET×MET bispecific antigen-binding protein containing an anti-MET antibody and an Fc domain containing one or more pairs or groups of mutations selected from the following: 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 aforementioned Fc domain mutations and other mutations within the antibody variable domain useful herein are intended to be within the scope of this disclosure.

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

[0118] Characteristics of binding proteins 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 approximately 230 nM when measured by surface plasmon resonance at 25°C or 37°C using, for example, the assay format specified in Example 3 of U.S. Patent Publication No. 2018-0134794 or a substantially similar assay. DThe present invention includes an anti-MET antibody that binds to monomeric human MET (e.g., hMET.mmh). According to one embodiment, anti-MET antibodies useful herein are those measured by surface plasmon resonance, such as using the assay format specified in Example 3 of U.S. Patent Publication No. 2018-0134794, or a substantially similar assay, with K levels of less than approximately 230 nM, less than approximately 200 nM, less than approximately 150 nM, less than approximately 100 nM, less than approximately 50 nM, less than approximately 25 nM, less than approximately 20 nM, less than approximately 10 nM, less than approximately 8 nM, less than approximately 6 nM, less than approximately 5 nM, less than approximately 4 nM, or less than approximately 3 nM. D Then, at 37°C, it binds to monomeric human MET.

[0119] In some embodiments, the binding protein has a dissociation half-life (t) greater than approximately 1 minute when measured by surface plasmon resonance at 25°C or 37°C, for example, using the assay format specified in Example 3 of U.S. Patent Publication No. 2018-0134794 or a substantially similar assay. 1 / 2 ) is an antibody or its antigen-binding fragment that binds to monomeric human MET (e.g., hMET.mmh). In certain embodiments, such an anti-MET antibody, when measured by surface plasmon resonance, for example using the assay format specified in Example 3 of U.S. Patent Publication No. 2018-0134794 or a substantially similar assay, has a t duration of more than about 1 minute, more than about 2 minutes, more than about 4 minutes, more than about 6 minutes, more than about 8 minutes, more than about 10 minutes, more than about 12 minutes, more than about 14 minutes, more than about 16 minutes, more than about 18 minutes, or more than about 20 minutes. 1 / 2 Then, at 37°C, it binds to monomeric human MET.

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

[0121] In some embodiments, the binding protein has a dissociation half-life (t) greater than approximately 4 minutes when measured by surface plasmon resonance at 25°C or 37°C, for example, using the assay format specified in Example 3 of U.S. Patent Publication No. 2018-0134794 or a substantially similar assay. 1 / 2 ) is an antibody or its antigen-binding fragment that binds to dimeric human MET (e.g., hMET.mmh) at t 1 / 2 Then, at 37°C, it binds to the dimerized human MET.

[0122] In some embodiments, the binding protein has a dissociation half-life (t) greater than approximately 10 minutes when measured by surface plasmon resonance at 25°C or 37°C, for example, using the assay format specified in Example 5 of U.S. Patent Publication No. 2018-0134794 or a substantially similar assay. 1 / 2) is an antibody or antigen-binding fragment that binds to dimeric human MET (e.g., hMET.mmh). According to one embodiment, a MET×MET bispecific antigen-binding protein useful herein is measured by surface plasmon resonance using, for example, the assay format specified in Example 5 of U.S. Patent Publication No. 2018-0134794 or a very similar assay, and has a duration of about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 200 minutes, about 300 minutes, about 400 minutes, about 500 minutes, about 600 minutes, about 700 minutes, about 800 minutes, about 900 minutes, about 1000 minutes, or about 1100 minutes or more. 1 / 2 Then, at 37°C, it binds to the dimerized human MET.

[0123] In some embodiments, the binding protein is an antibody or its antigen-binding fragment, such as a MET×MET bispecific antigen-binding protein, which blocks the interaction between HGF and MET in an in vitro ligand-binding assay, for example. MET×MET bispecific antigen-binding proteins useful herein can block HGF binding to cells expressing human MET, and in the absence of HGF signaling, induce minimal or no MET activation. For example, MET×MET bispecific antigen-binding proteins that exhibit 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 the MET agonist activity observed in a cell-system MET activity reporter assay using a monospecific antibody containing D1 or D2 alone are useful herein.

[0124] In one embodiment, the antibody or fragment is a human monoclonal antibody or its antigen-binding fragment that binds to MET, and the antibody or fragment exhibits one or more of the following characteristics: (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 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, ( ii) comprising an LCVR having the amino acid sequence of SEQ ID NO: 138 or a substantially similar sequence 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 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 at least 90%, at least 95%, at least 98% It also includes an HCDR1 domain having a substantially similar sequence with 99% sequence identity, and includes 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 an HCDR2 domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and includes the amino acid sequence of SEQ ID NO: 140 or at least 90%, at least 95%,(v) A multispecific antigen-binding molecule comprising an LCDR1 domain having a substantially similar sequence with at least 98% or at least 99% sequence identity, and an LCDR2 domain having the amino acid sequence of SEQ ID NO: 142 or a substantially similar sequence 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 to MET and a second binding specificity to a tumor-specific antigen, (vi) a multispecific antigen-binding molecule comprising a first binding specificity to one epitope of MET and a second binding specificity to a second epitope of MET, and (vii) a K of less than approximately 230 nM when measured by surface plasmon resonance at 25°C or 37°C. D (viii) When bound to monomeric human MET (e.g., hMET.mmh), and measured by surface plasmon resonance at 25°C or 37°C, the K is less than approximately 3 nM. D It binds to the dimeric human MET, (ix) blocking the binding of HGF to MET, and (x) suppressing tumor growth in subjects with cancer and increasing survival rates.

[0125] In one embodiment, the antibody or fragment is a human monoclonal antibody or its antigen-binding fragment that blocks HGF bound to MET, and the antibody or fragment exhibits one or more of the following characteristics: (i) 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 an HCVR having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. (ii) comprising an LCVR having the amino acid sequence of SEQ ID NO: 138 or a substantially similar sequence 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 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 at least 90%, at least 95%, at least 98% or less It comprises an HCDR1 domain having a substantially similar sequence with at least 99% sequence identity, and 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 an HCDR2 domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and the amino acid sequence of SEQ ID NO: 140 or at least 90%, at least 95%,(v) A multispecific antigen-binding molecule comprising an LCDR1 domain having a substantially similar sequence with at least 98% or at least 99% sequence identity, and an LCDR2 domain having the amino acid sequence of SEQ ID NO: 142 or a substantially similar sequence 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 to MET and a second binding specificity to a tumor-specific antigen, (vi) a multispecific antigen-binding molecule comprising a first binding specificity to one epitope of MET and a second binding specificity to a second epitope of MET, and (vii) a K of less than approximately 230 nM when measured by surface plasmon resonance at 25°C or 37°C. D (viii) When bound to monomeric human MET (e.g., hMET.mmh), and measured by surface plasmon resonance at 25°C or 37°C, the K is less than approximately 3 nM. D It binds to the dimer human MET and (ix) suppresses tumor growth in subjects with cancer and increases survival rate.

[0126] In certain embodiments, the binding protein is a MET × MET bispecific antibody or its antigen-binding fragment, where 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 or fragment of human MET, either in its native form or produced by recombinantly. In some embodiments, 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 cell-based MET activity reporter assays. In some embodiments, the bispecific antigen-binding molecule exhibits a degree of MET agonist activity in cell-based MET activity reporter assays that is less than 10% of the MET agonist activity of a monovalent antigen-binding molecule containing D1 or D2 alone. In some embodiments, the bispecific antigen-binding molecule promotes the degradation of cell surface-expressed MET. In some embodiments, the bispecific antigen-binding molecule inhibits the growth of tumors with MET gene alterations or promotes tumor reduction. In some embodiments, bispecific antigen-binding molecules inhibit tumor growth or promote tumor reduction, as tumor growth is driven by autocrine HGF signaling.

