Drug conjugates of cMET monoclonal binding agents and their uses

Abstract

Disclosed herein are novel monoclonal cMET-binding agents conjugated with pyrrolobenzodiazepine toxins, compositions thereof, and uses thereof for treating cancer.

Description

Technical Field

[0001] Some embodiments of the present invention relate to pyrrolobenzodiazepines Toxin (pyrrolobenzodiazepinetoxin) conjugated cMET binding agents, compositions thereof and uses thereof. Background Art

[0002] cMET protein (sometimes referred to as MET or hepatocyte growth factor receptor (hepatocyte growth factor receptor, HGFR)) is a protein encoded by the MET gene (MET proto-oncogene, receptor tyrosine kinase) in humans. cMET is a one-way cell surface receptor with tyrosine kinase activity. The primary single-chain precursor protein of the MET translation product is cleaved after translation to produce α and β subunits, which are disulfide-linked to form mature cell surface cMET receptors. cMET is expressed in cells of epithelial origin as well as stem cells, progenitor cells and other cell types (e.g., various cancer cell types). Hepatocyte growth factor / scatter factor (hepatocyte growth factor / Scatter Factor, HGF / SF) and its splicing isoforms (NK1, NK2) have been identified as ligands for cMET.

[0003] cMET is considered to be essential for normal embryonic development, organogenesis and wound healing. Abnormal cMET expression and / or activity is associated with certain neoplastic disorders and cancers (e.g., renal cancer, liver cancer, gastric cancer, breast cancer and brain cancer), where cMET is involved in tumor growth, angiogenesis and metastasis. Overexpression of cMET and its autocrine activation through co-expression of its ligands are also involved in tumorigenesis.

[0004] Provided herein are novel anti-cMET binding agents (eg, monoclonal antibodies) conjugated to a cytotoxic payload, pharmaceutical compositions thereof, and methods of use thereof. Summary of the invention

[0005] In some aspects, provided herein are binder-drug conjugates comprising a binder and a payload, wherein the payload comprises a pyrrolobenzodiazepine toxin, and the binding agent specifically binds to mesenchymal epithelial transition factor (cMET). In some embodiments, the payload comprises a linking group, wherein the pyrrolobenzodiazepine The toxin is covalently linked to a linker, and the linker is covalently linked to a binding agent. In some embodiments, the binding agent is a monoclonal antibody or an antigen-binding portion thereof.

[0006] In some aspects, provided herein are binding agent-drug conjugates comprising a binding agent and a payload, wherein the binding agent comprises: (i) two or more of CDR-L1, CDR-L2, and CDR-L3 which are polypeptide sequences of light chain complementarity determining regions (CDR-L), wherein CDR-L1 is selected from the amino acid sequences of SEQ ID NO: 1 to 15, CDR-L2 is selected from the amino acid sequences of SEQ ID NO: 16 to 25, and CDR-L3 is selected from the amino acid sequences of SEQ ID NO: 26 to 36, and (ii) two or more of CDR-H1, CDR-H2, and CDR-H3 which are polypeptide sequences of heavy chain complementarity determining regions (CDR-H), wherein CDR-H1 is selected from the amino acid sequences of SEQ ID NO: 50 to 61, CDR-H2 is selected from the amino acid sequences of SEQ ID NO: 62 to 78, and CDR-H3 is selected from the amino acid sequences of SEQ ID NO: 79 to 93; and the payload comprises pyrrolobenzodiazepine toxin and a linker; wherein the pyrrolobenzodiazepine toxin is covalently linked to the linker, the linker is covalently linked to the binding agent, and the binding agent specifically binds to the extracellular domain of mesenchymal epithelial transition factor (cMET).

[0007] In certain embodiments, the pyrrolobenzodiazepine toxin comprises a structure of Formula I:

[0008] [Chemical 1]

[0009]

[0010] Wherein:

[0011] Both Z1 and Z2 are N;

[0012] Both Z3 and Z4 are C;

[0013] [Chemical 2]

[0014] Double dashed line represents a single bond or a double bond;

[0015] n is from 1 to 12;

[0016] R3 and R4 are each independently H or C 1-4 alkoxy; and

[0017] R1 and R2 are each independently selected from H, C 1-5 alkyl, C3-6 cycloalkyl, C 2-5 alkenyl, and phenyl optionally substituted by R5,

[0018] wherein:

[0019] R5 is selected from -NH2, -NHR6, and a piperazinyl group substituted by R7 having the following structure:

[0020] [Chemical formula 3]

[0021]

[0022] R6 contains a linking group, and

[0023] R7 is H or C 1-5 alkyl;

[0024] X1 is empty, a protecting group, or contains a linking group;

[0025] X2 is empty, a protecting group, or contains a linking group;

[0026] only one of X1, X2, R1, and R2 contains a linking group; and

[0027] Y1 and Y2 are each independently empty, OH, or SO3H;

[0028] provided that:

[0029] [Chemical formula 4]

[0030] (i) when X1 contains a linking group, Z1 Z3 is N-C,

[0031] (ii) when X2 contains a linking group, Z2 Z4 is N-C,

[0032] (iii) when X1 contains a protecting group, Z1 Z3 is N-C, and

[0033] (iv) when X2 contains a protecting group, Z2 Z4 is N-C,

[0034] where empty indicates the absence of that moiety or the presence of one or more hydrogens to satisfy the required valency.

[0035] In some embodiments of the pyrrolobenzodiazepine toxins having the structure of Chemical formula I, n is 3 or 5. In some embodiments of the pyrrolobenzodiazepine toxins having the structure of Chemical formula I, both R3 and R4 are -O-CH3. In some embodiments of the pyrrolobenzodiazepine In certain embodiments of the toxin, both R1 and R2 are methyl or both R1 and R2 are -CH=CH-CH3. In the pyrrolobenzodiazepine having the structure of Formula I In some embodiments of the toxin, R2 is cyclopropyl. In the pyrrolobenzodiazepine having the structure of Formula I In certain embodiments of the toxin, R2 is a phenyl group substituted with 4-methylpiperazin-1-yl or a phenyl group substituted with R5, where R5 is -NHR6 and R6 contains a linker.

[0036] [Chemical Formula 5]

[0037] In the pyrrolobenzodiazepine having the structure of Formula I In some embodiments of the toxin, X1 is empty, Y1 is empty, Z1 Z3 is N=C, X2 is empty, Y2 is empty, and Z2 Z4 is N=C. In the pyrrolobenzodiazepine having the structure of Formula I In certain embodiments of the toxin, X1 contains a linker, Y1 is OH, Z2 Z4 is N=C, X2 is empty, and Y2 is empty. In the pyrrolobenzodiazepine having the structure of Formula I In certain embodiments of the toxin, X1 contains a linker, Y1 is OH, Z2 Z4 is N-C, X2 is a protecting group, and Y2 is OH.

[0038] In the pyrrolobenzodiazepine having the structure of Formula I In certain embodiments of the toxin, the linker is connected to the pyrrolobenzodiazepine through a carbamate group or an amide group toxin.

[0039] In certain embodiments, the linker contains the structure of Formula A:

[0040] [Chemical Formula 6]

[0041]

[0042] where the asterisk indicates the point of connection to the pyrrolobenzodiazepine toxin; the wavy line indicates the point of connection to the binder; m is from 1 to 20; q is from 0 to 10; and E is a linker. In certain embodiments of the linker of Formula A, m is 4 or 8 and q is 0, 1, or 2. In some embodiments, m is 8 and q is 2.

[0043] In certain embodiments, the linker contains the structure of Formula B:

[0044] [Chemical Formula 7]

[0045]

[0046] wherein the asterisk indicates the point of attachment to the pyrrolobenzodiazepine toxin; the wavy line indicates the point of attachment to the binder; E comprises a linker; v is from 0 to 10; and u is 0 or 1; wherein when u is 1, t is from 1 to 10. In certain embodiments of the linker of Formula B, v is 1. In certain embodiments of the linker of Formula B, u is 1 and t is 8. In certain embodiments of the linker of Formula B, u is 0 and v is 4.

[0047] In certain embodiments of the linkers of Formulas A and B, the binder is linked to E by a thioether bond formed between a cysteine thiol residue of the binder and E. In some embodiments, E comprises the structure of Formula C:

[0048] [Chemical Formula 8]

[0049]

[0050] wherein the wavy line indicates the point of attachment to the binder and the double asterisk indicates the point of attachment to the linker.

[0051] In some embodiments of the pyrrolobenzodiazepine toxin having the structure of Formula I, the protecting group has the following structure (D):

[0052] [Chemical Formula 9]

[0053]

[0054] wherein the asterisk indicates the point of attachment to the pyrrolobenzodiazepine toxin; and w is from 1 to 5. In some embodiments, w is 2. In some embodiments, the protecting group is a cleavable protecting group.

[0055] In certain aspects, provided herein are binder-drug conjugates comprising a binder and a payload, wherein the binder specifically binds to the extracellular domain of mesenchymal epithelial transition factor (cMET), the payload is covalently linked to the binder, and the payload comprises a structure selected from Formulas II, III, V, VI, and VII, wherein Formula II comprises the following structure:

[0056] [Chemical Formula 10]

[0057]

[0058] wherein m is 8 and the wavy line indicates the point of attachment to the binder;

[0059] Formula III comprises the following structure:

[0060] [Chemical Formula 11]

[0061]

[0062] Where m is 8, p is 2 or 3, X2 is a protecting group, and the wavy line indicates the connection point to the binder;

[0063] Chemical formula V contains the following structure:

[0064] [Chemical Formula 12]

[0065]

[0066] Where m is 8, and the wavy line indicates the connection point to the binder;

[0067] Chemical formula VI contains the following structure:

[0068] [Chemical Formula 13]

[0069]

[0070] Where t is 8, v is 1, and the wavy line indicates the connection point to the binder;

[0071] And chemical formula VII contains the following structure:

[0072] [Chemical Formula 14]

[0073]

[0074] Where the wavy line indicates the connection point to the binder.

[0075] In certain embodiments, the protecting group of X2 has the following structure (D):

[0076] [Chemical Formula 15]

[0077]

[0078] Where the asterisk indicates the connection point to the payload; and w is from 1 to 5.

[0079] In certain aspects, provided herein are conjugate-drug conjugates comprising a monoclonal antibody or an antigen-binding portion thereof and a payload, wherein the monoclonal antibody or the antigen-binding portion thereof comprises a CDR-L1 comprising an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, and 14; a CDR-L2 comprising an amino acid sequence selected from SEQ ID NO: 17, 19, 21, 23, and 25; a CDR-L3 comprising an amino acid sequence selected from SEQ ID NO: 27, 29, 31, 33, and 35; a CDR-H1 comprising an amino acid sequence selected from SEQ ID NO: 51, 53, 55, 57, and 59; a CDR-H2 comprising an amino acid sequence selected from SEQ ID NO: 63, 65, 67, 69, 73, and 75; and a CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 80, 82, 84, 86, 88, 91, and 93.

[0080] In certain embodiments, the conjugate of the conjugate-drug conjugate described herein comprises: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10 or 14; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 71; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 88.

[0081] In certain embodiments, the conjugate of the conjugate-drug conjugate described herein comprises: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9 or 15; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 24; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70 or 78; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87.

[0082] In some embodiments, the binder comprises a light chain variable region comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 37 to 44. In some embodiments, the binder comprises a light chain variable sequence having at least 90% or 100% sequence identity to any one of the amino acid sequences of SEQ ID NO: 45 to 49. In some embodiments, the binder comprises a light chain variable sequence having at least 90% sequence identity to any one of SEQ ID NO: 37 to 49, wherein the light chain variable sequence has 1 to 10 or 1 to 5 amino acid modifications selected from amino acid addition, amino acid deletion, and amino acid substitution.

[0083] In some embodiments, the binder comprises a heavy chain variable region comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 94 to 103. In some embodiments, the binder comprises a heavy chain variable region having at least 90% or 100% sequence identity to any one of the amino acid sequences of SEQ ID NO: 104 to 108. In some embodiments, the binder comprises a heavy chain variable region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NO: 94 to 108, wherein the heavy chain variable sequence has 1 to 10 or 1 to 5 amino acid modifications selected from amino acid addition, amino acid deletion, and amino acid substitution.

[0084] In certain embodiments, the binder comprises a light chain variable sequence having at least 90% sequence identity to any one of SEQ ID NO: 37 to 49, and a heavy chain variable sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NO: 94 to 108.

[0085] In certain embodiments, the binder, monoclonal antibody, or antigen-binding portion thereof is chimeric or humanized. For example, in some embodiments, the binder comprises one or more humanized or human framework regions and / or one or more murine framework regions.

[0086] In certain embodiments, the binder, monoclonal antibody, or antigen-binding portion thereof comprises: a heavy chain and a light chain, each having CDRs identical to the corresponding CDRs of the heavy chain and light chain of the antibody produced by the hybridoma cell line F6B1P3D12 deposited with the ATCC on March 20, 2019.

[0087] In some embodiments, the binder that specifically binds to cMET or a portion thereof comprises the antigen-binding portion of an antibody or a single-chain antibody. For example, in certain embodiments, the binder of the binder-drug conjugate comprises the Fab, Fab’, F(ab’)2, Fv, or scFV fragment of an antibody.

[0088] In certain embodiments, the binding agent specifically binds to mammalian cMET. In certain embodiments, the binding agent specifically binds to human cMET, monkey cMET, and / or rat cMET. In some embodiments, the binding agent specifically binds to the extracellular domain of wild-type or variant cMET. In some embodiments, the binding agent that specifically binds to cMET is a binding agent that induces internalization and / or degradation of cMET on human cancer cells. In some embodiments, the binding agent that specifically binds to cMET is a binding agent that is not a cMET agonist. Thus, a cMET binding agent that is not a cMET agonist is a binding agent that, upon binding to cell surface cMET, substantially does not induce signal transduction through cell surface cMET.

[0089] In certain aspects, provided herein are pharmaceutical compositions comprising a binding agent-drug conjugate as described herein, and a pharmaceutically acceptable excipient, diluent, additive, or carrier.

[0090] In certain aspects, provided herein are methods of treating a subject having a neoplastic disorder or cancer, the method comprising administering to the subject having or suspected of having a neoplastic disorder or cancer a therapeutically effective amount of a binding agent-drug conjugate as described herein. In certain embodiments, the binding agent-drug conjugate blocks, inhibits, ameliorates, eliminates, or retards the growth, viability, or metastasis of cancer. In certain embodiments, the binding agent-drug conjugate induces death, necrosis, or apoptosis of some or all of the cancer. In certain embodiments, the neoplastic disorder or cancer comprises carcinoma, sarcoma, neuroblastoma, glioblastoma, myeloma, lymphoma, melanoma, or solid or soft tissue tumors. In certain embodiments, the neoplastic disorder or cancer comprises bladder cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer (e.g., exocrine pancreatic cancer and pancreatic neuroendocrine cancer), esophageal cancer, liver cancer, hepatocellular carcinoma, hypopharyngeal cancer, lung cancer, adenocarcinoma, ovarian cancer, or kidney cancer. In certain embodiments, the neoplastic disorder or cancer comprises pancreatic adenocarcinoma, colorectal adenocarcinoma, small intestinal malignancy, cholangiocarcinoma, non-small cell lung cancer (NSCLC), thyroid cancer, esophageal or esophagogastric junction (EGJ) cancer, gastric adenocarcinoma, hepatocellular carcinoma of the liver, head and neck squamous cell carcinoma, female genital tract malignancy, breast cancer, small cell lung cancer, ovarian surface epithelial carcinoma, retroperitoneal or peritoneal sarcoma, prostatic adenocarcinoma, neuroendocrine tumor, gastrointestinal stromal tumor, glioblastoma, or non-epithelial ovarian cancer. In certain embodiments, the cancer that can be treated by the methods described herein is a cancer comprising cells (e.g., malignant or neoplastic cells) that express a cMET polypeptide (e.g., cMET, e.g., expressed on the cell surface).

[0091] Certain aspects of the technology are further described in the following description, examples, claims, and figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] The figures illustrate embodiments of the technology and are not restrictive. For clarity and ease of illustration, the figures are not drawn to scale, and in some instances, multiple aspects may be shown in an exaggerated or enlarged manner to facilitate understanding of a particular embodiment.

[0093] Figure 1 Figure 1 Shows a summary of the workflow for generating monoclonal antibodies (exemplary binders) that specifically bind to cMET. Lead monoclonal Ab P3D12 was generated from mice immunized with the recombinant full extracellular domain of cMET fused to human Fc.

[0094] Figure 2 Figure 2 Shows the immunization protocol for generating monoclonal antibodies (exemplary binders) that specifically bind to cMET. As indicated, mice were initially immunized by intraperitoneal injection (i.p.) with 100 μg of human cMET-Fc fusion protein (cMET-Fc) in Freund's complete adjuvant (CFA) or 50 to 100 μg of cMET peptide conjugated to KLH. cMET-Fc contains the extracellular domain of human cMET fused to the Fc portion of the antibody. The cMET peptides were strategically selected from a portion of the cMET extracellular domain. As indicated, the immunized mice received one or more booster immunizations containing 25 or 50 μg of cMET-Fc or peptide in incomplete Freund's adjuvant (IFA). Some mice received multi-site repeated immunizations (RIMMS). The immunizations included Met-Fc fusions, peptides, conventional, and RIMMS. Spleens from the immunized mice were obtained and fused with a suitable fusion partner. Over 20,000 hybridoma clones were obtained and screened.

[0095] Figure 3 Figure 3 Shows the 3D structure of the MET SEMA domain binding to the Fab (5D5 Fab) of the agonist Met-mAb antibody and the HGFβ subunit (HGFβ or HGFb). The bottom arrow indicates the position of the portion of cMET used to design peptide 3.

[0096] Figure 4 Figure 4 ​​​​​​​​Shows the characterization results from an exemplary fusion (Fusion 6B1, plate 3). Anti-cMET hybridomas were selected, in part, based on the presence of specific binding to cMET as determined by ELISA (see column labeled "MET Binding ELISA OD450nm") and the ability to induce cMET internalization on human cancer cell lines as measured by flow cytometry (see column labeled "FACS Geometric Mean"). FACS geometric means below the negative control indicate cMET internalization. The arrow indicates the lead hybridoma F6B1P3D12.

[0097] Figure 5 Figure 5 Shows the results of the MET degradation assay. Anti-cMET antibodies isolated from the indicated wells (x-axis) were tested and selected based on their ability to induce degradation of cMET on human cancer cell lines as measured by Mesoscale (MSD) cMET protein quantification. The relative values of Met degradation are indicated on the y-axis as % control (percentage of control). Values below 100% control (negative control level) indicate cMET internalization and degradation. Degradation indicates not only internalization but also lysosomal trafficking, which is an important property of antibody-drug conjugates. The arrow indicates the results for the lead hybridoma F6B1P3D12.

[0098] Figure 6 Figure 6 Shows the results of the phosphorylation-ERK assay (P-ERK assay), which measures the agonist activity of anti-cMET antibodies by indirectly measuring the phosphorylation of ERK induced by the binding of anti-cMET antibodies to cMET on the cell surface. The amount of phosphorylated ERK detected in cell lysates after treatment of live cells with anti-cMET antibodies is shown (as % of control, y-axis). Anti-cMET antibodies generated from a variety of anti-cMET hybridomas (x-axis) were tested at 6 μg / ml or 30 μg / ml (as indicated on the x-axis) and selected based on their inability to induce significant phosphorylation of ERK (i.e., inability to induce proliferation, i.e., lack of agonist activity). The arrow indicates the lead monoclonal antibody (mAb) P3D12.

[0099] [Figure 7-1] Figure 7 shows the results of binding of six cMET monoclonal antibodies (mAbs) to human, monkey (Cynomolgus Macaque, "Cyno"), dog, rat, and mouse cMET as determined by ELISA. All monoclonal antibodies bind to human and monkey cMET. P3D12 shows significant cross-reactivity with rat cMET. Multiple concentrations of each antibody are indicated on the x-axis. The relative binding amount (OD450 nm) is indicated on the y-axis. Figure 7-1 provides the results for three monoclonal antibodies.

[0100] ​​​​[Figure 7-2] Figure 7-2 provides the results of three additional monoclonal antibodies.

[0101] Figure 8 Figure 8 A shows the alignment of the amino acid sequences of the light chain variable regions of nine mouse monoclonal anti-cMET antibodies, whose names are shown to the left of each sequence. The SEQ ID NO: is shown to the right of each sequence. The amino acid sequences of the light chain variable regions of LC F6B1P1E2 and F6BP3E2 are 100% identical. Additionally, the amino acid sequences of the light chain variable regions of LC F6B1P3D12 and F6B1P3E9 are 100% identical.

[0102] Figure 8 B shows the alignment of the amino acid sequences of the heavy chain variable regions of nine mouse monoclonal anti-cMET antibodies, whose names are shown to the left of each sequence. The SEQ ID NO: is shown to the right of each sequence. The amino acid sequences of the heavy chain variable regions of F6B1P3D12H7913 and F6B1P3E9 are 100% identical. Additionally, the amino acid sequences of the heavy chain variable regions of F6B1P1E2H7819 and F6BP3E2 are 100% identical.

[0103] Figure 9 Figure 9 A and 9B show the results of in vivo xenograft mouse models evaluating the potency of the indicated anti-cMET antibody-drug conjugates (ADCs) using the MKN45 tumor model (cMET+ gastric cancer model) in nude mice. Animals were treated once with 2.5 mg / kg (9A) or 5.0 mg / kg (9B) of the ADC. The potency of each drug-conjugated anti-cMET binder was compared to PBS or an irrelevant non-targeting monoclonal antibody (IgG-ADC). Tumor volume (y-axis) was measured at multiple time points (y-axis, days (post-inoculation)) after inoculation. Inhibition of tumor growth indicates positive potency. The anti-cMET binders and non-targeting control monoclonal antibody (IgG) were conjugated to monomethyl auristatin F (MMAF).

[0104] Figure 10 Figure 10 ​​​​​​Shows the results of a phospho-ERK assay (P-ERK assay), which measures the agonist activity of an anti-cMET antibody by indirectly measuring the phosphorylation of ERK induced by the binding of the anti-cMET antibody to cMET on the cell surface. Shown is the amount of phosphorylated ERK detected in cell lysates (shown as % of control, y-axis) after treatment of live cells with the anti-cMET antibody. As indicated on the x-axis, the constant regions of the heavy and light chains of an isolated murine monoclonal antibody designated P3D12 were replaced with the antibody constant regions of human IgG1 (P3D12(hIgG1)) or the antibody constant regions of human IgG2 (P3D12(hIgG2)). As indicated on the x-axis, each antibody was tested at 0.00064 μg / ml, 0.0032 μg / ml, 0.016 μg / ml, 0.08 μg / ml, 0.4 μg / ml, 2 μg / ml, 10 μg / ml, and 50 μg / ml. "Control" represents an untreated negative control. HGF (EC90) is a positive control and represents cells treated with hepatocyte growth factor (HGF), the natural ligand of the cMET receptor. This data indicates that the human IgG2 isotype does not display detectable agonist activity.

[0105] Figure 11 Figure 11 Shows the results of a MET degradation assay. Degradation is a measure of cMET internalization following antibody binding. The ability of chimeric anti-cMET antibodies to induce cMET degradation on human cancer cell lines was tested as measured by Mesoscale (MSD) cMET protein quantification. The relative value of Met degradation is indicated on the y-axis as % control (percentage of control). Values below 100% control indicate internalization and degradation of cMET. As indicated on the x-axis, chimeric antibodies were generated by replacing the constant regions of the heavy and light chains of an isolated murine monoclonal antibody designated P3D12 (P3D12(mouse)) with the antibody constant regions of human IgG1 (P3D12(hIgG1)) or the antibody constant regions of human IgG2 (P3D12(hIgG2)). As indicated on the x-axis, each antibody was tested at 0.00064 μg / ml, 0.0032 μg / ml, 0.016 μg / ml, 0.08 μg / ml, 0.4 μg / ml, 2 μg / ml, 10 μg / ml, and 50 μg / ml. The chimeric cMET antibodies of P3D12 showed similar internalization / degradation activity to the parental murine P3D12 antibody.

[0106] Figure 12 Figure 12 Shows a flow chart of the method development for testing and selecting lead anti-cMET monoclonal binders.

[0107] Figure 13 Figure 13 ​​​​​​Alignment of five humanized light chain variable regions of the humanized form of murine anti-cMET clone P3D12 is shown, with the name and SEQ ID NO: shown to the left of each sequence. The murine anti-cMET mAb was humanized using five independent methods. Two methods gave the same result, and thus four different light chains are shown.

[0108] Figure 14 Figure 14 Alignment of five humanized heavy chain variable regions of the humanized form of murine clone P3D12 is shown, with the name and SEQ ID NO: shown to the left of each sequence. The murine anti-cMET mAb was humanized using five independent methods. Two methods gave the same result, and thus, there are four different light chains (see Figure 13 ) and four different heavy chains, which can form 16 different combinations of binders.

[0109] Figure 15 Figure 15 Results of an in vivo xenograft mouse model are shown that tested the potency of the indicated humanized anti-cMET antibody-drug conjugate (ADC) at 2.5 mg / kg (2.5 mpk) or 5 mg / kg (5 mpk), as demonstrated using the MKN45 tumor model (cMET+ gastric cancer model). Animals were treated once with the indicated ADC at 2.5 or 5.0 mg / kg. The potency of each anti-cMET binder was compared to PBS or the non-targeting monoclonal antibody rituximab (Retux), which is an anti-cancer monoclonal antibody targeting CD20, which is mainly present on the surface of immune system B cells. Tumor volume (y-axis) was measured at multiple time points (y-axis) after inoculation. Inhibition of tumor growth indicates positive potency. The anti-cMET binders were conjugated to monomethyl auristatin F (MMAF).

[0110] Figure 16 Figure 16 ​​​​​​Shows the binding of the anti-cMET monoclonal binder hD12, which has heavy and light chain variable regions of SEQ ID NO: 108 and 47 and a human IgG2 constant region, and a negative control antibody that does not bind cMET (Denosumab), to cMET-Fc and mutant cMET (E168D) Fc recombinant fusion proteins. The E168D mutation is a somatic mutation present in small cell lung cancer (SCLC). The mutation is located in the Sema domain and results in constitutive activation of the cMET receptor. The abundance of somatic mutations in cMET is very low. E168D occurs in 0.8% to 3% of SCLC patients. Binding ELISA was performed using the extracellular domain of human cMET or E168D cMET fused to human IgG1 Fc. The cMET protein was coated on the plate overnight, and then the samples were titrated and detected with goat anti-human IgG (H+L)-HRP. The EC50 was determined using a sigmoidal dose-response fit.

[0111] Figure 17 Figure 17 A shows the results of an ELISA-based cMET binding assay. Briefly, the ability of five representative antibody-drug conjugates (i.e., hD12-VI, hD12-II, hD12-VII, hD12-IV, and hD12-XI (see antibody nomenclature explained in Example 3)) to bind to human cMET coated on the plate was tested at increasing concentrations (x-axis, antibody-drug conjugate concentration (μg / ml)). The relative binding strength is indicated by luminescence (y-axis). A negative control antibody that does not bind cMET (Denosumab) was conjugated to each of five different payloads (i.e., payloads of chemical formulas VI, II, IV, VII, and XI) and tested as a negative control (denoted as denosumab-VI, denosumab-II, denosumab-IV, denosumab-VII, and denosumab-XI). Figure 17 B shows Figure 17 the IC50 values of each antibody tested in A.

[0112] Figure 18-1 Figures 18A to 18D show the results of cytotoxicity assays of five representative antibody-drug conjugates (hD12-VI, hD12-II, hD12-VII, hD12-IV, and hD12-XI) on four cell lines (SNU-1, which does not express cMET (Figure 18A); SNU-16, which moderately expresses cMET (Figure 18B); SNU-620, which highly expresses cMET (Figure 18C); and MKN-45, which highly expresses cMET (Figure 18D)) that express different amounts of cMET on their cell surface. The percentage survival is indicated on the y-axis, and the amount of antibody-drug conjugate added is indicated on the x-axis (pM).

[0113] ​​​​Figure 18-2 The results are summarized in FIG. 18E, which shows the comparative IC50 values.

[0114] Figure 19-1 FIGS. 19A-19E show the results of cytotoxicity assays of five representative antibody-drug conjugates (hD12-VI, hD12-II, hD12-VII, hD12-IV, and hD12-XI) on five cell lines that express different amounts of cMET on their cell surface (H441, medium cMET expression (FIG. 19A); H1373, medium cMET expression (FIG. 19B); H1975, medium cMET expression (FIG. 19C); SNU-5, high cMET expression (FIG. 19D); and H1573, medium cMET expression (FIG. 19E)). Figure 19-1 FIGS. 19A-19D are provided.

[0115] Figure 19-2 FIG. 19E is provided here. The percent survival is indicated on the y-axis and the amount of antibody-drug conjugate added is indicated on the x-axis (pM). The results are summarized in FIG. 19F, which shows the comparative IC50 values.

[0116] Figure 20-1 FIG. 20 shows the results of an in vivo xenograft study. H1975 tumor cells (medium cMET expression) were injected into mice and treated with hD12-XI (FIGS. 20A, 20B, and 20C), hD12-VI (FIGS. 20A, 20B, and 20D), hD12-II (FIGS. 20A, 20B, and 20E), hD12-VII (FIGS. 20A, 20B, and 20F), and hD12-IV (FIGS. 20A, 20B, and 20G), and the tumor volume (y-axis, FIGS. 20A and 20C-20G) or body weight (FIG. 20B) was determined over time (i.e., days, x-axis). A negative control antibody (denosumab) conjugated to each of five different payloads (i.e., VI, II, IV, VII, and XI) was tested as a negative control. Figure 20-1 FIG. 20A is provided.