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

[0128] The antibodies and antigen-binding fragments described herein bind specifically to MET and modulate the interaction between MET and HGF. MET×MET bispecific antibodies may bind to MET with high or low affinity. In certain embodiments, the antibody is a blocking antibody, where the antibody binds to MET and blocks the interaction between MET and HGF. In some embodiments, the blocking antibodies of this disclosure block the binding of HGF to MET. In some embodiments, the blocking antibodies are useful for treating subjects with cancer. They may be used to inhibit the proliferation of tumor cells in the subject. They may be used alone or as adjunctive therapy in conjunction with other therapeutic parts or modalities known in the art for treating cancer. In certain embodiments, a MET×MET bispecific antibody that binds to MET with low affinity is used as a multispecific antigen-binding molecule, where the first binding specificity binds to MET with low affinity, and the second binding specificity binds to different epitopes of MET or tumor-specific antigens.

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

[0130] Non-limiting and 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 constant of a human MET×MET bispecific antibody to human MET were determined by surface plasmon resonance and measured 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×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 inhibition of MET overexpressing cell proliferation by anti-MET antibodies and MET×MET bispecific antibodies. Example 10 of US-2018-0134794 demonstrates that a MET×MET bispecific antibody induces MET degradation and inhibits the phosphorylation of both MET and ERK. US-2018-0134794 also provides several examples of tumor growth inhibition or tumor reduction induced by MET×MET bispecific antibodies, both in vivo and in vitro.

[0131] Unless otherwise specified, the term “antibody” as used herein is understood to encompass antibody molecules containing two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., “complete antibody molecules”) and their antigen-binding fragments. As used herein, terms such as “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody 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 terms “antigen-binding fragment” or “antibody fragment” of an antibody refer to one or more fragments of an antibody that possess the ability to specifically bind to MET. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term “antigen-binding fragment” refers to a polypeptide or fragment of a multispecific antigen-binding molecule. In such embodiments, the term “antigen-binding fragment” includes, for example, the extracellular domain of HGF that specifically binds to MET. Antibody antigen-binding fragments can be obtained from a complete antibody molecule using any suitable standard technique, such as protein digestion techniques or recombinant gene manipulation techniques, which involve the manipulation and expression of DNA encoding the antibody variable domain and (optionally) a constant domain. 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. DNA can be sequenced and manipulated, for example, by chemical or molecular biological techniques, to position one or more variable domains and / or constant domains in a preferred configuration, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

[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 that mimic the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide) or the restricted FR3-CDR3-FR4 peptide. Other manipulated molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapies (SMIPs), and shark variable IgNAR domains are also included in the expression “antigen-binding fragment” as used herein.

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

[0134] In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen-binding fragment of the antibody of this disclosure include (i)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 This includes. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly bound to each other or bound 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 result in a mobile or semi-mobile binding between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies of this disclosure are bound to each other and / or one or more monomer V H Or V LNon-covalent association with the domain (e.g., via disulfide bonds) may include homodimers or heterodimers (or other polymers) of either the variable domain configuration or the constant domain configuration listed above.

[0135] Similar to fully antibody molecules, antigen-binding fragments may be monospecific or polyspecific (e.g., bispecific). A polyspecific antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any form of polyspecific antibody, including examples of bispecific antibody forms disclosed herein, may be adapted for use with the antigen-binding fragments of the disclosed antibodies using conventional techniques available in the art.

[0136] The anti-MET antibodies and MET×MET bispecific antibodies and antibody fragments useful herein include proteins having amino acid sequences that differ from the amino acid sequence of the described antibodies but 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 the biological activity of the described antibody. Similarly, the antibody-coding DNA sequences of this disclosure include sequences encoding antibodies or antibody fragments that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequence, but are essentially biologically equivalent to the antibodies or antibody fragments of this disclosure.

[0137] Two antigen-binding proteins or antibodies are considered biologically equivalent or pharmaceutical substitutes if, under similar experimental conditions, they do not show significant differences in the rate and range of absorption when administered in the same molar dose, either as a single or multiple doses. Some antibodies may be considered biologically equivalent or pharmaceutical substitutes if their absorption range is equivalent but their absorption rates are not. However, if this difference in absorption rate is intentional, reflected in the labeling, and is not considered medically important to the specific drug being tested (for example, not essential for achieving effective drug concentration in the body during chronic use), then those antibodies may be considered equivalent or pharmaceutical substitutes.

[0138] In one embodiment, the two antigen-binding proteins are biologically equivalent if there are no clinically significant differences in their safety, purity, or efficacy.

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

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

[0141] Biological homogeneity can be demonstrated by in vivo and in vitro methods. Methods for measuring biological homogeneity include, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum or other biological fluids; (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 appropriate acute pharmacological effect of an antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that demonstrate the safety, efficacy, bioavailability, or bioequivalence of an antibody.

[0142] Bioequivalent variants of the antibodies disclosed herein can be constructed, for example, by various substitutions of residues or sequences, or by deletion of terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or substituted with other amino acids to prevent the formation of unnecessary or inaccurate intramolecular disulfide crosslinks during regeneration. In other circumstances, bioequivalent antibodies may include antibody variants that include amino acid changes that alter the glycosylation properties of the antibody, such as mutations that eliminate or remove glycosylation.

[0143] Anti-MET antibodies and MET×MET bispecific antibodies useful herein may include, for example, an Fc domain containing one or more mutations that increase or decrease the antibody's ability to bind to acidic FcRn receptors compared to neutral pH. For example, this disclosure includes an Fc domain with C H 2 or C HThe antibody contains a MET×MET bispecific antibody with mutations in three regions, where the mutations increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to animals. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 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 position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F and / or 308P). In yet another embodiment, modifications include modifications of 265A (e.g., D265A) and / or 297A (e.g., N297A).

[0144] For example, anti-MET antibodies and MET×MET bispecific antibodies containing an Fc domain containing one or more pairs or groups of mutations selected from the following groups are useful: 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 N4 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 disclosure includes a MET×MET bispecific antibody containing an Fc domain with an S108P mutation in the hinge region of IgG4 that promotes dimer stabilization. All possible combinations of the aforementioned Fc domain mutations and other mutations in the antibody variable domains provided herein in Table 1 are intended to be within the scope of the disclosure.

[0145] The anti-MET antibodies and MET×MET bispecific antibodies useful in this specification are chimeric heavy chain constant (C) H ) can include the region, Chimera C H The region is C of one or more immunoglobulin isotypes. H It contains segments derived from the region. For example, an antibody may contain C1, C2, or C4 molecules derived from human IgG1, human IgG2, or human IgG4 molecules. H In combination with some or all of the three domains, C derived from human IgG1, human IgG2, or human IgG4 molecules. H Chimeric C containing part or all of two domains H It may include a region. According to a particular embodiment, the antibody is a chimeric C having a chimeric hinge region. HThe region may include an upper hinge amino acid sequence (amino acid residues at EU numbering positions 216-227) derived from the human IgG1 hinge region, human IgG2 hinge region, or human IgG4 hinge region, combined with an upper hinge sequence (amino acid residues at EU numbering positions 228-236) derived from the human IgG1 hinge region, human IgG2 hinge region, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region includes amino acid residues derived from the human IgG1 upper hinge or human IgG4 upper hinge and amino acid residues derived from the human IgG2 lower hinge. Chimeric C as described herein H Antibodies containing the region exhibit modified Fc effector function without negatively affecting the therapeutic or pharmacokinetic properties of the antibody, in certain embodiments. (See, for example, USSN. 14 / 170,166, filed January 31, 2014, the disclosure of which is incorporated herein by reference in its entirety.)