[0117] Figure 20-2 Figure 20-2 FIG. 20B is provided.

[0118] Figure 20-3 Figure 20-3 FIG. 20C is provided.

[0119] Figure 20-4 Figure 20-4 FIG. 20D is provided.

[0120] Figure 20-5 Figure 20-5 FIG. 20E is provided. ​​​​​​​​​​​

[0121] Figure 20-6 Figure 20-6 Figure 20F is provided.

[0122] Figure 20-7 Figure 20-7 Figure 20G is provided.

[0123] Figure 21-1 Figure 21 shows the results of an in vivo xenograft study. Mice were injected with H1373 tumor cells (moderate cMET expression) and treated with hD12-XI (Figure 21A), hD12-VI (Figure 21B), hD12-II (Figure 21C), hD12-VII (Figure 21D), and hD12-IV (Figure 21E), and tumor volume (y-axis) was determined over time (i.e., days, x-axis). A negative control antibody conjugated to II (denosumab) was tested as a negative control. Figure 21-1 Figures 21A and 21B are provided. Figure 21-2 Figure 21-2 Figure 21C is provided.

[0124] Figure 21-3 Figure 21-3 Figure 21D is provided.

[0125] Figure 21-4 Figure 21-4 Figure 21E is provided.

[0126] Figure 22 Figure 22 Shows the body weight (y-axis) of the treated mice in Figure 21 over time (x-axis).

[0127] Figure 23 Figure 23 Shows the serum concentrations (y-axis) of hD12-XI, hD12-VI, hD12-II, hD12-VII, hD12-IV, and denosumab-II in mice after i.v. injection. The time (hours) after injection is shown on the x-axis. The concentration of each antibody-drug conjugate was determined by ELISA.

[0128] Figure 24 Figure 24 ​​​​​​​​​​​​​​​​Shows the results of an ELISA-based cMET binding assay, which displays the relative affinities of hD12 and four variants of hD12 (hD12-T289C, hD12-V442C, and hD12-V282C) covalently linked to Payload II for plate-bound human cMET. hD12-II is randomly linked to the payload at random thiols. hD12-T289C-II, hD12-V442C-II, and hD12-V282C-II are site-specifically linked to the payload at the positions of the variant cysteine residues (i.e., T289C, V442C, and V282C, respectively). The ability of the antibody-drug conjugates to bind to plate-bound human cMET is tested at increasing concentrations (x-axis, antibody-drug conjugate concentration (μg / ml)). The relative binding strength is indicated by luminescence (y-axis). A negative control antibody (denosumab-II) was tested as a negative control. Figure 24 Panel B shows Figure 24 the IC50 values for each of the antibodies tested in A.

[0129] Figure 25-1 Figure 25 shows the results of cytotoxicity assays of the antibody conjugates (i.e., hD12-II, hD12-T289C-II, hD12-V442C-II, and hD12-V282C-II) on four cell lines that express different amounts of cMET on their cell surfaces (SNU-1, which does not express cMET (Figure 25A); SNU-16, which moderately expresses cMET (Figure 25B); SNU-620, which highly expresses cMET (Figure 25C); MKN-45, which highly expresses cMET (Figure 25D); and N87, which lowly expresses cMET (Figure 25E)). Figure 24 Percent survival is indicated on the y-axis, and the amount of antibody-drug conjugate added is indicated on the x-axis (pM). The results are summarized in Figure 25F, which shows the comparative IC50 values. The concentrations of the antibody-drug conjugates tested are shown in Figure 25G. Figure 25-1 Figure 25A is provided.

[0130] Figure 25-2 Figure 25-2 Figure 25B is provided.

[0131] Figure 25-3 Figure 25-3 Figure 25C is provided.

[0132] Figure 25-4 Figure 25-4 Figure 25D is provided.

[0133] Figure 25-5 Figure 25-5 Figure 25E is provided.

[0134] ​​​​​​​​​​Figure 25-6 ] Figure 25-6 Figures 25F and 25G are provided.

[0135] [ Figure 26 ] Figure 26 A shows that in a high cMET expression cell line (i.e., SNU-5), Figure 24 Results of cytotoxicity assays of antibody conjugates of hD12-II, hD12-T289C-II, hD12-V442C-II, and hD12-V282C-II. Percent survival is indicated on the y-axis, and the amount of antibody drug conjugate added is indicated on the x-axis (pM). The results are summarized in Figure 26 In B, comparative IC50 values ​​are shown. The concentrations of the antibody drug conjugates tested are shown in Figure 26 C.

[0136] [ Figure 27-1 ] FIG27 shows three cell lines (H1373, FIG27A ; H1573, FIG27B ; and H1975, FIG27C ) expressing moderate amounts of cMET on their cell surfaces. Figure 24 The results of the cytotoxicity assay of antibody conjugates (i.e. hD12-II, hD12-T289C-II, hD12-V442C-II and hD12-V282C-II). Percent survival is indicated on the y-axis, and the amount of the added antibody drug conjugate is indicated on the x-axis (pM). The concentration of the antibody drug conjugate tested is shown in Figure 27D. The results are summarized in Figure 27E, which shows the IC50 values ​​for comparison. Figure 27-1 Figure 27A is provided.

[0137] [ Figure 27-2 ] Figure 27-2 Figure 27B is provided.

[0138] [ Figure 27-3 ] Figure 27-3 Figure 27C is provided.

[0139] [ Figure 27-4 ] Figure 27-4 Figures 27D and 27E are provided.

[0140] [ Figure 28-1Figure 28 shows the results of in vivo xenograft studies. Mice were injected with H1975 tumor cells (moderate cMET expression) and treated with the indicated concentrations of hD12-V442C-II (Figure 28A), hD12-T289C-II (Figure 28B), and hD12-V282C-II (Figure 28C). Mice were treated with randomly conjugated hD12-II as a positive control. Mice were treated with vehicle (PBS) or denosumab-II as negative controls. Tumor volume (y-axis) was determined over time (i.e., days, x-axis). Figure 28-1 Figure 28A is provided.

[0141] Figure 28-2 Figure 28-2 Figure 28B is provided.

[0142] Figure 28-3 Figure 28-3 Figure 28C is provided.

[0143] Figure 29 Figure 29 Shows the body weight (y-axis) of the treated mice in Figure 28 over time (x-axis).

[0144] Figure 30 Figure 30 Shows the serum concentrations (y-axis) of hD12-V442C-II (Figure 28A), hD12-T289C-II (Figure 28B), and hD12-V282C-II (Figure 28C) in rats after i.v. injection. Time (hours) after injection is indicated on the x-axis. The concentration of each antibody-drug conjugate was determined by ELISA.

[0145] Figure 31 Figure 31 Shows a table Figure 30 summarizing the pharmacokinetic data obtained from the

[0146] Figure 32 Figure 32 Shows the results of in vivo patient-derived xenograft (PDX) studies using human primary gastric cancer tissue ( Figure 32 A), human primary colorectal cancer tissue ( Figure 32 B), and human head and neck cancer tissue ( Figure 32 C). The percentage of tumor growth inhibition (TGI%) is shown on the y-axis, and the concentration of the administered antibody conjugate (hD12-T289C-II) is shown on the x-axis. TGI% was calculated as described in Example 12.

[0147] Figure 33-1 ​​​​​​​​​​​​​Figure 33A shows immunohistochemical staining of a cross-section of a gastric cancer PDX model GA3121 against human cMET protein. Figure 33B shows an enlarged view of the area shown in the white inset of Figure 33A.

[0148] Figure 33-2 Figure 33C shows the results of tumor growth inhibition in an in vivo patient-derived xenograft (PDX) model GA3121. Each data point represents 1 group consisting of 10 mice. Mice were treated with a vehicle (PBS) as a negative control or sekukinumab-II. The tumor volume (y-axis) was determined over time (i.e., days, x-axis). Detailed Description

[0149] In some embodiments, disclosed herein are conjugate-drug conjugates comprising a binder (e.g., a monoclonal antibody or an antigen-binding portion thereof) and a payload (e.g., a cytotoxic payload). In some embodiments, the binder is a novel monoclonal antibody or an antigen-binding portion thereof that specifically binds to cMET. In some embodiments, the payload comprises a pyrrolobenzodiazepine (PBD) toxin and a specific linker. The novel conjugate-drug conjugates disclosed herein can be used to treat cancer and / or neoplastic disorders.

[0150] The binders disclosed herein are novel, both in terms of the amino acid sequence of their antigen-binding regions (e.g., heavy and light chain variable regions) and in terms of their functional properties. For example, the anti-cMET binders described herein have a unique combination of properties not present in other cMET antibodies. First, the anti-cMET binders described herein do not induce significant signal transduction (e.g., receptor tyrosine kinase activity) from the cMET receptor upon binding. Thus, upon binding, the cMET binders described herein do not induce undesired oncogenic activity upon binding (e.g., growth, proliferation, metastasis, or angiogenesis). Second, the anti-cMET binders described herein can induce cMET degradation and are internalized after binding. The advantage of this property is that any toxic payload conjugated to the anti-cMET binder is brought inside the target cell, thereby reducing off-target, non-specific toxicity of the payload. This function also allows control or regulation of the activity of the toxic payload. For example, in some embodiments, the toxic payload described herein is substantially inactive until it contacts intracellular proteases. Third, certain anti-cMET binders described herein cross-react with non-human primates, rats, and / or mice, which allows testing and optimization of antibody-drug conjugates using non-human animal models. Fourth, the anti-cMET binders described herein are soluble, exhibit an extended half-life in vivo, and are stable upon storage.

[0151] ​Numerous cytotoxic payloads are known which can be conjugated to an antibody using known linkers to produce an antibody-drug conjugate (ADC) (e.g., see US2014 / 0120118, US2014 / 0127239, US2016 / 0250344, US2016 / 0250345 and Tiberghien, et al., (2016) ACS Medicinal Chemistry Letters 7(11):983-987). However, after conjugation to the payload, the biochemical function of the antibody binder often changes. Similarly, after conjugation to the antibody, the biochemical properties of the payload are not always predictable. For example, depending on the type of linker used and the location at which the linker is attached to the antibody, the in vivo activity of known payloads can vary widely, from lethal to no therapeutic effect. Thus, it is extremely challenging, time-consuming, and requires a great deal of inventive effort to produce an antibody-drug conjugate by combining a specific cMET binder with an ideal linker, a selected toxic payload, and an optimal conjugation site of the linker to the antibody to provide optimal delivery of the toxin while substantially limiting adverse events (e.g., off-target toxicity) and maintaining the desired biological functional properties of the binder. The ADCs shown herein provide a unique combination of toxin, linker, and a novel cMET binder that results in highly efficient ADCs that provide optimal therapeutic effects while substantially reducing or eliminating off-target toxicity.

[0152] cMET

[0153] MET is used synonymously with the term "cMET" herein. cMET is also known as hepatocyte growth factor receptor (HGFR). Human cMET (e.g., SEQ ID NO:109) contains an immature polypeptide sequence of 1390 amino acids and, numbered from the N-terminus to the C-terminus, includes the following: an N-terminal single sequence of amino acids 1 to 24, an extracellular domain of human cMET of approximately amino acids 24 to 932, a transmembrane domain of approximately amino acids 933 to 955, and a cytoplasmic domain of approximately amino acids 956 to 1390. Methods for identifying the leader sequence, extracellular domain, transmembrane domain, and cytoplasmic domain of the cMET receptor are known, and any suitable method can be used to identify such domains or regions within a cMET polypeptide sequence from a suitable mammalian species. Human cMET polypeptides can include a variety of known variants (e.g., see, e.g., the URL: http: / / www.uniprot.org / uniprot / P08581 accessed on May 5, 2016, the cMET variants and alternative sequences disclosed therein are incorporated herein by reference). Non-limiting examples of naturally occurring variants of human cMET include amino acid substitutions at positions 143, 150, 156, 168, 238, 316, 320, 375, 385, 773, 970, 991, and / or 992 of human cMET (SEQ ID NO:109). In some embodiments, cMET or the cMET extracellular domain contains an E to D substitution at position 168 of human cMET, referred to herein as E168D. In some embodiments, cMET or the cMET extracellular domain contains an N to S substitution at position 375 of human cMET, referred to herein as N375S.

[0154] In some embodiments, cMET is mammalian cMET. In some embodiments, cMET is primate cMET. In some embodiments, cMET is human cMET. In some embodiments, cMET is monkey cMET. In some embodiments, cMET is rodent cMET (e.g., rat and / or mouse). In some embodiments, cMET is canine cMET (e.g., dog cMET). Non-limiting examples of mammalian cMET are provided in Example 5 and / or the Sequence Listing of the present application. In certain embodiments, the extracellular domain of cMET comprises the N-terminal portion of the cMET polypeptide that is typically expressed on the cell surface of a complete mammalian cell. The extracellular domain of cMET may comprise two or more polypeptide chains derived from the MET translation product. In certain embodiments, the extracellular domain of cMET can be expressed in a soluble and / or non-membrane-bound form lacking the cytoplasmic and / or transmembrane domains. In certain embodiments, the extracellular domain of cMET is expressed, isolated, and / or purified as a fusion protein. For example, the extracellular domain of mammalian cMET can be engineered and expressed as a fusion protein comprising the Fc portion of an immunoglobulin (e.g., cMET-Fc). In certain embodiments, cMET and / or the extracellular domain of cMET comprises one or more amino acid additions, deletions, or substitutions. The cMET polypeptide can be at least 80%, at least 85%, at least 90%, or at least 95% of the cMET polypeptides disclosed herein. In certain embodiments, the cMET polypeptide comprises a portion (e.g., a subsequence) of the cMET protein. In some embodiments, a portion of cMET comprises the extracellular domain of cMET or a portion thereof.

[0155] Binder

[0156] In certain embodiments, the binder comprises or consists of one or more polypeptides or one or more proteins that specifically bind to cMET or a portion thereof. In some embodiments, the binder comprises or consists of a protein that specifically binds to cMET or a portion thereof. The binder typically comprises at least one antigen-binding portion (i.e., binding portion). The antigen-binding portion of the binder is the portion that specifically binds to the antigen. In certain embodiments, the binding portion of the binder comprises or consists of a single polypeptide (e.g., a single-chain antibody). In some embodiments, the binding portion of the binder comprises or consists of two polypeptides. In some embodiments, the binding portion of the binder comprises 2, 3, 4, or more polypeptides or consists of the same. In some embodiments, the binder comprises one or more structural portions (e.g., a backbone, a structural polypeptide, a constant region, and / or a framework region). In some embodiments, the binder or its binding portion is linked to a substrate (e.g., a polymer, an inorganic material, silicon, beads, etc.).

[0157] The binder may comprise one antigen-binding portion or multiple antigen-binding portions. For example, sometimes a binder comprising one binding portion is referred to as monovalent. Sometimes a binder comprising two binding portions is referred to as bivalent. In some embodiments, the binder comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more binding portions. In certain embodiments, all of the binding portions of the multivalent binder bind to the same antigen. In certain embodiments, all of the binding portions of the multivalent binder comprise one or more polypeptide sequences having at least 90%, at least 95%, at least 99%, or 100% identity.

[0158] In certain embodiments, two or more binding portions of the binder bind to different antigens. Sometimes such binders are referred to as bispecific or multispecific binders (e.g., antibodies). Thus, in certain embodiments, the binder comprises a first antigen-binding portion that specifically binds to cMET or a portion thereof and a second antigen-binding portion that specifically binds to another antigen (e.g., a polypeptide that is not cMET or a portion thereof). In some embodiments, the binder that specifically binds to cMET is covalently or non-covalently linked to another binder that does not specifically bind to cMET or a portion thereof. In certain embodiments, the binder that specifically binds to cMET comprises a second binder that specifically binds to another antigen.

[0159] In some embodiments, the binder comprises an antibody or a portion thereof (e.g., its binding portion). In certain embodiments, the binder comprises or consists of a suitable antibody or the antigen-binding portion of an antibody. In some embodiments, the binder is an antibody (e.g., a monoclonal antibody and / or a recombinant antibody). The binder or antibody can be produced, manufactured, or made by suitable methods. In some embodiments, the binder is monoclonal. In some embodiments, the binder is a monoclonal antibody derived from a suitable species. Certain non-limiting examples of the binder include monoclonal antibodies, chimeric antibodies, antibody-binding fragments (e.g., the antigen-binding portion of an antibody), CDR-grafted antibodies, humanized antibodies, human antibodies, or a portion thereof. Human antibodies can be obtained by any suitable method. For example, human antibodies can be obtained from transgenic animals engineered to produce fully human antibodies. In certain embodiments, the binder is not polyclonal, is not a polyclonal antibody, and the term "binder" does not refer to a polyclonal antibody.

[0160] In some embodiments, the binding agent is obtained, generated, acquired, isolated, and / or purified from a suitable species. In some embodiments, the binding agent is obtained, generated, acquired, isolated, and / or purified from, for example, rabbits, goats, horses, cows, rats, mice, fish, birds, or llamas. In some embodiments, the binding agent is obtained, generated, acquired, isolated, and / or purified from birds (e.g., chickens or bird eggs). In some embodiments, the binding agent is obtained, generated, acquired, isolated, and / or purified from plants (e.g., recombinant binding agents produced by genetically modified plants). In some embodiments, the binding agent is obtained, generated, acquired, isolated, and / or purified from a suitable mammal. In certain embodiments, the suitable mammal is a genetically altered mammal (e.g., a transchromosomal or transgenic mammal) that has been modified to produce an antibody comprising a human heavy chain and / or a human light chain or a portion thereof. In some embodiments, the binding agent is produced, acquired, isolated, or purified from prokaryotic or eukaryotic cells (e.g., recombinant binding agents produced by genetically modified cells). In some embodiments, the binding agent is produced, acquired, isolated, or purified from viruses (e.g., recombinant binding agents produced by genetically modified viruses). The binding agent can be expressed, isolated, and / or purified from a suitable expression system, non-limiting examples of which include suitable bacterial, phage, insect, viral, plant, or mammalian expression systems. For example, a nucleic acid encoding the binding agent can be introduced into a suitable mammalian cell line, which expresses the binding agent and secretes it into the cell culture medium.

[0161] In certain embodiments, the binding agent is not found in nature and is not naturally occurring. For example, in certain embodiments, the binding agent is artificially produced in an animal by administering an emulsified mixture comprising an exogenous recombinant antigen, a potent adjuvant, and typically mineral oil and / or a detergent to induce an artificial immune response against the exogenous recombinant antigen (e.g., cMET, cMET-Fc).

[0162] In certain embodiments, a monoclonal antibody or monoclonal binding agent is a substantially homogeneous population of the binding agent or its binding fragment, wherein each individual binding agent in the population is substantially identical and / or binds to the same epitope, except for possible variants that may arise during the production of the monoclonal binding agent. In some embodiments, such variants are typically absent or may be present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each binding agent in a monoclonal binding agent population typically binds to a single determinant of the antigen. Monoclonal binding agents are typically not contaminated with other immunoglobulins. One or more different monoclonal binding agents can be purposefully added to a composition to form a mixture.

[0163] The modifier "monoclonal" should not be construed as requiring that the binder be produced by any particular method. Monoclonal binders can be produced by any suitable method. For example, in certain embodiments, monoclonal antibodies are prepared by the hybridoma method described by Kohler et al. (1975) Nature, 256:495 or variations thereof. In some embodiments, monoclonal binders are prepared by suitable recombinant DNA methods. For example, monoclonal antibodies can be prepared or altered by the methods described in U.S. Patent No. 5,225,539 and / or Daugherty et al. (1991) Nucleic Acids Research 19(9):2471-2476 or variations thereof. Monoclonal binders can be prepared by screening a recombinant library using, for example, a suitable expression system (e.g., a phage display expression system). In some embodiments, monoclonal binders are isolated from a phage library of binders by using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-628 and / or Marks et al. (1991) J. Mol Biol, 222:581-597 or variations thereof.

[0164] In certain embodiments, the binder comprises one or more structural or framework portions, sometimes referred to as a scaffold. The binder can comprise a scaffold, non-limiting examples of which include scaffolds derived from: antibodies, the Z domain of protein A, γ-B crystallin, ubiquitin, cystatin, Sac7d, triple-helical coiled coils, lipocalin, ankyrin repeat motifs, the SH3 domain of Fyn, the Kunitz domain of a suitable protease inhibitor, fibronectin domains, nucleic acid polymers, etc., portions thereof, or combinations thereof. In some embodiments, the binder does not comprise a scaffold. In certain embodiments, the binder comprises one or more structural portions of a mammalian antibody.

[0165] In certain embodiments, the binder comprises one or more constant regions (e.g., constant regions derived from an antibody (e.g., a mammalian antibody)). In certain embodiments, the binder comprises the constant region of an antibody light chain and / or the constant region of an antibody heavy chain. In mammalian antibodies, there are at least two types of immunoglobulin light chains, called lambda (λ) and kappa (κ). The binder can comprise any suitable constant region of an antibody, or one or more portions thereof. In some embodiments, the binder comprises the λ light chain constant region or a portion thereof. In some embodiments, the binder comprises the κ light chain constant region or a portion thereof. In some embodiments, the binder comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the polypeptide sequence of the constant region of a mammalian antibody light chain or a portion thereof. In some embodiments, the binder comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the polypeptide sequence of the constant region of a human antibody light chain. In some embodiments, the binder does not comprise a light chain constant region.

[0166] In certain embodiments, the binder comprises the constant region of an antibody heavy chain. In mammals, antibodies can have at least five types / categories of Ig heavy chains, designated IgA, IgD, IgE, IgG, and IgM, which are determined by the presence of distinct heavy chain constant regions or portions thereof (e.g., CH1, CL, CH2, CH3 domains). The binder can comprise any suitable heavy chain constant region or a portion thereof. In some embodiments, the binder comprises the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, or one or more portions thereof. In some embodiments, the binder comprises one or more heavy chain constant regions, or a portion thereof, of the IgM, IgD, IgA, or IgE isotype.

[0167] Unless otherwise specified herein, the numbering of amino acid residues in antibody constant regions is according to the EU numbering system described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78 - 85 (1969). PMID: 5257969.

[0168] In some embodiments, the binder comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity or 100% identity to the polypeptide sequence of the constant region of a mammalian antibody heavy chain or a portion thereof. In some embodiments, the binder comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity or 100% identity to the polypeptide sequence of the constant region of a human antibody heavy chain. In some embodiments, the binder comprises one or more additions, deletions, and / or modifications to the constant region. Sometimes the binder is modified to alter the antibody class or isotype of the binder. In some embodiments, the binder comprises one or more additions, deletions, and / or modifications (one or more amino acid substitutions, deletions, or additions) to alter one or more functions of the binder, such as eliminating, enhancing, or reducing serum half-life, Fc receptor binding, complement binding (e.g., C1q binding), glycosylation, sialylation, cytotoxicity, antibody-dependent cell-mediated phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), etc. In some embodiments, the binder does not comprise one or more portions of the heavy chain constant region or the light chain constant region. In some embodiments, the binder does not comprise the heavy chain constant region.

[0169] In some embodiments, the binder comprises or consists of one or more variable regions of an antibody or a portion thereof. In some embodiments, the binder comprises one or more light chain variable regions or a portion thereof. In some embodiments, the binder comprises one or more heavy chain variable regions or a portion thereof. In certain embodiments, the binder comprises at least one light chain variable region and at least one heavy chain variable region. The light chain variable region and the heavy chain variable region can be on the same or different polypeptides. In certain embodiments, the antigen-binding portion of the binder consists of one or more heavy chain variable regions. In certain embodiments, the antigen-binding portion of the binder consists of one or more light chain variable regions. In certain embodiments, the antigen-binding portion of the binder consists of one or more light chain variable regions and one or more heavy chain variable regions.

[0170] In some embodiments, the binding agent comprises Fab, Fab’, F(ab’)2, Fv fragments, single-chain Fv (scFv), diabody (Dab), synbody, etc. and / or combinations or portions thereof, or consists of them. In some embodiments, the binding agent is Fab, Fab’, F(ab’)2, Fv fragments, single-chain Fv (scFv), diabody (Dab), synbody, etc. and / or combinations or portions thereof (see, e.g., U.S. Patent Nos. 6,099,842 and 5,990,296). In some embodiments, the binding agent comprises a single-chain polypeptide containing one or more antigen-binding portions. For example, a single-chain binding agent can be constructed by linking a heavy-chain variable region or its antigen-binding portion to a light-chain variable region or its antigen-binding portion using a linker (e.g., an amino acid, a polypeptide linker) by recombinant molecular biology methods. Similar to the parental two-chain monoclonal binding agent, such single-chain binding agents generally exhibit specificity and affinity for the antigen. The binding agent typically comprises engineered regions, such as CDR-grafted or humanized portions. In certain embodiments, the binding agent is a complete two-chain immunoglobulin, and in other embodiments, the binding agent is a Fab monomer or a Fab dimer.

[0171] The nucleic acid encoding the polypeptide of the binder or a part thereof can be cloned, subcloned, rearranged or modified by methods known to those skilled in the art through suitable cloning procedures for recombinant expression, and then expressed using a suitable expression system (for example, see Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Methods in molecular biology, edited by Benny K.C. Lo, Springer Science & Business Media, 2004; Antibody Engineering, Vol. 1, Roland E. Kontermann, Stefan Dübel, Edition 2, Publisher Springer Science & Business Media, 2010; Antibody Phage Display: Methods and Protocols, Biomed Protocols, Vol. 178 of Methods in molecular biology, Editors Philippa M. O’Brien, Robert Aitken, Springer Science & Business Media, 2004).

[0172] In mammals, the variable region of the heavy chain and the variable region of the light chain of an antibody each contribute three CDRs (complementary determining regions), commonly referred to as CDR1, CDR2, and CDR3, which are separated and / or flanked by framework regions (such as FR1, FR2, FR3, and FR4). As used herein, the term "CDR" refers to the amino acid sequence of a polypeptide identified as a complementary determining region. In certain embodiments, the definitive description of the CDR polypeptide sequence and the identification of the residues comprising the binding site of a binder are accomplished by discerning the structure of the binder and / or discerning the structure of the binder-antigen complex. In certain embodiments, this can be accomplished by any suitable method, such as X-ray crystallography and / or computer modeling. In certain embodiments, multiple analytical methods can be employed to identify or approximate the CDR sequences of a binder or antibody. For example, suitable methods can be used to identify the amino acid sequence and / or location of CDRs in the polypeptide sequence of a binder, antibody, binding portion thereof, or variable region thereof, non-limiting examples of such methods include: the Kabat system (e.g., see Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, and Johnson, G. and Wu, T.T. (2000) Nucleic Acids Research 28(1):214-8) and / or the Chothia numbering scheme (e.g., Chothia & Lesk, (1987) J. Mol. Biol, 196:901-917; Chothia et al. (1989) Nature 342:878-883; and Al-Lazikani et al. (1997) JMB 273, 927-948). In some embodiments, the AbM method and / or the contact method can be used to identify the amino sequence and / or location of CDRs of an antibody. "AbM" defines the use of an integrated computer program suite for modeling antibody structures produced by Oxford Molecular Group (see, e.g., Martin et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:9268-9272; "AbM TM"A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). AbM defines the use of a combination of a knowledge database and an ab initio method (such as those described by Samudrala et al. (1999) Proteins, Structure, Function and Genetics, Suppl, 3:194-198 and Xia Y, et al. (2000) J Mol Biol. 300(1):171-85) to model the tertiary structure of antibodies from primary sequences. In certain embodiments, the contact definition is based on the analysis of available complex crystal structures (see, for example, MacCallum et al. (1996) J. Mol. Biol 5:732-45).

[0173] In some embodiments, the binder and / or the antigen-binding portion of the binder comprises at least 2, at least 3, at least 4, at least 5, or at least 6 CDRs. In some embodiments, the binder comprises 3 to 60 CDRs (e.g., for a binder having multiple antigen-binding portions). In some embodiments, the binder comprises 3 to 12 CDRs. In some embodiments, the antigen-binding portion of the binder comprises 1 to 6 CDR polypeptide sequences.

[0174] In certain embodiments, the binder and / or the antigen-binding portion of the binder comprises one, two, or three CDRs of the light chain variable region. In some embodiments, the light chain variable region of the binder comprises one or more CDRs (e.g., one, two, three, or more CDRs). The amino acid sequences representing the CDRs in the light chain variable region of an antibody or binder are designated CDR-L1, CDR-L2, and CDR-L3, and are numbered in order from the amino-terminal (N-terminal) to the carboxyl-terminal (C-terminal) direction of the light chain variable region (i.e., L1, L2, and L3). For example, in a polypeptide representing the light chain variable region of a binder, CDR-L1, when present, is the most N-terminal light chain CDR; CDR-L3, when present, is the most C-terminal light chain CDR; and CDR-L2, when present, is located (i) between CDR-L1 and CDR-L3, (ii) on the N-terminal side of CDR-L3, or (iii) on the C-terminal side of CDR-L1 in the light chain variable region or binding portion of the binder. The terms "CDR-L1", "CDR-L2", and "CDR-L3" refer in part to the amino acid sequences of polypeptides identified as or disclosed herein as the complementarity determining regions of a binder (e.g., the CDRs of the light chain variable region). Non-limiting examples of the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are provided in Tables 1 to 3, respectively. The light chain variable region or antigen-binding portion of the binder described herein may comprise any combination of CDR-L1, CDR-L2, and CDR-L3 disclosed herein, wherein the binder retains specific binding to cMET or a portion thereof. In certain embodiments, the light chain variable region or antigen-binding portion of the binder described herein comprises a single light chain CDR comprising an amino acid sequence having at least 70% identity to a CDR-L3 selected from Table 3.