[0146] B. Positron emitter and chelate portion Suitable positron emitters include, but are not limited to, those that form stable complexes with chelate moieties and have a physical half-life suitable for immunoPET imaging. Exemplary positron emitters include: 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Y is one example, but it is not limited to these. A suitable positron emitter also includes, 76 Br and 124 I, and prosthetic groups, for example, 18 Examples include those introduced by F, but are not limited to those that directly bind to MET-binding proteins.

[0147] The chelate moieties described herein include a chemical moiety covalently bound to a MET-binding protein, such as a MET×MET bispecific antibody, which can chelate with a positron emitter, i.e., react with a positron emitter to form a coordination chelate complex. Appropriate moieties include those that enable efficient loading of a specific metal and form a sufficiently stable metal chelating complex for in vivo diagnostic applications, such as immunoPET imaging. Exemplary chelate moieties include those that minimize positron emitter dissociation and accumulation in bone minerals, plasma proteins, and / or bone marrow deposits to an extent suitable for diagnostic applications.

[0148] An example of a chelate portion is a positron emitter. 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Examples include, but are not limited to, those that form stable complexes with Y. The entirety of these is incorporated by reference in the exemplary chelate moieties: 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 Examples include, but are not limited to, those described in Chem., 26(12):2579(2015); WO2015 / 140212A1; and U.S. Patent No. 5,639,879.

[0149] The exemplary chelate moieties include desferrioxamine (DFO) (also known as deferoxamine), 1,4,7,10-tetraacetic acid (DOTA), diethyleneethriaminpentaacetic 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), H4 Octapa, H6 Hosupa, H2 Dedopa, H5 Dekapa, H2 Azapa, HOPO, DO2A, 1,4,7,10-Tetrakis(carbamoylmethyl)-1,4,7,10-Tetraazacyclododecane (DOTAM), 1,4,7-Triazacyclono Nan-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 (Cyclen), 1,4,8,11-tetraazacyclododecane (Cyclam), octadente Examples of chelating agents include, but are not limited to, to chlorotate chelating agents, hexadentate chelating agents, phosphonate-based chelating agents, macrocyclic chelating agents, chelating agents containing macrocyclic terephthalamide ligands, bifunctional chelating agents, fusalinin C and fusalinin C derivative chelating agents, triacetylfusalinin C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), and ferrichrome A (FCHA).

[0150] In some embodiments, the chelate portion is covalently bound to a MET-binding protein, such as an antibody or its antigen-binding fragment, via a linker portion that covalently binds the chelate portion of the chelate portion to the binding protein. In some embodiments, these linker portions are formed from a reaction between a reaction portion of the MET-binding protein, such as cysteine ​​or lysine of an antibody, and a reaction portion bound to a chelating agent, such as a p-isothiocyanatobenyl group and a reaction portion provided in the binding method described below. In addition, such linker portions optionally include chemical groups used to modulate the polarity, solubility properties, steric interactions, rigidity, and / or length between the chelate portion and the MET-binding protein.

[0151] C. Preparation of radiolabeled MET-binding protein complexes Radiolabeled anti-MET antibodies or MET×MET bispecific antibody complexes can be prepared by (1) reacting a MET-binding protein, such as a MET×MET bispecific antibody, with a molecule containing a positron emitter chelator and a moiety that is reactive to a desired conjugation site of the MET-binding protein, and (2) loading the molecule with a desired positron emitter.

[0152] Suitable conjugation sites include, but are not limited to, lysine and cysteine, both of which may be, for example, natural or engineered, and may be located, for example, on the heavy or light chain of an antibody. Cysteine ​​conjugation sites include, but are not limited to, those obtained from mutations, insertions, or reductions of antibody disulfide bonds. Methods for producing cysteine-engineered antibodies include, but are not limited to, those disclosed in WO2011 / 056983. Site-directed conjugation methods can also be used to direct the binding reaction of an antibody to a specific site, 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, as well as those described in J.Clin.Immunol., 36:100 (2016), which is incorporated herein by reference in whole. A suitable moiety that is reactive to the desired conjugation site generally enables efficient and easy binding of MET-binding proteins, such as antibodies, to positron-emitting chelating agents. Moieties that are reactive to lysine and cysteine ​​sites contain electrophiles known to those skilled in the art. In certain embodiments, when the desired conjugation site is lysine, the reactive moiety is an isothiocyanate, such as a p-isothiocyanatobenyl group or a reactive ester. In certain embodiments, when the desired conjugation site is cysteine, the reactive moiety is maleimide.

[0153] When the chelating agent is desferrioxamine (DFO) (also known as deferoxamine), preferred reactive moieties include, but are not limited to, isothiocyanatobenzyl groups, n-hydroxysuccinimide esters, 2,3,5,6-tetrafluorophenol esters, n-succinimidyl-S-acetylthioacetate, and those described in BioMed Research International, Vol 2014, Article ID 203601, which are incorporated herein by reference. In certain embodiments, the MET-binding protein is an antibody, and the molecule comprising the positron-emitting chelating agent and the moiety reactive to the conjugation site is p-isothiocyanatobenzyl-desferrioxamine (p-SCN-Bn-DFO). [ka]

[0154] The loading of the positron emitter is achieved, for example, by carrying out the method described in the examples provided herein, or substantially similar, by incubating the MET-bound protein chelating complex with the positron emitter for a time sufficient to allow partial chelating of the positron emitter to the chelating agent.

[0155] D. Exemplary Embodiments of the Complex The disclosure includes radiolabeled antibody complexes comprising a human MET, for example, an antibody or antigen-binding fragment thereof that binds to an anti-MET antibody or a MET×MET bispecific antibody, and a positron emitter. Furthermore, the disclosure includes radiolabeled antibody complexes comprising an anti-MET antibody or a MET×MET bispecific antibody, a chelate moiety, and a positron emitter.

[0156] In some embodiments, the chelate portion is 89It contains a chelating agent that 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 is 89 The protein is Zr. In some embodiments, less than 1.0% of the MET-binding protein binds to the positron emitter, less than 0.9% of the MET-binding protein binds to the positron emitter, less than 0.8% of the MET-binding protein binds to the positron emitter, less than 0.7% of the MET-binding protein binds to the positron emitter, less than 0.6% of the MET-binding protein binds to the positron emitter, less than 0.5% of the MET-binding protein binds to the positron emitter, less than 0.4% of the MET-binding protein binds to the positron emitter, less than 0.3% of the MET-binding protein binds to the positron emitter, less than 0.2% of the MET-binding protein binds to the positron emitter, and less than 0.1% of the MET-binding protein binds to the positron emitter.

[0158] In some embodiments, the chelate-to-antibody ratio of the conjugate is 1 to 2. As used herein, “chelate-to-antibody ratio” is the mean chelate-to-antibody ratio and is a measure of the chelate load per antibody. This ratio is similar to the “DAR,” i.e., the drug-antibody ratio used by those skilled in the art to measure the drug load per antibody for antibody-drug conjugates (ADCs), and in the case of the conjugates described herein relating to iPET imaging, the chelate-to-antibody ratio is similar to the method described herein and other methods known in the art for determining DAR, e.g., Wang et al., Antibody-Drug Conjugates, The 21 stThis can be confirmed using the information provided in Century Magic Bullets for Cancer (2015). In some embodiments, the chelate partial antibody ratio is 1.0–4.0, or about 1.0–3.0, or about 1.0–2.0. In some embodiments, the chelate partial antibody ratio is about 1.26, for example, about 1.3.