[0175] In certain embodiments, the light chain variable region or antigen-binding portion of the binding agent described herein comprises an amino acid sequence having at least 70% identity to a CDR-L3 selected from Table 3, and any other suitable CDR-L2 and / or CDR-L1 polypeptide sequences, wherein the binding agent retains specific binding to cMET or a portion thereof. In certain embodiments, the light chain CDRs of the light chain variable region or antigen-binding portion of the binding agent consist of CDR-L3 and CDR-L2, wherein CDR-L3 comprises an amino acid sequence having at least 70% identity to a CDR-L3 selected from Table 3, and CDR-L2 comprises an amino acid sequence having at least 70% identity to a CDR-L2 selected from Table 2. In certain embodiments, the light chain variable region or antigen-binding portion of the binding agent described herein comprises an amino acid sequence having at least 70% identity to a CDR-L3 selected from Table 3 and an amino acid sequence having at least 70% identity to a CDR-L2 selected from Table 2, and any other suitable CDR-L1 polypeptide sequence, wherein the binding agent retains specific binding to cMET or a portion thereof. In certain embodiments, the light chain variable region or antigen-binding portion of the binding agent described herein comprises three light chain CDRs consisting of an amino acid sequence having at least 70% identity to a CDR-L3 selected from Table 3, an amino acid sequence having at least 70% identity to a CDR-L2 selected from Table 2, and an amino acid sequence selected with at least 70% identity to CDR-L1 of Table 1. In certain embodiments, the light chain variable region or antigen-binding portion of the binding agent described herein comprises: an amino acid sequence having at least 70% identity to a CDR-L3 selected from Table 3, an amino acid sequence having at least 70% identity to a CDR-L2 selected from Table 2, and an amino acid sequence having at least 70% identity to a CDR-L1 selected from Table 1, wherein the binding agent retains specific binding to cMET or a portion thereof.

[0176] In some embodiments, the binding agent comprises one or more light chain CDRs having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to any of the CDR sequences listed in Table 1, 2, or 3. In some embodiments, the binding agent or the antigen-binding portion of the binding agent comprises a CDR-L1 having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to any of the sequences shown in Table 1. In some embodiments, the binding agent or the antigen-binding portion of the binding agent comprises a CDR-L1 of any of the sequences shown in Table 1.

[0177] [Table 1]

[0178] CDR-L1 sequence

[0179]

[0180] The clone names cited in Tables 1 to 10 represent the fusion number (F), the plate number (P) of the 96-well plate from which the clone was derived, and the well number (A1 to H12). Thus, for example, clone F6AP12F12 is derived from fusion 6A, plate 12, well F12. The fusion number of each clone corresponds to the fusion shown in Figure 2 the fusion shown in

[0181] In some embodiments, the binder or the antigen-binding portion of the binder comprises a CDR-L2 having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to any of the sequences shown in Table 2. In some embodiments, the binder or the antigen-binding portion of the binder comprises a CDR-L2 of any of the sequences shown in Table 2.

[0182] [Table 2]

[0183] CDR-L2 sequence

[0184]

[0185] In some embodiments, the binder or the antigen-binding portion of the binder comprises a CDR-L3 having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to any of the sequences shown in Table 3. In some embodiments, the binder or the antigen-binding portion of the binder comprises a CDR-L3 of any of the sequences shown in Table 3.

[0186] [Table 3] CDR-L3 sequence

[0187]

[0188] In some embodiments, the binder or the antigen-binding portion of the binder comprises a light chain variable region having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence of Table 4. In some embodiments, the binder or the antigen-binding portion of the binder comprises the light chain variable region sequence of Table 4.

[0189] [Table 4]

[0190] Light chain variable sequence

[0191]

[0192] In some embodiments, the binder or the antigen-binding portion of the binder comprises a humanized light chain variable region having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the sequences of Table 5. In some embodiments, the binder or the antigen-binding portion of the binder comprises the humanized light chain variable region sequence of Table 5.

[0193] [Table 5]

[0194] Humanized P3D12 light chain

[0195]

[0196] In certain embodiments, the binder and / or the antigen-binding portion of the binder comprises one, two, or three CDRs of the heavy-chain variable region. In some embodiments, the heavy-chain variable region comprises one or more CDRs (e.g., one, two, three, or more CDRs). The amino acid sequences representing the CDRs in the heavy-chain variable region of an antibody or binder are designated CDR-H1, CDR-H2, and CDR-H3, which are numbered in sequence in the direction from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) of the heavy-chain variable region (i.e., H1, H2, and H3). For example, in a polypeptide representing the heavy-chain variable region of a binder, CDR-H1, when present, is the most N-terminal CDR; CDR-H3, when present, is the most C-terminal CDR; and CDR-H2, when present, is located (i) between CDR-H1 and CDR-H3, (ii) on the N-terminal side of CDR-H3, or (iii) on the C-terminal side of CDR-H1. The terms "CDR-H1", "CDR-H2", and "CDR-H3" refer in part to the amino acid sequences of polypeptides identified as or disclosed herein as the complementarity-determining regions of a binder (e.g., the CDRs of the heavy-chain variable region of a binder). Non-limiting examples of the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are provided in Tables 6 to 8, respectively. The heavy-chain variable region or antigen-binding portion of the binder described herein may comprise any combination of CDR-H1, CDR-H2, and CDR-H3 disclosed herein, provided that the binder retains specific binding to cMET or a portion thereof. In certain embodiments, the heavy-chain variable region or antigen-binding portion of the binder described herein comprises a single heavy-chain CDR consisting of an amino acid sequence having at least 70% identity to a CDR-H3 selected from Table 8. In certain embodiments, the heavy-chain variable region or antigen-binding portion of the binder described herein comprises an amino acid sequence having at least 70% identity to a CDR-H3 selected from Table 8 and any other suitable CDR-H2 and / or CDR-H1 polypeptide sequences, provided that the binder retains specific binding to cMET or a portion thereof. In certain embodiments, the heavy-chain CDRs of the heavy-chain variable region or antigen-binding portion of the binder consist of CDR-H3 and CDR-H2, wherein CDR-H3 comprises an amino acid sequence having at least 70% identity to a CDR-H3 selected from Table 8, and CDR-H2 comprises an amino acid sequence having at least 70% identity to a CDR-H2 selected from Table 7. In certain embodiments, the heavy-chain variable region or antigen-binding portion of the binder described herein comprises an amino acid sequence having at least 70% identity to a CDR-H3 selected from Table 8 and an amino acid sequence having at least 70% identity to a CDR-H2 selected from Table 7, and any other suitable CDR-H1 polypeptide sequences, provided that the binder retains specific binding to cMET or a portion thereof.In certain embodiments, the heavy chain variable region or antigen-binding portion of the binding agent described herein comprises three heavy chain CDRs, which are composed of an amino acid sequence having at least 70% identity with a CDR-H3 selected from Table 8, an amino acid sequence having at least 70% identity with a CDR-H2 selected from Table 7, and an amino acid sequence having at least 70% identity with CDR-H1 of Table 6. In certain embodiments, the heavy chain variable region or antigen-binding portion of the binding agent described herein comprises: an amino acid sequence having at least 70% identity with a CDR-H3 selected from Table 8, an amino acid sequence having at least 70% identity with a CDR-H2 selected from Table 7, and an amino acid sequence having at least 70% identity with a CDR-H1 selected from Table 6, wherein the binding agent retains specific binding to cMET or a portion thereof.

[0197] In some embodiments, the binding agent comprises one or more heavy chain CDRs having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with any of the CDRs of Table 6, 7 or 8. In some embodiments, the binding agent or the antigen-binding portion of the binding agent comprises a CDR-H1 having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with any of the sequences shown in Table 6. In some embodiments, the binding agent or the antigen-binding portion of the binding agent comprises a CDR-H1 of any of the sequences shown in Table 6.

[0198] [Table 6]

[0199] CDR-H1 sequences

[0200]

[0201] In some embodiments, the binding agent or the antigen-binding portion of the binding agent comprises a CDR-H2 having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with any of the sequences shown in Table 7. In some embodiments, the binding agent or the antigen-binding portion of the binding agent comprises a CDR-H2 of any of the sequences shown in Table 7.

[0202] [Table 7] CDR-H2 sequences

[0203]

[0204] In some embodiments, the binder or the antigen-binding portion of the binder comprises a CDR-H3 having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to any of the sequences shown in Table 8. In some embodiments, the binder or the antigen-binding portion of the binder comprises a CDR-H3 of any of the sequences shown in Table 8.

[0205] [Table 8] CDR-H3 Sequences

[0206]

[0207] In some embodiments, the binder or the antigen-binding portion of the binder comprises a heavy chain variable region having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the sequences of Table 9. In some embodiments, the binder or the antigen-binding portion of the binder comprises the heavy chain variable region sequences of Table 9.

[0208] [Table 9]

[0209] Heavy Chain Variable Sequences

[0210]

[0211]

[0212] In some embodiments, the binder or the antigen-binding portion of the binder comprises a humanized heavy chain variable region having at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the sequences of Table 10. In some embodiments, the binder or the antigen-binding portion of the binder comprises the humanized heavy chain variable region sequences of Table 10.

[0213] [Table 10]

[0214] Humanized P3D12 Heavy Chain

[0215]

[0216] In some embodiments, the binder or the antigen-binding portion of the binder comprises: a CDR-L3 that comprises an amino acid sequence having at least 70%, at least 75%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the amino acid sequences of SEQ ID NOs: 26 to 36 (e.g., a CDR-L3 sequence selected from Table 3); and a CDR-H3 that comprises an amino acid sequence having at least 70%, at least 75%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the amino acid sequences of SEQ ID NOs: 79 to 93 (e.g., a CDR-H3 sequence selected from Table 8). In some embodiments, the binder or the antigen-binding portion of the binder comprises: a CDR-L3 that comprises an amino acid sequence having at least 70%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 34 or 35; and a CDR-H3 that comprises an amino acid sequence having at least 70%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 87, 88, 92, or 93.

[0217] In some embodiments, the binder or the antigen-binding portion of the binder comprises: CDR-L3, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any of the amino acid sequences of SEQ ID NOs: 26 to 36 (e.g., a CDR-L3 sequence selected from Table 3); CDR-L2, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any of the amino acid sequences of SEQ ID NOs: 16 to 25 (e.g., a CDR-L2 sequence selected from Table 2); CDR-H3, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any of the amino acid sequences of SEQ ID NOs: 79 to 93 (e.g., a CDR-H3 sequence selected from Table 8); and CDR-H2, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any of the amino acid sequences of SEQ ID NOs: 62 to 78 (e.g., a CDR-H2 sequence selected from Table 7). In some embodiments, the binder or the antigen-binding portion of the binder comprises: CDR-L3, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 34 or 35; CDR-L2, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 24 or 25; CDR-H3, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 87, 88, 92 or 93; and CDR-H2, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 70, 71 or 78.

[0218] In some embodiments, the binder or the antigen-binding portion of the binder comprises: CDR-L3, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 26 to 36 (e.g., a CDR-L3 sequence selected from Table 3); CDR-L2, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 16 to 25 (e.g., a CDR-L2 sequence selected from Table 2); CDR-L1, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 15 (e.g., a CDR-L1 sequence selected from Table 1); CDR-H3, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 79 to 93 (e.g., a CDR-H3 sequence selected from Table 8); CDR-H2, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 62 to 78 (e.g., a CDR-H2 sequence selected from Table 7); and CDR-H1, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 50 to 61 (e.g., a CDR-H1 sequence selected from Table 6). In some embodiments, the binder or the antigen-binding portion of the binder comprises: CDR-L3, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 34 or 35; CDR-L2, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 24 or 25; CDR-L1, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 9, 10 or 15; CDR-H3, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 87 or 88; CDR-H2, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 70, 71 or 78; and CDR-H1, which comprises an amino acid sequence having at least 70%, at least 90% or 100% identity to the amino acid sequence of SEQ ID NO: 58 or 59.

[0219] In some embodiments, the binder or the antigen-binding portion of the binder comprises: a heavy-chain variable region comprising an amino acid sequence having at least 70%, at least 75%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 94 to 108 (e.g., a heavy-chain variable region selected from Tables 9 and 10); and a light-chain variable region comprising an amino acid sequence having at least 70%, at least 75%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 37 to 49 (e.g., a light-chain variable region selected from Tables 4 and 5). In some embodiments, the binder or the antigen-binding portion of the binder comprises: a heavy-chain variable region comprising an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 104 to 108 (e.g., a heavy-chain variable region selected from Table 10); and a light-chain variable region comprising an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 45 to 49 (e.g., a light-chain variable region selected from Table 5).

[0220] The abbreviations "abb", "sdr", "fra", "ven.", and "cdr" used herein are explained as follows. The abbreviation "cdr" or "CDR" refers to a complementary determining region. The abbreviation "abb" refers to an abbreviated CDR, as described, for example, in Padlan et al. (1995) FASEB J 9:133-139. In some embodiments, the abbreviated CDRs are residues 27D to 34, 50 to 55, and 89 to 96 in the light chain, and residues 31 to 35B, 50 to 58, and 95 to 101 in the heavy chain, which are grafted onto a suitable human framework. Key framework residues are generally retained. The abbreviation "sdr" refers to "specificity determining residues" as described, for example, in Padlan et al. (1995), which are residues that are thought to be involved in antigen binding. The abbreviation "fra" refers to the "Frankenstein method", as described, for example, in Wu and Kabat (1992) Mol Immunol 29:1141-1146. The abbreviation "ven" refers to "Veneering", as described, for example, in Padlan (1991), Mol Immunol 28:489-498.

[0221] The terms "percent identical" or "percent identity" refer to sequence identity between two amino acid sequences. Identity can be determined by comparing positions in each sequence, which may be aligned for purposes of comparison. A molecule is identical at a position when the equivalent position in the compared sequences is occupied by the same amino acid. A molecule may be said to be homologous (similar) at that position when the equivalent site is occupied by the same or a similar amino acid residue (e.g., similar in steric and / or electronic nature). The percent homology, similarity or identity is a function of the number of identical or similar amino acids at positions shared by the compared sequences. The percent homology, similarity or identity is a function of the number of identical or similar amino acids at positions shared by the compared sequences. A variety of alignment algorithms and / or programs can be used, including FASTA, BLAST or ENTREZ. FASTA and BLAST are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be utilized, for example, with the settings used. ENTREZ is available through the National Center for Biotechnology Information (National Library of Medicine, National Institutes of Health, Bethesda, Md.). In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.

[0222] Other alignment techniques are described in the following: Methods in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. In some embodiments, an alignment program that allows gaps in the sequence is used to align sequences. Smith-Waterman is an algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Additionally, the GAP program using the Needleman and Wunsch alignment method can be used to align sequences. Another search strategy is to use the MPSRCH software that runs on a MASPAR computer. MPSRCH scores sequences using the Smith-Waterman algorithm on a massively parallel computer. This method improves the ability to obtain distantly related matches and, in particular, tolerates small gaps and nucleotide sequence errors. Amino acid sequences encoded by nucleic acids can be used to search both protein and DNA databases.