[0159] In certain embodiments, the chelate portion is p-isothiocyanatobenzyl-desferrioxamine, and the positron emitter is 89 It is Zr. In another specific embodiment, the chelate moiety is p-isothiocyanatobenzyl-desferrioxamine, and the positron emitter is 89 It is Zr, and the chelate portion versus antibody ratio of the complex is 1-2.

[0160] In some embodiments, antigen-binding proteins that bind to MET are provided herein, and the antigen-binding proteins that bind to MET are covalently bound to one or more portions having the following structure: -LM Z In the formula, L is the chelate moiety, M is the positron emitter, and z is independently 0 or 1 in each occurrence, with at least one of z being 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 chelate, M is a cation 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 as follows: [ka]

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

[0163] In some embodiments, k is an integer between 1 and 2. In some embodiments, k is 1. In some embodiments, k is 2.

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

[0165] Furthermore, the compound of formula (III) [ka] 89 Methods for synthesizing radiolabeled antibody complexes, including contact with Zr, are also included in this disclosure, 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] Compound of formula (III) and 89 The products of the reaction with Zr are also provided herein.

[0167] Compounds of formula (III) are provided herein, [ka] In the formula, A is an antibody or its antigen-binding fragment that binds to MET, and k is an integer from 1 to 30. In some embodiments, k is 1 or 2.

[0168] (i) an antibody or antigen-binding fragment thereof that binds to MET, and (ii) one or more chelate moieties are provided herein.

[0169] In some embodiments, the chelate portion includes the following: [Chemistry] [Chemistry] is a covalent bond to an antibody or an antigen-binding fragment thereof.

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

[0171] In some embodiments, compositions are provided herein that include a conjugate having the following structure: A-L k wherein A is a protein that binds to MET, L is a chelate 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 radioactivity suitable for clinical PET imaging. In some embodiments, the amount of chelated positron emitter is an amount sufficient to provide a specific radioactivity of from about 1 to about 50 mCi per 1 to 50 mg of the protein that binds to MET.

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

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

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

[0175] In some embodiments, the antibody or antigen-binding fragment thereof competes with a reference antibody comprising a complementarity determining region (CDR) of the HCVR for binding to human MET, the HCVR is an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1, and has a CDR of the LCVR, and the LCVR has the amino acid sequence shown in Table 1. In some embodiments, the reference antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pairs described 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. In some embodiments, the antibody or antigen-binding fragment thereof does not increase or decrease MET binding to its ligand.

[0177] [[ID=]] In some embodiments, the antibody or its antigen-binding fragment comprises a complementation-determining region (CDR) of HCVR, where HCVR has 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 LCVR has an amino acid sequence of SEQ ID NO: 138. In certain embodiments, the isolated antibody contains 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.

[0178] In some embodiments, the antibody is a human monoclonal antibody or its antigen-binding fragment that specifically binds to human MET, and the antibody or its antigen-binding fragment includes 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 its antigen-binding fragment that specifically binds to human MET, and the antibody or its antigen-binding fragment includes 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 its antigen-binding fragment that specifically binds to human MET, and the antibody or its antigen-binding fragment includes (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 its antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in any one of the heavy chain variable region (HCVR) sequences listed in Table 1, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light chain variable region (LCVR) sequences shown in Table 1.

[0182] In some embodiments, the antibody or its antigen-binding fragment includes: (a) 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) 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) 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) LCDR1 domain having the amino acid sequence of sequence number 140, (e) LCDR2 domain having the amino acid sequence of Sequence ID No. 142, and (f) The LCDR3 domain having the amino acid sequence of sequence number 144.

[0183] In some embodiments, the antibody or its antigen-binding fragment 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.

[0184] In some embodiments, the antibody or its antigen-binding fragment comprises a CDR of HCVR, where HCVR has 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 the CDR of LCVR has the amino acid sequence of SEQ ID NO: 138.

[0185] In some embodiments, the antibody or its antigen-binding fragment is an anti-MET antibody containing a CDR in the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 82 / 138. In some embodiments, the antibody or its antigen-binding fragment is an anti-MET antibody containing 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 its antigen-binding fragment is an anti-MET antibody containing a CDR in the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 18 / 138. In some embodiments, the antibody or its antigen-binding fragment is an anti-MET antibody containing 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 its antigen-binding fragment is an anti-MET antibody containing a CDR in the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 58 / 138. In some embodiments, the antibody or its antigen-binding fragment is an anti-MET antibody containing 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 its antigen-binding fragment is a MET×MET bispecific antigen-binding protein comprising a CDR in the D1-HCVR amino acid sequence of SEQ ID NO: 58 and a CDR in the D2-HCVR amino acid sequence of SEQ ID NO: 82. In some embodiments, the MET×MET bispecific antigen-binding protein further comprises a CDR in the LCVR amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody or its antigen-binding fragment is a MET×MET bispecific antigen-binding protein comprising a D1-HCVR amino acid sequence of SEQ ID NO: 58 and a D2-HCVR amino acid sequence of SEQ ID NO: 82. In some embodiments, the MET×MET bispecific antigen-binding protein further comprises the LCVR amino acid sequence of SEQ ID NO: 138.

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

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

[0191] In some embodiments, the radiolabeled antibody conjugate comprises an antibody or an antigen-binding fragment thereof that binds to MET, a chelating moiety, and a positron emitter, wherein the antibody or the 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 of Using Radiolabeled Immunoconjugates In certain aspects, the present disclosure provides diagnostic and therapeutic methods of using the radiolabeled antibody conjugates of the present disclosure.

[0193] According to one aspect, the present disclosure provides a method of detecting MET in a tissue, the method comprising administering to the tissue the 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 that expresses MET, the method comprising administering to the tissue the 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 a tumor cell line. In one embodiment, the tissue is contained within a tumor lesion of a subject.

[0195] In one embodiment, the disclosure provides a method for measuring the response to anti-MET therapy in subjects with cancer, wherein the response to therapy is measured by a change in MET expression relative to pre-therapy MET expression. The method according to this embodiment includes administering a radiolabeled antibody conjugate provided herein to a subject in need 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 anti-MET therapy.

[0196] In one embodiment, the present disclosure provides a method for determining whether a subject having a solid tumor is suitable for antitumor therapy including an inhibitor of the HGF / MET signaling pathway, the method comprising administering the radiolabeled antibody conjugate of the present disclosure to the subject 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 antitumor therapy including an inhibitor of the HGF / MET signaling pathway.

[0197] Useful antitumor therapies according to the methods disclosed herein may 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 antitumor therapy comprises an anti-MET antibody or its antigen-binding fragment, e.g., any one or more of the antibodies listed in Table 1. In some embodiments, the antitumor therapy comprises any one or more of the MET × MET bispecific antibodies, e.g., any one of the bispecific antibodies listed in Table 2. In some embodiments, the antibody, bispecific antibody, or its antigen-binding fragment is conjugated to a drug useful for treating cancer (i.e., an ADC). Exemplary anti-MET ADCs are disclosed in US-2018-0134794A1.

[0198] In one embodiment, the Disclosure identifies a candidate subject for antitumor therapy including an inhibitor of the HGF / MET signaling pathway, and the method comprises administering the radiolabeled antibody conjugate of the Disclosure to a subject having a tumor, localizing the administered radiolabeled antibody conjugate to the tumor by PET imaging, and determining that the subject is suitable for antitumor therapy including an inhibitor of the HGF / MET signaling pathway based on the presence of the radiolabeled antibody conjugate in the tumor.

[0199] In one embodiment, the Disclosure provides a method for predicting a response to a target antitumor therapy, the method comprising determining whether a tumor is MET-positive, and if the tumor is MET-positive, a positive response to the target antitumor therapy is predicted. In a particular embodiment, the tumor is determined to be MET-positive by administering the radiolabeled antibody conjugate of the 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.