[0223] In some embodiments, the binder or antigen-binding portion of the binder comprises one or more CDRs selected from the light chain variable regions of Tables 4 and 5. In some embodiments, the binder or antigen-binding portion of the binder comprises one or more CDRs selected from the heavy chain variable regions of Tables 9 and 10. In some embodiments, the binder or antigen-binding portion of the binder comprises one or more CDRs selected from the light chain variable regions of Tables 4 and 5 and one or more CDRs selected from the heavy chain variable regions of Tables 9 and 10. In certain embodiments, the binder or antigen-binding portion of the binder comprises CDR-L1, CDR-L2, and CDR-L3 of any light chain variable region each selected from Tables 4 and 5, and CDR-H1, CDR-H2, and CDR-H3 of any heavy chain variable region each selected from Tables 9 and 10. The amino acid sequences of the CDRs (e.g., CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) can be identified in the heavy or light chain variable regions disclosed herein by any suitable method described herein or known to those skilled in the art.

[0224] In certain embodiments, an agent that specifically binds to cMET comprises (i) CDR-L1, CDR-L2, and CDR-L3 which are polypeptide sequences of light chain complementarity determining regions (CDR-L), wherein CDR-L1 is selected from the amino acid sequences of SEQ ID NO: 1 to 15, CDR-L2 is selected from the amino acid sequences of SEQ ID NO: 16 to 25, and CDR-L3 is selected from the amino acid sequences of SEQ ID NO: 26 to 36; and (ii) CDR-H1, CDR-H2, and CDR-H3 which are polypeptide sequences of heavy chain complementarity determining regions (CDR-H), wherein CDR-H1 is selected from the amino acid sequences of SEQ ID NO: 50 to 61, CDR-H2 is selected from the amino acid sequences of SEQ ID NO: 62 to 78, and CDR-H3 is selected from the amino acid sequences of SEQ ID NO: 79 to 93.

[0225] In some embodiments, the agent comprises one or more suitable sequences selected from Tables 1 to 10, wherein the selected polypeptide sequence comprises 0 to 5, 1 to 5, 0 to 10, 1 to 10, 0 to 15, or 1 to 15 amino acid modifications, wherein the amino acid modifications can be amino acid additions, amino acid deletions, and / or amino acid substitutions. In some embodiments, the agent comprises one or more suitable sequences selected from Tables 4, 5, 9, or 10, wherein the selected polypeptide sequence comprises 0 to 5, 1 to 5, 0 to 10, 1 to 10, 0 to 15, or 1 to 15 amino acid modifications in the framework region or the constant region, wherein the amino acid modifications can be amino acid additions, amino acid deletions, and / or amino acid substitutions. In some embodiments, the amino acid modification is a conservative amino acid substitution. In some embodiments, the agents disclosed herein comprise one or more amino acid analogs, non-natural amino acids, or amino acid derivatives.

[0226] In certain embodiments, the agent or the antigen-binding portion of the agent comprises one or more framework regions (FR). Framework regions are typically located between the CDRs and / or flanking the CDR sequences in the variable regions of the heavy or light chains of an antibody or agent. In mammals, the variable region of the heavy chain typically comprises four framework regions, and the variable region of the light chain typically comprises four framework regions. Any suitable method can be used to identify one or more framework regions in an antibody, in the variable region of an antibody, or in an agent. The agent can comprise synthetic or naturally occurring framework regions, which, as discussed below, can be unmodified or modified (e.g., optimized).

[0227] In some embodiments, the binder or its antigen-binding portion is chimeric, grafted, and / or humanized. Chimeric, grafted, and / or humanized binders typically comprise a modified or replaced constant region and / or framework region while retaining the binding specificity for cMET or a portion thereof. In some embodiments, the binder or its antigen-binding portion comprises a constant region, a framework region, or a portion thereof derived from a human antibody. In some embodiments, the binder or its antigen-binding portion comprises a fully synthetic portion, one or more amino acids, or amino acid sequences that are not present in a native antibody molecule.

[0228] A naturally occurring framework region or a portion thereof can be obtained from any suitable species. In certain embodiments, the complementarity determining regions (CDRs) of the light and heavy chain variable regions of the binder or its antigen-binding portion are grafted into a framework region from the same or another species. For example, one or more framework regions of the binder can be derived from a rodent species (e.g., mouse or rat) or a primate species (e.g., human).

[0229] In certain embodiments, the CDRs of the light and / or heavy chain variable regions of the binder or its antigen-binding portion can be grafted into a consensus sequence human framework region. To generate a consensus sequence human framework region, in certain embodiments, the framework regions from multiple human heavy or light chain amino acid sequences can be aligned to identify the consensus sequence. In certain embodiments, the heavy or light chain framework region of an antibody or binder is replaced with one or more framework regions or portions thereof from different heavy or light chain variable regions. In some embodiments, the binder or its antigen-binding portion comprises one or more human framework regions. In certain embodiments, the binder or its antigen-binding portion comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 human framework regions. In some embodiments, the binder or its antigen-binding portion comprises one or more mouse framework regions. In certain embodiments, the binder or its antigen-binding portion comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse framework regions. In certain embodiments, the binder or its antigen-binding portion comprises one or more human framework regions and one or more mouse framework regions.

[0230] Methods for generating chimeric, humanized, and / or optimized antibodies or binding agents (e.g., by modification, replacement, or deletion of a framework region or a portion thereof) are known. Non-limiting examples of CDR grafting are described, for example, in the following: U.S. Patent No. 6,180,370; U.S. Patent No. 6,054,297; U.S. Patent No. 5,693,762; U.S. Patent No. 5,859,205; U.S. Patent No. 5,693,761; U.S. Patent No. 5,565,332; U.S. Patent No. 5,585,089; and U.S. Patent No. 5,530,101, as well as Jones et al. (1986) Nature 321:522-525; Verhoeyen et al. (1988) Science 239:1534-1536, and Winter (1998) FEBS Letts. 430:92-94. Other non-limiting examples of generating chimeric, grafted, and / or humanized binding agents include U.S. Patent No. 5,530,101; U.S. Patent No. 5,707,622; U.S. Patent No. 5,994,524; U.S. Patent No. 6,245,894; Queen et al. (1988) PNAS 86:10029-10033; Riechmann et al. (1988) Nature 332:323-327; Antibody Engineering: Methods and Protocols, Vol. 248 of Methods in molecular biology, edited by Benny K.C. Lo, Springer Science & Business Media, (2004); and Antibody Engineering, Vol. 1, Roland E. Kontermann, Stefan Dübel, Edition 2, Publisher Springer Science & Business Media, (2010). In some embodiments, humanization can be performed by exchanging one or more framework regions or a portion thereof (e.g., one or more amino acids) with one or more framework regions or a portion thereof from a human antibody. In certain embodiments, an antibody or binding agent can be humanized or grafted by transferring one or more CDRs (e.g., 1, 2, 3, 4, 5, or all 6 CDRs) from a donor binding agent (e.g., a murine monoclonal antibody) to a receptor binding agent (e.g., a human antibody) while retaining the binding specificity for the donor binding agent.In certain embodiments, methods of preparing chimeric, grafted, or humanized binding agents include making one or more amino acid substitutions, additions, or deletions in the constant or framework regions of the binding agent. In certain embodiments, techniques such as "reshaping," "hyperchimerization," or "veneering / resurfacing" can be used to generate humanized binding agents (e.g., see Vaswami et al. (1998) Annals of Allergy, Asthma, & Immunol. 81:105; Roguska et al. (1996) Prot. Engin. 9:895-904; and U.S. Patent No. 6,072,035). In some aspects, the binding agent is modified by the methods discussed above or by another suitable method to reduce immunogenicity (e.g., see Gilliland et al. (1999) J. Immunol. 62(6):3663-71).

[0231] In certain embodiments, the amino acid sequence of the binder is modified to optimize binding affinity to a target (e.g., cMET), species cross-reactivity, solubility, and / or function (e.g., agonist activity or its lack). In some embodiments, a particular combination of CDRs disclosed herein can be optimized to bind cMET, and / or the function or properties of the binder disclosed herein can be optimized. For example, the characteristic light chain variable region disclosed herein (e.g., the light chain variable region of SEQ ID NO:48) can be co-expressed using a suitable expression system with a library of heavy chain variable regions comprising CDR-H1 and CDR-H2 of the characteristic heavy chain variable region (e.g., the heavy chain variable region of SEQ ID NO:107), wherein CDR-H3 is replaced with a library of CDR-H3 sequences, which can comprise, for example, one or more CDR-H3 regions of Table 8. The resulting light chain / heavy chain binders can be screened for binding to cMET and / or a particular function. Optimized binders can be identified, and the amino acid sequence of CDR-H3 can be identified by suitable methods. The screening methods described above can be used to identify binders comprising a particular combination of CDRs, or particular optimized CDR sequences (e.g., CDR sequences comprising amino acid substitutions, additions, or deletions), which provide binders with improved binding specificity, binding affinity, and / or function. Such methods for screening and optimizing binders are known (e.g., see Portolano et al. (1993) Journal of Immunology 150:880-887; and Clarkson et al. (1991) Nature 352:624-628). These references teach methods for generating antibodies that bind a specific antigen by screening a library of complementary variable regions using a known variable light chain, a known variable heavy chain, or a portion thereof (e.g., its CDRs).

[0232] In certain embodiments, the binder is modified to eliminate or add glycosylation sites to optimize the affinity and / or function of the binder (see, e.g., Co et al. (1993) Mol. Immunol. 30:1361-1367). In some embodiments, the number and / or type of glycosylation sites in the binder are modified or altered. N-linked glycosylation sites are typically characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated X can be any amino acid residue other than proline. Substitution of the amino acid residues that give rise to this sequence provides potential new sites for the addition of N-linked carbohydrate chains. Alternatively, substitution that eliminates this sequence will remove existing N-linked carbohydrate chains. Rearrangement of N-linked carbohydrate chains is also provided in certain embodiments, where one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are generated. In some embodiments, the binder is modified by deleting one or more cysteine residues or substituting one or more cysteine residues with another amino acid (e.g., serine) compared to the unmodified binder. In certain embodiments, cysteine variants can be used to optimize expression, secretion, and / or solubility.

[0233] In certain embodiments, the binder is modified to include certain amino acid additions, substitutions, or deletions that are designed or intended to, for example, reduce the susceptibility of the binder to proteolysis, reduce the susceptibility of the binder to oxidation, increase the serum half-life, and / or confer or alter other physicochemical, pharmacokinetic, or functional properties of the binder.

[0234] In some embodiments, the binding agent specifically binds to mammalian cMET or a portion thereof. In some embodiments, the binding agent specifically binds to the extracellular domain or extracellular region of mammalian cMET or a portion thereof. In certain aspects, the binding agent specifically binds to wild-type cMET produced by cells of a mammalian that is unaltered (not genetically engineered) as it exists in nature. In certain aspects, the binding agent specifically binds to a naturally occurring cMET variant. In certain aspects, the binding agent specifically binds to cMET comprising one or more amino acid substitutions, additions, or deletions. In certain embodiments, the binding agent specifically binds to cMET produced and / or expressed on the cell surface of a human, non-human primate, dog, cat, or rodent (e.g., mouse or rat). In certain embodiments, the binding agent specifically binds to one or more cMET polypeptides or portions thereof having an amino acid sequence of any one of SEQ ID NOs: 109 to 113. In certain embodiments, the binding agent specifically binds to human cMET. In certain embodiments, the binding agent specifically binds to the extracellular domain of human cMET. In certain embodiments, the binding agent specifically binds to human cMET and / or its extracellular domain, wherein the human cMET comprises an E168 to D168 substitution (i.e., the E168D variant of cMET). In certain embodiments, the binding agent specifically binds to human cMET and / or its extracellular domain, wherein the human cMET comprises an N375 to S375 substitution (i.e., the N375S variant of human cMET).

[0235] The term "specifically binds" refers to a binding agent that preferentially binds to a target peptide relative to binding to other molecules or other peptides, as determined, for example, by a suitable in vitro assay (e.g., Elisa, immunoblotting, flow cytometry, etc.). The specific binding interaction is distinguishable from a non-specific binding interaction by about 2-fold or more, typically about 10-fold or more, and sometimes about 100-fold or more, 1000-fold or more, 10,000-fold or more, 100,000-fold or more, or 1,000,000-fold or more.

[0236] In some embodiments, the binder that specifically binds to cMET or a portion thereof is a binder that binds to cMET or a portion thereof (e.g., the extracellular domain of cMET) with the following binding affinity constant (KD): equal to or less than 100 nM, equal to or less than 50 nM, equal to or less than 25 nM, equal to or less than 10 nM, equal to or less than 5 nM, equal to or less than 1 nM, equal to or less than 900 pM, equal to or less than 800 pM, equal to or less than 750 pM, equal to or less than 700 pM, equal to or less than 600 pM, equal to or less than 500 pM, equal to or less than 400 pM, equal to or less than 300 pM, equal to or less than 200 pM, or equal to or less than 100 pM. In some embodiments, the binder that specifically binds to cMET or a portion thereof is a binder that binds to human cMET or a portion thereof (e.g., the extracellular domain of human cMET) with the following binding affinity constant (KD): equal to or less than 100 nM, equal to or less than 50 nM, equal to or less than 25 nM, equal to or less than 10 nM, equal to or less than 5 nM, equal to or less than 1 nM, equal to or less than 900 pM, equal to or less than 800 pM, equal to or less than 750 pM, equal to or less than 700 pM, equal to or less than 600 pM, equal to or less than 500 pM, equal to or less than 400 pM, equal to or less than 300 pM, equal to or less than 200 pM, or equal to or less than 100 pM. In some embodiments, the binder that specifically binds to cMET or a portion thereof is a binder that specifically binds to cMET or a portion thereof from a non-human species (e.g., non-human primate, or rodent; e.g., mouse or rat) with the following binding affinity constant (KD): equal to or less than 100 nM, equal to or less than 50 nM, equal to or less than 25 nM, equal to or less than 10 nM, equal to or less than 5 nM, equal to or less than 1 nM, equal to or less than 900 pM, equal to or less than 800 pM, equal to or less than 750 pM, equal to or less than 700 pM, equal to or less than 600 pM, equal to or less than 500 pM, equal to or less than 400 pM, equal to or less than 300 pM, equal to or less than 200 pM, or equal to or less than 100 pM. In certain embodiments, the binders disclosed herein specifically bind to human cMET or a portion thereof and specifically bind to cMET or a portion thereof from a non-human primate. In certain embodiments, the binders disclosed herein specifically bind to human cMET or a portion thereof and specifically bind to cMET or a portion thereof from a rodent (e.g., mouse or rat).In certain embodiments, the binder (i) specifically binds to human cMET or a portion thereof (e.g., the extracellular domain of human cMET) with a KD of 10 nM or lower, or 1 nM or lower, and (ii) specifically binds to rat or mouse cMET or a portion thereof (e.g., the extracellular domain of rat or mouse cMET) with a KD of 100 nM or lower, 90 nM or lower, 80 nM or lower, 70 nM or lower, 60 nM or lower, 50 nM or lower, 40 nM or lower, 30 nM or lower, 20 nM or lower, or 10 nM or lower.