[0200] In one embodiment, the present disclosure provides a method for predicting a response to antitumor therapy in a subject having a solid tumor, the method comprising determining whether the tumor is MET-positive, and if the tumor is MET-positive, a positive response in the subject is predicted. In a particular embodiment, the tumor is determined to be MET-positive by administering the 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.

[0201] In one embodiment, the Disclosure provides a method for detecting a MET-positive tumor of a target. The method according to this embodiment comprises administering the radiolabeled antibody conjugate of the Disclosure to a target and determining the localization of the radiolabeled antibody conjugate by PET imaging, wherein 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 antitumor therapy, for example, whether the tumor shrinks or stops growing.

[0202] According to one embodiment, the present disclosure provides a method for determining the size of a target MET-positive tumor. The method according to this embodiment includes administering the radiolabeled antibody conjugate of the present disclosure to a target and visualizing the radiolabeled antibody conjugate by PET imaging, thereby determining the size of the tumor.

[0203] This specification also provides a method for determining the presence of MET-expressing cells in a subject. This method includes administering a radiolabeled anti-MET antibody conjugate or a MET×MET bispecific antibody conjugate as described herein to the subject and visualizing MET expression by PET imaging.

[0204] In this specification, a method is provided for diagnosing and treating a subject having a tumor, the method comprising administering a radiolabeled conjugate described herein to the subject, the localization of the radiolabeled antibody conjugate being imaged via PET imaging to determine whether the tumor is MET-positive, diagnosing the subject having a MET-positive tumor, and administering an antitumor therapy to the subject, comprising an inhibitor of the HGF / MET signaling pathway.

[0205] This specification provides a method for diagnosing a subject having a MET-expressing tumor, the method comprising administering a radiolabeled anti-MET antibody conjugate or a MET×MET bispecific antibody conjugate described herein to the subject, visualizing MET expression by PET imaging, and diagnosing the subject having a MET-expressing tumor when MET expression is visualized by PET imaging.

[0206] As used herein, the expression “subject in need” means 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 have a primary or metastatic tumor and / or be diagnosed 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 / chest tightness, splenomegaly, and elevated levels of cancer-related biomarkers (e.g., CA125). This expression includes subjects having a primary or engrafted tumor. In certain embodiments, this expression includes human subjects having 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, and / or in need of treatment for them. This term includes subjects with primary or metastatic tumors (advanced malignant tumors). In certain embodiments, the expression “subjects requiring it” 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, this expression includes subjects who have been treated with one or more previous alternative therapies, such as chemotherapy (e.g., carboplatin or docetaxel). In certain embodiments, the expression “subjects requiring it” includes patients with solid tumors that have been treated with one or more previous alternative therapies but have subsequently relapsed or metastasized.

[0207] In certain embodiments, the methods of this disclosure are used on subjects having 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 typically contain cysts or fluid areas. Solid tumors can be benign (not cancerous) or malignant (cancerous). For the purposes of this disclosure, the term “solid tumor” means malignant solid tumors. This term includes different types of solid tumors named by the cell type that forms them, i.e., sarcomas, carcinomas, and lymphomas. In certain embodiments, the term “solid tumor” includes cancers such as 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, tumors are 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 cancer, synovial sarcoma, thyroid cancer, and Wilms' tumor.

[0209] In one embodiment, the present disclosure provides a method for treating a target tumor. The method according to this embodiment comprises determining that the tumor is MET-positive and administering one or more doses of an inhibitor of the HGF / MET signaling pathway. In some embodiments, the inhibitor is an anti-MET antibody, a MET × MET bispecific antibody, or a drug conjugate thereof. In certain embodiments, the tumor is determined to be MET-positive by administering the radiolabeled antibody conjugate of the present disclosure to the target and by 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, terms such as “to treat” and “to treat” mean to alleviate symptoms, to eliminate the cause of symptoms, whether temporarily or permanently, to slow or inhibit tumor growth, to reduce tumor cell volume or tumor volume, to cause tumor shrinkage, necrosis and / or disappearance, to suppress or inhibit metastasis, to inhibit the growth of metastatic tumors, and / or to extend the survival of the subject.

[0211] In one embodiment, the Disclosure provides a method for monitoring the effectiveness of an antitumor therapy in a patient undergoing cancer treatment, the method comprising administering a radiolabeled antibody conjugate of the Disclosure to the patient, imaging the localization of the administered labeled conjugate in the tumor by PET imaging, and determining tumor growth, wherein a decrease from baseline in the radiolabeled signal indicates tumor reduction and the effectiveness of the antitumor therapy. In certain embodiments, the antitumor therapy comprises an inhibitor of the HGF / MET signaling pathway (e.g., a MET×MET bispecific antibody).

[0212] As used herein, the term “baseline” with respect to MET expression in a tumor means the numerical value of uptake of the radiolabeled complex in the subject before or at the time of administration of the dose of the antitumor therapy. Uptake of the radiolabeled complex 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 therapy comprises an inhibitor of the HGF / MET signaling axis.

[0213] To determine whether tumor reduction has occurred, the uptake of the radiolabeled complex is quantified at baseline and at one or more time points after administration of an HGF / MET signaling pathway inhibitor (e.g., a MET×MET bispecific antibody). For example, the uptake of the administered radiolabeled antibody complex (e.g., a radiolabeled MET×MET bispecific antibody) is quantified on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 22, 25, 29, and 36 after initial treatment with an HGF / MET signaling pathway inhibitor (e.g., a MET×MET bispecific antibody). Measurements may be taken on days 43, 50, 57, 64, 71, and 85, or at the end of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or any later. The difference between the uptake value at a specific point in time after the start of treatment and the baseline uptake value is used to establish whether there is a difference in the amount of tumor tissue (tumor reduction or progression). For example, a decrease from baseline in uptake in treatment with at least one dose of an HGF / MET signaling pathway inhibitor signifies tumor reduction and indicates the effectiveness of the antitumor 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 into a tumor. Upon administration, the radiolabeled antibody conjugate is localized within the tumor. The localized radiolabeled antibody conjugate is imaged by PET imaging, and the tumor's uptake of the radiolabeled antibody conjugate 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 complex can be administered in doses ranging from approximately 0.1 mg / kg to approximately 100 mg / kg of the subject's body weight, for example, approximately 0.1 mg / kg to approximately 50 mg / kg, or approximately 0.5 mg / kg to approximately 25 mg / kg, or approximately 0.1 mg / kg to approximately 1.0 mg / kg. [Examples]

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

[0217] Example 1: Generation of human antibodies against MET Human anti-MET antibodies, including those listed in Table 1, were prepared and characterized in their entirety as described in US-2018-0134794, which is incorporated herein by reference. Briefly, human antibodies against MET were generated using an immunogen containing a 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 from these mice have different heavy chain variable regions but essentially the same light chain variable domain.

[0218] The antibody immune response was monitored by MET-specific immunoassays. Once the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. 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 possessing both a human variable domain and a mouse constant domain) were obtained. Furthermore, as described in US2007 / 0280945, several fully human anti-MET antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells. The exemplary antibodies produced in this manner are shown as 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 shown in Table 1 above.

[0219] MET×MET bispecific antibodies were constructed from the anti-MET antibodies in Table 1. All anti-MET antibodies described herein contain the same (common) light chain (including 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). Furthermore, all bispecific antibodies illustrated 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, these bispecific antibodies differ from one another with respect to their D1 heavy chain variable region (HCVR) and heavy chain CDR (HCDR). Examples of these antibodies produced in this manner are shown as H4H14634D, H4H14635D, H4H14636D, H4H14637D, H4H14638D, H4H14639D, H4H14640D, H4H14641D, H4H16445D, H4H16446D, H4H16447D, H4H16448D, and H4H16449D, and the sequences of these antibodies are shown in Table 2 above. For example, the MET×MET bispecific antibody H4H14639D contains D1-HCVR (SEQ ID NO: 58) from the anti-MET antibody of H4H13306 and D2-HCVR (SEQ ID NO: 82) from the anti-MET antibody of H4H13312P2.