[0237] In certain embodiments, the binder comprises one or more functional features. Thus, the binder can be described structurally and functionally (e.g., by what it does, or by what it is capable of doing). The binders disclosed herein can specifically bind to the extracellular portion of cMET (e.g., the extracellular portion of cMET present on the cell surface). In some embodiments, the cell is a human cancer cell or a human tumor cell expressing cMET. In certain embodiments, the binders disclosed herein induce internalization of cMET after binding to cMET on the cell surface. The ability of the cMET binder to induce internalization and / or degradation of cMET provides an advantage over other cMET binders lacking this ability. The cMET binder-drug conjugate that induces internalization and / or degradation of cMET after binding provides for local intracellular delivery of a cytotoxic drug. Additionally, in some embodiments, the binder-drug conjugate is configured to release the PBD toxin from the binder only after internalization, e.g., by a lysosomal enzyme cleavage site incorporated into the linker. Thus, the binder-drug conjugates described herein can specifically deliver a toxin inside cancer cells expressing cMET in a subject while minimizing non-specific cytotoxicity to healthy cells. Thus, the anti-cMET binder-drug conjugates described herein provide for higher potency (e.g., target-specific cytotoxicity) and lower adverse side effects (e.g., lower non-specific cytotoxicity). Thus, in certain embodiments, the binder-drug conjugate comprises a binder that specifically binds to cMET on the cell surface of a cell and induces internalization of cMET after binding. In some embodiments, the binder specifically binds to cMET or a portion thereof and induces degradation of cMET. Thus, in certain embodiments, the binder-drug conjugate comprises a binder that specifically binds to cMET on the cell surface of a cell and induces internalization and / or degradation of cMET after binding. Internalization and / or degradation of cell surface-bound receptors induced by the binding of a ligand or binder is a known biological process that can be detected, measured, and / or quantified using suitable assays known in the art. Thus, the ability of a binder to induce internalization and / or degradation of cMET can be determined by using a suitable experimental assay without undue experimentation. Thus, in some embodiments, the binders described herein are binders that specifically bind to cMET or a portion thereof on the cell surface and induce internalization and / or degradation of cMET.

[0238] Activation of cMET by binding of cMET homologous ligands is associated with tumor growth, angiogenesis, and metastasis. Agonist anti-cMET antibodies typically mimic ligand binding by crosslinking the cMET receptor and inducing cMET activation. Thus, binders that bind cMET on the cell surface without activating the cMET receptor are more suitable for anticancer therapeutic applications. In some embodiments, the binder of the binder-drug conjugate specifically binds to cMET or a portion thereof on the cell surface and undetectably induces or promotes signal transduction (e.g., tyrosine kinase activity). In some embodiments, the binder of the binder-drug conjugate specifically binds to cMET or a portion thereof on the cell surface and substantially does not activate cMET (e.g., tyrosine kinase activity). In certain embodiments, the anti-cMET binders disclosed herein do not have detectable cMET agonist activity. In certain embodiments, the anti-cMET binder lacks agonist activity after binding cMET on the cell surface and / or is unable to induce or promote detectable tyrosine kinase activity after binding to cMET on the cell surface. In some embodiments, the anti-cMET binder is a cMET antagonist. In certain embodiments, the anti-cMET binder reduces, inhibits, decreases, blocks, or prevents signal transduction through the cMET receptor and / or reduces, inhibits, decreases, blocks, or prevents the cMET receptor from inducing or promoting detectable tyrosine kinase activity. In some embodiments, the anti-cMET binders disclosed herein reduce, inhibit, decrease, prevent, or block the binding of cMET to its native homologous ligand (e.g., hepatocyte growth factor or its isoforms).

[0239] In some embodiments, the binder comprises a label. As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, e.g., by incorporation of a labeled amino acid, or polypeptide linkage to a biotin moiety, which can be detected by a labeled avidin (e.g., streptavidin comprising a fluorescent marker or enzyme activity detectable by optical or colorimetric methods). In certain embodiments, the label or marker can be linked to the binder to produce a therapeutic or diagnostic agent. The binder can be linked covalently or non-covalently to any suitable label or marker. A variety of methods for labeling polypeptides and glycoproteins are known to those of skill in the art and can be used. Some non-limiting examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 125 I, 131I), fluorescent labels, enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, metal labels, chromophores, electrochemiluminescent labels, phosphorescent labels, quenchers (e.g., fluorophore quenchers), fluorescence resonance energy transfer (FRET) pairs (e.g., donor and acceptor), dyes, enzyme substrates, small molecules, mass tags, quantum dots, nanoparticles, biotin groups, predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal-binding domains, epitope tags), etc., or combinations thereof.

[0240] In some embodiments, the binder comprises a suitable carrier. The binder can be covalently or non-covalently linked to a suitable carrier. In certain embodiments, the carrier is a reagent or molecule that modifies or prolongs the in vivo half-life of the binder or improves its pharmacokinetic properties. Some non-limiting examples of carriers include polyethylene glycol, glycogen (e.g., by glycosylation of the binder), dextran, and the carriers or vehicles described in U.S. Patent No. 6,660,843, etc., or combinations thereof.

[0241] Load

[0242] PBD Toxin

[0243] In certain embodiments, the binder-drug conjugate comprises a binder as described herein (e.g., a binder that specifically binds to cMET) and a payload (e.g., a cytotoxic payload). The payload of the binder-drug conjugate is typically covalently linked to the binder. In some embodiments, the payload comprises pyrrolobenzodiazepine (PBD) toxins. In some embodiments, the payload comprises a linker or a suitable linker. In some embodiments, the payload comprises pyrrolobenzodiazepine (PBD) toxins and a linker. In certain embodiments, the payload comprises pyrrolobenzodiazepine (PBD) toxins and a linker, wherein the pyrrolobenzodiazepine toxin is covalently linked to the linker, and the linker is covalently linked to the binder as described herein.

[0244] Some non-limiting examples of PBD toxins and methods of making PBD toxins are described in the following patent application publications: US2011 / 0256157, WO / 2015 / 052322, US 2016 / 0106861, US2007 / 0072846, US2011 / 0201803, US2010 / 0113425, US2008 / 0167293, US2014 / 0127239, US2015 / 0158869, US2015 / 0344482, US2015 / 0111880, US2015 / 0315196, US2016 / 0015828, US2014 / 0088089, US2013 / 0035484, US2011 / 0196148, US2013 / 0028919, US2013 / 0059800, US2014 / 0274907, US2014 / 0275522, US2014 / 0234346, US2013 / 0266595, US2014 / 0302066, US2014 / 0286970, US2014 / 0294868, US2016 / 0144052, US2016 / 0031887, US2014 / 0120118, US2016 / 0250344, WO / 2017 / 137553, WO / 2017 / 137555, and WO / 2017 / 186894, the entire contents of which are incorporated herein by reference in their entirety.

[0245] In some embodiments, the pyrrolobenzodiazepine toxin comprises a structure of Formula I:

[0246] [Chemical Formula 16]

[0247]

[0248] wherein both Z1 and Z2 are N; both Z3 and Z4 are C;

[0249] [Chemical Formula 17]

[0250] Double dashed line represents a single bond or a double bond;

[0251] n is from 1 to 12; R3 and R4 are each independently H or C 1-4 alkoxy; and R1 and R2 are each independently selected from H, C 1-5 alkyl, C 3-6 cycloalkyl, C 2-5 alkenyl, and phenyl optionally substituted by R5, where R5 is selected from -NH2, -NHR6, and a piperazinyl group substituted by R7 having the following structure,

[0252] [Chemical Formula 18]

[0253]

[0254] wherein R6 contains a linking group, and R7 is empty or C 1-5 alkyl; X1 is empty, a protecting group or contains a linking group; X2 is empty, a protecting group or contains a linking group; only one of X1, X2, R1 and R2 contains a linking group; and Y1 and Y2 are each independently empty, OH or SO3; provided that:

[0255] [Chemical Formula 19]

[0256] (i) when X1 contains a linking group, is N-C, (ii) when X2 contains a linking group, is N-C, (iii) when X1 contains a protecting group, is N-C, and (iv) when X2 contains a protecting group, is N-C.

[0257] In certain embodiments, the PBD toxin contains only one linking group. For example, in Chemical Formula I, only one of X1, X2, R1 and R2 may contain a linking group. For example, in the case where X1 contains a linking group, X2, R1 and R2 do not contain a linking group.

[0258] In certain embodiments of the PBD toxin of Chemical Formula I, n is from 1 to 12. In certain embodiments of the PBD toxin of Chemical Formula I, n is from 1 to 10, 1 to 9, 1 to 7, 1 to 5 or 1 to 3. In certain embodiments of the PBD toxin of Chemical Formula I, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, n is 1, 3 or 5. In some embodiments, n is 3 or 5.

[0259] In certain embodiments of the PBD toxin of Chemical Formula I, R3 and R4 are independently C 1-4 alkoxy. In certain embodiments of the PBD toxin of Chemical Formula I, R3 and R4 are independently selected from -O-CH2CH3 or -O-CH3. In certain embodiments of the PBD toxin of Chemical Formula I, both R3 and R4 are -O-CH3.

[0260] In certain embodiments of the PBD toxin of Chemical Formula I, R1 and R2 are independently selected from H, C 1-5 alkyl, C3-C6 cycloalkyl and C 2-5Alkenyl. R1 and R2 may be the same or different. In some embodiments, R1 and R2 are independently selected from C1-C3 alkyl and C2-C3 alkenyl. In certain embodiments, R1 and R2 are independently selected from -CH2CH2CH3 and -CH3. In certain embodiments, both R1 and R2 are -CH2CH2CH3 or -CH3.

[0261] In certain embodiments of the PBD toxin of Formula I, R1 and R2 are independently selected from C3-C6 cycloalkyl and phenyl optionally substituted with R5, where R5 is selected from -NH2, -NHR6, and piperazinyl substituted with R7 having the following structure

[0262] [Chemical formula 20]

[0263]

[0264] where R6 contains a linker, and R7 is empty or C 1-5 alkyl. In certain embodiments, R1 and R2 are different and independently selected from: (i) C3-C6 cycloalkyl, and (ii) phenyl optionally substituted with R5, where R5 is selected from -NH2 and -NHR6, where R6 contains a linker. In certain embodiments, R1 and R2 are different and independently selected from: (i) C3 cycloalkyl, and (ii) phenyl substituted with -NH2 or -NHR6, where R6 contains a linker. In certain embodiments, R1 and R2 are different and independently selected from: (i) phenyl optionally substituted with R5, where R5 is selected from -NH2 and -NHR6, where R6 contains a linker, and (ii) piperazinyl substituted with R7 having the said structure, where R7 is empty or C1-C2 alkyl. In certain embodiments, R1 and R2 are different and independently selected from: (i) phenyl substituted with R5, where R5 is -NH2 and -NHR6, where R6 contains a linker, and (ii) piperazinyl substituted with R7 having the said structure,

[0265] [Chemical formula 21]

[0266]

[0267] where R7 is -CH3. In certain embodiments, R2 is phenyl substituted with 4-methylpiperazin-1-yl.

[0268] In certain embodiments of the PBD toxin of Formula I, X1 is empty, Y1 is empty, Z1Z3 is N═C, X2 is empty, Y2 is empty, and Z2Z4 is N═C. In certain embodiments of the PBD toxin of Formula I, X1 contains a linker, Y1 is OH, Z2Z4 is N═C, X2 is empty, and Y2 is empty. In certain embodiments of the PBD toxin of Formula I, X1 contains a linker, Y1 is OH, Z2Z4 is N-C, X2 is a protecting group, and Y2 is OH.

[0269] In some embodiments, the PBD toxin comprises the structure of Formula VII shown below:

[0270] [Chemical Formula 22]

[0271]

[0272] wherein X1 contains a linker.

[0273] In some embodiments, the PBD toxin comprises the structure of Formula VIII shown below:

[0274] [Chemical Formula 23]

[0275]

[0276] wherein X1 contains a linker.

[0277] In some embodiments, the PBD toxin comprises the structure of Formula IX shown below:

[0278] [Chemical Formula 24]

[0279]

[0280] wherein R6 contains a linker.

[0281] In some embodiments, the PBD toxin comprises the structure of Formula X shown below:

[0282] [Chemical Formula 25]

[0283]

[0284] wherein R6 contains a linker.

[0285] In some embodiments, the PBD toxin is linked (e.g., covalently linked) to a linker via a suitable bond, moiety, or group. In some embodiments, the PBD toxin is linked (e.g., covalently linked) to a linker via a carbonyl linkage or an amide linkage. In some embodiments, the PBD toxin is linked (e.g., covalently linked) to a linker via a carbamate group. In some embodiments, the PBD toxin is linked (e.g., covalently linked) to a linker via an amide group. Some non-limiting examples of linking the PBD toxin to a linker are described in US2017 / 0002096, US 2016 / 0331842, US2015 / 0250896, US2017 / 0080103, US2016 / 0136300, US2017 / 0152274, US2015 / 0209444, US2013 / 0274091, US2017 / 0095570, US2017 / 0157264, US2015 / 0125474, US2011 / 0256157, WO / 2015 / 052322, US2016 / 0106861, US2007 / 0072846, US2011 / 0201803, US2010 / 0113425, US2008 / 0167293, US2014 / 0127239, US2015 / 0158869, US2015 / 0344482, US2015 / 0111880, US2015 / 0315196, US2016 / 0015828, US2014 / 0088089, US2013 / 0035484, US2011 / 0196148, US2013 / 0028919, US2013 / 0059800, US2014 / 0274907, US2014 / 0275522, US2014 / 0234346, US2013 / 0266595, US2014 / 0302066, US2014 / 0286970, US2014 / 0294868, US2016 / 0144052, US2016 / 0031887, US2014 / 0120118, US2016 / 0250344, WO / 2017 / 137553, WO / 2017 / 137555, and WO / 2017 / 186894, the entire contents of which are incorporated herein by reference in their entirety.

[0286] As used herein, the term "empty" means the absence of the indicated moiety in the structure, however, the indicated moiety may be replaced or occupied by one or more hydrogen atoms to achieve the desired valence. Further, with reference to any structure shown herein, one or more hydrogens may be present to achieve the desired valence of the carbon, nitrogen, or oxygen atoms shown in the structure. Thus, one or more hydrogen atoms may be present unless explicitly indicated otherwise.