[0220] Example 2: Binding of MET×MET bispecific antibody H4H14639D to p-SCN-Bn-DFO The parent MET×MET bispecific antibody, H4H14639D, and the isotype control antibody were modified with the chelating agent p-SCN-bn-deferoxamine (DFO, also known as desferrioxamine, Macrocylics, catalog number: B-705) to make them suitable for immunoPET assays with radiolabeling, and then conjugated to the antibodies.

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

[0222] The concentration and the DFO ratio to antibody (DAR, chelating agent ratio to antibody) were subsequently measured by UV absorption spectroscopy. For absorbance measurements, the DFO-conjugated antibody was measured against the 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, complex 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 Calculation of antibody concentration

number

number

number

[0224] The final yield of DFO-Ab immune complexes was 61 mg.

[0225] The monomer purity of the DFO-Ab immunocomplex was analyzed by stereoexclusion high-performance liquid chromatography (SE-HPLC) using a Superdex 200 Increase 10 / 300 GL column (GE Healthcare, catalog number 28990944), an in-line UV absorbance detector monitored at 280 nm, and a PBS mobile phase of 0.75 mL / min (see Figure 1). The major elution peak at approximately 15 minutes corresponds to the monomer species. The DFO-Ab immunocomplex was also evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Invitrogen, Novex 4-20% Tris-Glycine Mini Gels, catalog number XP04200) against the unmodified bispecific antibody H4H14639D, performed according to the manufacturer's instructions (see Figure 2). The target binding equilibrium constant, i.e., the K of the DFO-Ab immunocomplex, was determined. Dを The analysis was performed using SPR (GE Healthcare, Biacore 8k), and bispecific antibody K D It was decided that it should be within 10%. [Table 3] [Table 4]

[0226] The bispecific antibody successfully bound to p-SCN-Bn-DFO via primary amine chemistry, as demonstrated 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. The SE-HPLC chromatogram showed a high monomeric product (99.6%) without detectable low molecular weight species. SDS-PAGE showed that the bispecific antibody remained unchanged after DFO binding.

[0227] Example 3: DFO-conjugated bispecific antibody 89 Zr Chelate To use ImmunoPET in an in vivo trial, we prepared a DFO-conjugated MET×MET bispecific antibody, H4H14639D, and a DFO-conjugated isotype control antibody. 89 It was labeled with Zr.

[0228] The DFO-conjugated antibody (250 ug) was initially divided into a total of 200 μL with 1 M HEPES, pH 7.4 (Teknova, catalog number: H1030). Separately, approximately 5 mCi (<150 uL) of the antibody was used. 89 A Zr-oxalic acid solution (3D Imaging, Little Rock AR) was neutralized and buffered to pH 6.8-7.4 to obtain a total volume of up to 1000 μL of 1 M HEPES, pH 7.4. DFO-Ab immunocomplexes and buffered 89 The Zr solution was combined and gently mixed with a pipette, then allowed to stand at room temperature for 45 minutes. Upon completion, the reaction mixture was rapidly buffered using a PD-10 column (GE Healthcare, catalog number: 17-0851-01) pre-prepared with 250 mM sodium acetate, pH 5.5 (Sigma-Aldrich, catalog number: 32319-1KG-R) according to the manufacturer's instructions. The concentration of the eluted product, called the DFO-Ab radioimmune complex, was measured at 280 nm and determined by UV absorption spectroscopy (Thermo Scientific NanoDrop 2000c, catalog number ND-2000c-US-CAN), calculated from the DFO contribution-adjusted primary sequence-based extinction coefficient using the following formula. Concentration in mg / mL = Absorption at 280 nm in AU ÷ 1.86 mL / mg 1 / cm

[0229] DFO-Ab radioimmunocomplexes 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 number: 5130-3215). Protein yield and specific activity of DFO-Ab radioimmunocomplexes were determined using the following equations: a. Protein yield in mg = concentration in mg / mL × mass of solution in grams b. SA in mCi / mg = Activity yield in mCi ÷ Mass of complex in mg

[0230] RCP, not incorporated 89 Zr and protein purity are measured using a Superdex 200 with an in-line UV280 absorption and gamma radiation detector (Agilent Technologies, Model 1260, consisting of Lablogic SCAN-RAM radioactive detectors) connected in series with a PBS mobile phase flow rate of 0.75 mL / min. The analysis was performed by stereoexclusion high-performance liquid chromatography (SE-HPLC) using an Increase 10 / 300 GL column (GE Healthcare, Cat. 28990944). Protein purity percentage was determined by comparing the relative integrals in the UV280 chromatogram of high molecular weight (HMW) species peaks (approximately 10–15 min) and the main peak (approximately 15–18 min). Low molecular weight species (approximately 18–25 min) were not observed. Radiation chromatography (gamma radiation) was used to identify unincorporated proteins. 89 Radiochemical purity was determined by a relative comparison of the Zr peak (approximately 25 minutes) and the integral of the main peak for %HMW species.

[0231] The IR of the DFO-Ab radioimmune complex was measured at 2.0 × 10⁷ cells / mL and 0.5 × 10⁷ cells. 7Cells / mL were measured by a cell binding assay requiring two 500 μL aliquots, A and B, of EBC-1 cells (JCRB number JCRB0820) in B. DFO-Ab radioimmuneconectote (20 ng) was added to aliquot A and incubated at 37°C in 5% CO2 for 45 minutes. Both aliquots A and B were centrifuged at 1500 rpm for 5 minutes (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 described above. Each cell pellet (A and B) was washed twice with 1 mL of fresh cell culture medium, centrifuged at 1500 rpm for 5 minutes between each wash. The supernatant was collected from the washes. The final activity of all components (500 μL of cell culture medium, supernatant, and each cell pellet resuspended in four wash supernatants) was measured using a gamma counter (Perkin Elmer Wizard2; model number 2470-0020). IR was determined by dividing the sum of the activities of both pellets by the sum of the activities of all components, multiplied by 100%. This process was tested against a nonspecific DFO-Ab radioimmune complex (n=1), and the IR was determined to be 2.8%.

[0232] The assay results (n=5) of the DFO-Ab radioimmune complexes generated above are recorded in Table 5. In particular, the mean RCP was 94.6±1.2%, indicating that it was not incorporated. 89 The Zr content was 2.7±1.9%, and the protein purity was 98.3±1.5%. Representative chromatograms are shown in Figures 3 and 4, respectively. The SA range was 11.9–21.3 mCi / mg, which was 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 bispecificity and DFO-bound METxMET bispecificity Equilibrium dissociation constant (K) for hMET.mmh binding to an anti-METxMET bispecific mAb (H4H14639D) or DFO (H4H14639D-DFO) bound to a purified anti-METxMET bispecific mAb (H4H14639D) or DFO (H4H14639D-DFO). D The value was determined using a real-time surface plasmon resonance biosensor with a Biacore T-200 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody to capture purified H4H14639D or H4H14639D-DFO. This Biacore binding test was performed in a buffer consisting of 0.01 M HEPES (pH 7.4), 0.15 M NaCl, and 0.05% v / v surfactant P20 (HBS-P electrophoresis buffer). Different concentrations of hMET with a C-terminal myc.myc hexahistidine tag, prepared in HBS-ET electrophoresis buffer, were injected into the captured antibody at a flow rate of 50 μL / min. Binding of hMET.mmh to the captured monoclonal antibody was monitored for 5 minutes, and dissociation of hMET.mmh in HBS-ET electrophoresis buffer was monitored for 10 minutes. The coupling dynamics experiment was conducted at 25°C. (Scubber) By fitting real-time sensorgrams to a 1:1 coupled model using 2.0c curve fitting software, dynamic association (K a ) and dissociation (K d The rate constant was determined. The bond dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 The following velocity constants were used to calculate the following: K D (M=k) d / k a , and t 1 / 2 (min)=0.693 / k d / 60

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

[0235] Example 5: In vivo distribution of radiolabeled MET×MET bispecific antibodies Different tumor xenografts were selected for imaging in immunodeficient mice based on MET expression levels: EBC1 (high MET), NCI-H441 (moderate MET), and NCI-H358 (low MET). 5 × 10⁶ SCID mice were subjected to imaging. 6 The tumor cells were transplanted and allowed to grow for 10-14 days. Then, mice were given 0.1 mg / kg of 89 Zr-DFO-H4H14639D was administered, and the amount of unlabeled, unbound antibody was increased to achieve final protein doses of 0.1, 0.5, and 5 mg / kg. Control animals received 0.1 mg / kg 89 A Zr-DFO isotype control antibody and an unlabeled, unbound antibody were administered at a final protein dose of 0.5 mg / kg. PET imaging was performed on days 0, 1, 4, and 6. In vivo distribution was observed on day 6.

[0236] PET / CT images were acquired using the Sofie Biosciences G8 PET / CT (Sofie Biosciences and Perkin Elmer). Before image acquisition, the instrument was used. 89 Pre-calibration was performed for Zr detection. The energy window ranged from 150 to 650 keV with a reconfigured resolution of 1.4 mm at the center of the field of view. Mice, induced anesthetized with isoflurane, were held under a continuous flow of isoflurane during imaging. Static 10-minute images were acquired using G8 acquisition software and then reconstructed using pre-configured settings. Image data was corrected for attenuation and other parameters. CT images were acquired after PET acquisition and then aligned with the PET images. Images were processed using VivoQuant post-processing software (inviCRO). Created using Imaging Services.

[0237] For the biological distribution test, mice were ( 89Six days after administration of Zr-DFO-H4H14639D, the animals were euthanized, and blood was collected via cardiac puncture. Tumor and normal tissue were excised, placed in a counter, and weighed. Then, in CPM... 89 Zr counting data was collected by measuring samples on an automated gamma counter (Wizard 2470, Perkin Elmer). The percentage of gram per injection dose (%ID / g) was calculated for each sample using standards prepared from the injection material.

[0238] The imaging results are: 89 Zr-DFO-H4H14639D was shown to be specifically localized to MET-expressing tumor xenografts (Figures 5-7), which is further demonstrated by in vivo distribution data. 89 The blocking doses for Zr-DFO-H4H14639D are 0.1 and 0.5 mg / kg. 89 Compared to low doses of Zr-DFO-H4H14639D, increased blood uptake (% ID / g) and decreased tumor uptake (% ID / g) were observed in NCI-H441 (medium MET) and NCI-H358 (low MET) tumors (Figure 8). 89 Overall tumor uptake of Zr-DFO-H4H14639D shows a good correlation with relative MET expression (Figure 9).

[0239] Example 6: MET x MET bispecific antibody binding ability (ABC) by saturated 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 89Antibody binding (ABC) assays were performed in EBC-1, NCI-H441, and NCI-H358 cell lines using Zr-DFO-H4H14639D as a radioligand. Radioligand preparation (DFO antibody binding and subsequent Zr 89 Examples of (radioactive labeling) are described above. All six experiments were carried out in a similar manner with the help of the Hamilton-Stallett liquid processing system. Briefly, cells were first collected, analyzed for viability (confirmed to be >90% by trypan blue exclusion staining), and then placed in complete medium in a 2-5 x 10⁶ state. 6 This yielded cells / mL. Next, 100µL (200,000-500,000 cells, N) was added along overlapping or quadrupled rows in a V-bottom 96-well plate ("cell plate"). c Alicocoated the radiolabeled antibody. The cell plates were kept at 4°C until the radiolabeled antibody was administered in a later step. In a second V-bottom 96-well plate ("mAb plate"), 150 μL of radiolabeled antibody (0.10–0.12 mg / mL) was alicocoated across rows of column 1 and single wells of column 12 as an internal transfer control. Columns 2–9 were then serially diluted with cold medium at a factor of 2.8. Next, 50 μL of the mAb plate was pressed onto the cell plates. The cell plates were incubated at 4°C for 45 minutes with gentle agitation. After incubation, the wells from columns 1–9 were first gently mixed by pipette to create a rapid cell suspension, and then 30 μL (or 20% of the total) was collected from each well into flip-cap tubes representing the total amount of antibody administered. The remaining cell plates were centrifuged at 150 g for 5 minutes, and the supernatant was removed and discarded. Next, the cell plate was washed with 200 μL of cold medium, rotated again in five aspiration / dispensing cycles, then the supernatant was removed and discarded. After repeating this washing process two more times, the cells were resuspended in 200 μL of cold buffer (10% FBS in PBS, v / v). Of the 200 μL, 180 μL was collected from each well and dispensed into flip-cap tubes representing cell-binding antibodies. The activity of the total antibody sample was measured using a gamma counter (Hidex Automatic). Measurements were taken using a Gamma Counter (model 425-601). Based on serial dilutions (divided into 5 parts for sampling) starting from the initial antibody concentration, a calibration curve was created from the total antibody dose samples using the number of pairs of antibodies detected per T. Using the calibration curve, the number of conjugated fractions (multiplied by 1.38 for sampling) was converted to the mass (or concentration) of conjugated antibody B.

[0240] Internal transition comparison Internal transfer of radioactive ligands was also analyzed at the highest dose concentration for 45 minutes at 4°C. If significant, the fraction of internally transferred antibody was used to scale ABC accordingly. For the internal transfer control, the well content (200 μL) from terminal column 12 was 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 minutes. After 10 minutes of incubation, the Eppendorf tube was centrifuged at 150 g for 5 minutes. The supernatant was removed without disturbing the cell pellet and set aside for counting. The cell pellet was washed with 1 mL of cold buffer (10% FBS in PBS, v / v), and the supernatant was aspirated, spun, and removed 10 times during the wash. The activity of the supernatant after cell pellet removal and the washed supernatant was measured using a gamma counter. The fractions were internalized and calculated as the ratio of the cell pellet activity to the sum of the total activities of I, cell pellet, removed supernatant, and washed supernatant.

[0241] ABC of Comp1: 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 / washing protocol, nonspecific binding was determined to be a negligible component for all runs and was not considered as part of the analysis. Internal migration was determined to have a small contribution to the bound radioactivity and was also not considered as part of the analysis.

[0242]

number

[0243] Formula 2:

number

[0244] Since H4H14639D has multiple binding forms, the dataset was fitted using the Hill-Langmuir equation (Equation 3). The 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. Internal transfer of radioactive ligands was determined to have a non-negligible contribution to the determination of the bound radiation dose and was therefore compensated for.

[0245] Equation 3 (regarding 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] The ABC value using Comp1, an anti-Met, single-arm / monovalent format antibody, is a reasonable estimate of the Met receptor copy number (i.e., a 1:1 antibody against the receptor). However, the ABC value using the antibody H4H14639D is not expected to deductively indicate a 1:1 Met receptor copy number.

[0248] The embodiments and examples described above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize or confirm them by using only common experiments, specific compounds, materials, and numerous equivalents of procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims. The present invention provides, for example, the following items: (Item 1) A radiolabeled antibody conjugate comprising an antibody that binds to MET or its antigen-binding fragment, a chelate moiety, and a positron emitter. (Item 2) The complex comprises an antibody or antigen-binding fragment that binds to MET, and the antibody or antigen-binding fragment is covalently bound to one or more parts of formula (A), -LM Z (A) The complex described in item 1, wherein L is a chelate, M is a positron emitter, and z is independently 0 or 1 in each occurrence, with at least one of z being 1. (Item 3) The complex according to item 1 or item 2, wherein the chelate portion contains desferrioxamine. (Item 4) The aforementioned positron emitter 89 A complex that is Zr, as described in any of items 1-3. (Item 5) -LM, [ka] In the formula, Zr is the positron emitter 89 A complex that is Zr, as described in any of items 1-4. (Item 6) A complex according to any one of items 1 to 5, wherein an antibody or its antigen-binding fragment is covalently bound to one, two, or three parts of formula (A). (Item 7) The aforementioned antibody is as follows: (i) comprising 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 an HCVR having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, (ii) comprising an LCVR having the amino acid sequence of SEQ ID NO: 138, or a substantially similar sequence 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR3 domain having the amino acid sequence of SEQ ID NO: 144, or a substantially similar sequence 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 an HCDR1 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, or 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 at least 90%, at least The present invention includes an HCDR2 domain having a substantially similar sequence with 95%, at least 98%, or at least 99% sequence identity, the amino acid sequence of SEQ ID NO: 140, or an LCDR1 domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR2 domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. (v) A multispecific antigen-binding molecule that includes a first binding specificity to MET and a second binding specificity to tumor-specific antigens, (vi) A multispecific antigen-binding molecule that includes a first binding specificity to one epitope of MET and a second binding specificity to a second epitope of MET, (vii) When measured by surface plasmon resonance at 25°C or 37°C, K is less than approximately 230 nM D It binds to monomeric human MET (e.g., hMET.mmh), (viii) When measured by surface plasmon resonance at 25°C or 37°C, K is less than approximately 3 nM. D Then it binds to the dimer human MET, (ix) Block the binding of HGF to MET, and (x) A complex according to any one of items 1 to 6, having one or more properties selected from the group consisting of (x) suppressing tumor growth and increasing survival in subjects with cancer. (Item 8) The complex according to any one of items 1 to 7, wherein the antibody comprises three heavy chain complementarity-determining regions (HCDRs) in its heavy chain variable region (HCVR), the 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, and the light chain variable region (LCVR) comprises three light chain complementarity-determining regions (LCDRs), the LCVR having the LCVR amino acid sequence of SEQ ID NO: 138. (Item 9) The antibody is a complex according to any one of items 1 to 8, wherein the antibody comprises three CDRs in the HCVR of SEQ ID NO: 18. (Item 10) The antibody is a complex according to any one of items 1 to 8, wherein the antibody comprises three CDRs in the HCVR of SEQ ID NO: 58. (Item 11) The antibody is a complex according to any one of items 1 to 8, wherein the antibody comprises three CDRs in the HCVR of SEQ ID NO: 82. (Item 12) The antibody is a complex according to any one of items 1 to 11, wherein the antibody comprises three CDRs in the LCVR of SEQ ID NO: 138. (Item 13) The aforementioned antibody (i) the first antigen-binding domain (D1), and (ii) comprising a second antigen-binding domain (D2), D1 specifically binds to the first epitope of human MET. D2 is a complex described in any one of items 1-12 that specifically binds to the second epitope of human MET. (Item 14) (i) D1 contains the CDR in the HCVR amino acid sequence of SEQ ID NO: 58, (ii) The complex according to item 13, wherein D2 contains the CDR in the HCVR amino acid sequence of sequence number 82. (Item 15) (i) D1 contains the set HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of the amino acid sequence 60-62-64-140-142-144, (ii) The complex described in item 13, wherein D2 contains the set HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of the amino acid sequence 84-86-88-140-142-144. (Item 16) A method for imaging tissue expressing MET, comprising administering a radiolabeled antibody complex described in any one of items 1 to 15 to the tissue, and visualizing MET expression by positron emission tomography (PET) imaging. (Item 17) A method for identifying a target MET-expressing tumor, comprising administering a radiolabeled antibody complex described in any one of items 1 to 15 to the target, and imaging the radiolabeled antibody complex via positron emission tomography (PET), wherein the localization of the radiolabeled antibody complex in the target indicates a MET-expressing tumor. (Item 18) A method for treating a subject with a solid tumor, the following: (a) Determine that the solid tumor is MET-positive, (b) A method comprising administering one or more doses of the HGF / MET signaling pathway inhibitor to the subject in need thereof. (Item 19) Step (a) is as follows: (i) Administering a radiolabeled antibody conjugate as described in any one of items 1 to 15 to the subject in need thereof. (ii) The method of item 19, comprising imaging the localization of the radiolabeled antibody complex in the tumor by positron emission tomography (PET) imaging, wherein the presence of the radiolabeled antibody complex in the tumor indicates that the tumor is MET-positive. (Item 20) The method according to item 19, wherein the subject is administered 0.1 to 10 mg / kg of the radiolabeled antibody conjugate. (Item 21) The method according to item 19 or item 20, wherein the radiolabeled antibody conjugate is administered to the subject subcutaneously or intravenously. (Item 22) The method according to 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) The method according to any one of items 18 to 22, wherein step (a) is performed before treating the subject with an inhibitor of the HGF / MET signaling pathway. (Item 24) below: (a) After treating the subject with at least one dose of an inhibitor of the HGF / MET signaling pathway, administer the radiolabeled antibody conjugate. (b) The method according to any one of items 18 to 23, further comprising imaging the localization of the radiolabeled antibody complex in the tumor by PET imaging, wherein a decrease from baseline in the area of ​​localization of the radiolabeled antibody complex in the tumor indicates tumor reduction. (Item 25) The method according to 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) The method according to 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, liver cancer, lung cancer (e.g., non-small cell lung cancer [NSCLC]), lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, synovial sarcoma, thyroid cancer, and Wilms' tumor. (Item 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) The method according to 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) The method according to any one of items 18 to 28, wherein the inhibitor of the HGF / MET signaling pathway is a MET × MET bispecific antibody or its antigen-binding fragment. (Item 30) The MET×MET bispecific antibody or its antigen-binding fragment is as follows: (i) The HCVR amino acid sequence of Sequence ID No. 58 contains the first antigen-binding domain (D1) comprising the CDR, and (ii) The method according to item 29, wherein the HCVR amino acid sequence of sequence number 82 includes a second antigen-binding domain (D2) containing the CDR. (Item 31) The method according to item 30, wherein the MET×MET bispecific antibody or its antigen-binding fragment contains the CDR in the LCVR amino acid sequence of SEQ ID NO: 138. (Item 32) The method according to item 30, wherein D1 contains the heavy chain variable region (HCVR) of SEQ ID NO: 58 and D2 contains the HCVR of amino acid SEQ ID NO: 82. (Item 33) A compound of formula (III), [ka] A compound in which A is an antibody or antigen-binding fragment that binds to MET, and k is an integer from 1 to 30. (Item 34) The compounds listed in item 33, wherein k is 1 or 2. (Item 35) (i) an antibody or its antigen-binding fragment that binds to MET, and (ii) an antibody complex comprising one or more chelate moieties. (Item 36) The chelate portion is as follows: [ka] During the ceremony, [ka] The antibody complex described in item 35, wherein the bond is covalent to the antibody or its antigen-binding fragment. (Item 37) The antibody complex according to item 35 or item 36, wherein the complex has a chelate portion for an antibody of 1.0 to 3.0. (Item 38) The antibody conjugate described in any one of items 35 to 37, wherein the chelate portion versus antibody ratio is approximately 1.3.

Claims

[Claim 1] The invention as shown in the drawings.

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