[0287] Linker

[0288] In some embodiments, the payload comprises a linker that facilitates, in part, the linkage between the binder and the PBD toxin. In certain embodiments, any suitable linker can be used to link the PBD toxin to the binder. Some non-limiting examples of linkers and methods of preparing linkers are described in WO / 2015 / 052322, US2015 / 0158869, US2015 / 0344482, US 2014 / 0127239, US2017 / 0002096, US2016 / 0331842, US2015 / 0250896, US2017 / 0080103, US2016 / 0136300, US2017 / 0152274, US2015 / 0209444, US2013 / 0274091, US2017 / 0095570, US2017 / 0157264, and US2015 / 0125474, which are incorporated herein by reference in their entirety. In some embodiments, the linker comprises a C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkoxy, one or more amino acids or amino acid derivatives, a peptide containing 1 to 20 amino acids, a phenyl group, a suitable polymer (e.g., polyethylene glycol), or a combination thereof.

[0289] In some embodiments, the linker comprises a structure of Formula A:

[0290] [Chemical Formula 26]

[0291]

[0292] where the asterisk indicates the point of attachment of the linker to the pyrrolobenzodiazepine toxin, the wavy line indicates the point of attachment of the linker to the binder, m is from 0 to 20, q is from 0 to 10, and E is the linker. In some embodiments of the linker of Formula A, m is from 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 to 8, or 4 or 8. In some embodiments of the linker of Formula A, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments of the linker of Formula A, q is from 1 to 10, 1 to 8, 1 to 6, or 1 to 4. In some embodiments of the linker of Formula A, q is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments of the linker of Formula A, q is 0, 1, or 2. 18. In some embodiments of the linker of Formula A, m is 8 and q is 2.

[0293] In some embodiments, the linker comprises a structure of Formula B:

[0294] [Chemical 27]

[0295]

[0296] wherein the asterisk indicates the point of attachment of the linker to the pyrrolobenzodiazepine toxin, the wavy line indicates the point of attachment of the linker to the binder, v is from 0 to 10, and u is 0 or 1, wherein when u is 1, t is from 1 to 10, and E is the linker. In some embodiments of the linker of Formula B, v is from 1 to 10, from 1 to 8, from 1 to 4, or from 0 to 4. 21. In some embodiments of the linker of Formula B, v is selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments of the linker of Formula B, when u is 1, t is from 1 to 8, from 1 to 5, from 1 to 4, or from 2 to 5. In some embodiments of the linker of Formula B, when u is 1, t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments of the linker of Formula B, t is 8, u is 1, and v is 2. In some embodiments of the linker of Formula B, u is 0, and v is 4.

[0297] The linker E of Formulas A and B can comprise any suitable bond, linker or moiety, some non-limiting examples of which include disulfide bonds, thioether bonds, thioester bonds, amide bonds, amines, ketones, carboxylate ethers, carbamates, esters, thioesters, etc., or combinations thereof. In certain embodiments, E comprises a covalent linkage between the linker and the binder. In some embodiments, E comprises a covalent bond. In some embodiments, E comprises a reacted moiety that remains after a suitable conjugation reaction. A variety of conjugation reactions are known in the art, any of which can be used to covalently link the linkers disclosed herein to the binders disclosed herein. Any suitable conjugation chemistry can be used to covalently link the linker to the binder randomly or site-specifically, some non-limiting examples of which include the conjugation reactions described in the following: Shan S. Wong (published June 18, 1991) Chemistry of Protein Conjugation and Cross-Linking, CRC Press; Greg T. Hermanson (copyright 2013) Bioconjugate Techniques, Third Edition, Elsevier Inc.; and Thiol-X Chemistries in Polymer and Materials Science, RSC Polymer Chemistry Series No. 6 (2013) edited by Andrew B. Lowe and Christopher N. Bowman, RSC Publishing; WO / 2015 / 052322; US2015 / 0158869; US2015 / 0344482; US2014 / 0127239; US2017 / 0002096; US2016 / 0331842; US2015 / 0250896; US2017 / 0080103; US2016 / 0136300; US2017 / 0152274; US2015 / 0209444; US2013 / 0274091; US2017 / 0095570; US2017 / 0157264 and US 2015 / 0125474, the entire contents of which are incorporated herein by reference in their entirety.Other non-limiting examples of conjugating a payload or a linker to a binder include: reacting an amine or amino group with an N-hydroxysuccinimide (NHS) ester, succinimidyl succinate, succinimidyl succinamide, succinimidyl propionate, succinimidyl carbonate, carbonyloxyimidazole, nitrophenyl carbonate, trichlorophenyl carbonate, tresylate, maleic anhydride, methyl maleic anhydride, imidoester, pentafluorophenyl (PFP) ester, hydroxymethylphosphine, ethylene oxide, or any other carbonyl moiety; reacting a carboxyl moiety with a carbodiimide; reacting a thiol moiety with maleimide, haloacetyl, pyridyl disulfide, ortho-pyridyl disulfide, and / or vinyl sulfone; reacting an aldehyde moiety with hydrazine or acylhydrazine; reacting any non-selective group with diazirine and / or aryl azide; reacting a hydroxyl moiety with isocyanate; reacting a hydroxylamine moiety with a carbonyl moiety; and the like, and combinations thereof.

[0298] Thus, E is generally defined chemically for conjugating a linker to a binder. In some embodiments, E comprises a suitable moiety configured to link a linker to a binder. In some embodiments, the linker is covalently linked to the binder via a suitable thiol-thiol reaction, such as by using a maleimide or pyridyl disulfide reactive group that reacts with reduced cysteine to form a stable thioether bond. Other non-limiting examples of reactive thiol-reactive moieties include: haloacetyl, aziridine, acryloyl, arylating agent, vinyl sulfone, pyridyl disulfide, and TNB-thiol. In certain embodiments, the binder is linked to E via a thioether bond formed between a cysteine thiol residue (e.g., a thiol) of the binder and E. Thus, in certain embodiments, E comprises a disulfide bond or a thioether bond. In some embodiments, for example, in the case of covalently linking a binder to a linker using a maleimide reaction, E comprises a structure of formula C:

[0299] [Chemical formula 28]

[0300]

[0301] where the wavy line indicates the point of attachment to the binder and the double asterisk (**) indicates the point of attachment to the linker. In certain embodiments, the double asterisk of formula C represents a thioether bond.

[0302] The payload, linker can be conjugated to any suitable amino acid of the binder randomly or site - specifically. In some embodiments, the payload, linker is conjugated to one or more suitable cysteines of the binder. In some embodiments, the payload, linker is conjugated to one or more suitable lysine residues of the binder. In certain embodiments, one or more amino acids of the binder are replaced with amino acids suitable for conjugation to the payload, linker. Some non - limiting examples of amino acids that can be replaced with amino acid residues containing a thiol group or lysine residues include A118, S119, S239, V282, T289, N361, and V422 of IgG2, S115, S252, V289, T306, and N384 of IgG1, or the corresponding positions in IgG1, IgG2, IgG3, or IgG4. Incorporating cysteine into the antibody by mutagenesis allows for direct conjugation of the payload, linker to specific sites on the antibody, for example, via disulfide bonds or thioether bonds. For example, one or more amino acids of the binder can be replaced with cysteine, where the cysteine can be used for site - specific conjugation of the payload, linker using suitable chemical reactions. Any suitable amino acid in the antibody constant region can be mutated to cysteine or lysine for site - specific conjugation to the payload, linker. The stability of the antibody - drug conjugate produced by site - specific conjugation can be evaluated by methods known in the art.

[0303] In some embodiments, the linker contains a suitable enzyme cleavage site. In certain embodiments, the enzyme cleavage site contains an enzyme recognition site for a mammalian protease. Thus, in some embodiments, the linker or a portion thereof can be cleaved by a mammalian protease. The linker can be cleaved by an enzyme present at or near the target site (e.g., at or near the cMET protein). The enzyme present at or near the target site can be intracellular, membrane - bound, membrane - associated, or extracellular (e.g., secreted). For example, the linker can be configured to be cleaved by a cell - surface protease, a secreted protease, or an intracellular protease (e.g., a lysosomal protease). Some non - limiting examples of enzyme cleavage sites include protease recognition sites for lysosomal cysteine proteases and / or lysosomal aspartic proteases. Some non - limiting examples of lysosomal proteases include cathepsin B, C, H, I, J, K, L, M, N, O, P, S, T, and X and cathepsin D, E, F, G, and / or cathepsin A (carboxypeptidase A).

[0304] Protecting Group

[0305] In some embodiments, the PBD toxin comprises a suitable protecting group. Some non-limiting examples of protecting groups and methods for preparing protecting groups are described in the following patent application publications: US 2011 / 0256157, WO / 2015 / 052322, US2011 / 0201803, US2008 / 0167293, US2014 / 0127239, US2015 / 0158869, US2015 / 0344482, US2015 / 0315196, US2015 / 0315196, US2014 / 0302066, US2006 / 0264622, and US2015 / 0133435, the entire contents of which are incorporated herein by reference in their entirety.

[0306] In some embodiments, the protecting group comprises a structure of Formula D below:

[0307] [Chemical Formula 29]

[0308]

[0309] wherein the asterisk indicates the point of attachment to the pyrrolobenzodiazepine toxin; and w is from 0 to 10. In some embodiments, w is from 0 to 8, from 0 to 6, from 0 to 4, from 1 to 10, from 1 to 8, from 1 to 5, or from 1 to 4. In certain embodiments, w is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, w is 2.

[0310] In some embodiments, the protecting group is removable. In certain embodiments, the protecting group can be cleaved using suitable chemical methods.

[0311] In some embodiments, the payload comprises a structure of Formula II:

[0312] [Chemical Formula 30]

[0313]

[0314] wherein m is 8, and the wavy line indicates the point of attachment to the binder.

[0315] In some embodiments, the payload comprises a structure of Formula III:

[0316] [Chemical Formula 31]

[0317]

[0318] wherein m is 8, p is 1 or 3, X2 is empty or a protecting group, and the wavy line indicates the point of attachment to the binder. In certain embodiments, the payload comprises a structure of Formula IV:

[0319] [Chemical formula 32]

[0320]

[0321] Wherein the wavy line indicates the point of attachment to the binder.

[0322] In some embodiments, the payload comprises a structure of Chemical formula V':

[0323] [Chemical formula 33]

[0324]

[0325] Wherein m is 8, E is a suitable linking group, and the wavy line indicates the point of attachment to the binder. In some embodiments, E comprises the succinamide moiety of Structure C:

[0326] [Chemical formula 34]

[0327]

[0328] Wherein the wavy line indicates the point of attachment to the binder, and the double asterisk indicates the point of attachment to the payload of Chemical formula V. The payload of Chemical formula V comprising a linking group of Structure C is sometimes referred to herein as Chemical formula XI.

[0329] In some embodiments, the payload comprises a structure of Chemical formula VI:

[0330] [Chemical formula 35]

[0331]

[0332] Wherein t is 8, v is 1, and the wavy line indicates the point of attachment to the binder.

[0333] In some embodiments, the payload comprises a structure of Chemical formula VII:

[0334] [Chemical formula 36]

[0335]

[0336] Wherein the wavy line indicates the point of attachment to the binder.

[0337] In some embodiments, the binder-drug conjugate comprises a payload and a binder, the payload comprising a structure selected from any one of Formulas II, III, IV, V, VI, VII, and XI, and the binder comprising: CDR-L1 having an amino acid sequence selected from SEQ ID NO:2, 4, 6, 8, 10, 12, and 14; CDR-L2 having an amino acid sequence selected from SEQ ID NO:17, 19, 21, 23, and 25; CDR-L3 having an amino acid sequence selected from SEQ ID NO:27, 29, 31, 33, and 35; CDR-H1 having an amino acid sequence selected from SEQ ID NO:51, 53, 55, 57, and 59; CDR-H2 having an amino acid sequence selected from SEQ ID NO:63, 65, 67, 69, 71, 73, and 75; and CDR-H3 having an amino acid sequence selected from SEQ ID NO:80, 82, 84, 86, 88, 91, and 93.

[0338] In some embodiments, the binder-drug conjugate comprises a payload and a binder, the payload comprising a structure selected from any one of Formulas II, III, IV, V, VI, VII, and XI, and the binder comprising: CDR-L1 having the amino acid sequence of SEQ ID NO:10 or 14; CDR-L2 having the amino acid sequence of SEQ ID NO:21; CDR-L3 having the amino acid sequence of SEQ ID NO:35; CDR-H1 having the amino acid sequence of SEQ ID NO:59; CDR-H2 having the amino acid sequence of SEQ ID NO:71; and CDR-H3 having the amino acid sequence of SEQ ID NO:88.

[0339] In some embodiments, the binder-drug conjugate comprises a payload and a binder, the payload comprising a structure selected from any one of Formulas II, III, IV, V, VI, VII, and XI, and the binder comprising: CDR-L1 having the amino acid sequence of SEQ ID NO:9; CDR-L2 having the amino acid sequence of SEQ ID NO:24; CDR-L3 having the amino acid sequence of SEQ ID NO:34; CDR-H1 having the amino acid sequence of SEQ ID NO:58; CDR-H2 having the amino acid sequence of SEQ ID NO:70; and CDR-H3 having the amino acid sequence of SEQ ID NO:87.

[0340] In some embodiments, the conjugate-drug conjugate comprises a payload and a conjugate, the payload comprising a structure selected from any one of Formulas II, III, IV, V, VI, VII, and XI, and the conjugate comprising: a light chain variable region having at least 90% sequence identity with an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 37 to 44, and a heavy chain variable region having at least 90% sequence identity with an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 94 to 103.

[0341] In some embodiments, the conjugate-drug conjugate comprises a payload and a conjugate, the payload comprising a structure selected from any one of Formulas II, III, IV, V, VI, VII, and XI, and the conjugate comprising: a light chain variable region having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 37 to 44, and a heavy chain variable region having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 94 to 103.

[0342] In some embodiments, the conjugate-drug conjugate comprises a payload and a conjugate, the payload comprising a structure selected from any one of Formulas II, III, IV, V, VI, VII, and XI, and the conjugate comprising: a light chain variable region having at least 90% sequence identity with an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 45 to 49, and a heavy chain variable region having at least 90% sequence identity with an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 104 to 108.

[0343] In some embodiments, the conjugate-drug conjugate comprises a payload and a conjugate, the payload comprising a structure selected from any one of Formulas II, III, IV, V, VI, VII, and XI, and the conjugate comprising: a light chain variable region containing an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 45 to 49, and a heavy chain variable region containing an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 104 to 108.

[0344] Pharmaceutical Composition

[0345] In some embodiments, the composition or pharmaceutical composition comprises the conjugate-drug conjugate described herein. In some embodiments, the pharmaceutical composition comprises the conjugate-drug conjugate and a pharmaceutically acceptable excipient, diluent, additive, or carrier.

[0346] A pharmaceutical composition can be formulated for a suitable route of administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous (s.c.), intradermal, intramuscular, intraperitoneal, and / or intravenous (i.v.) administration. In certain embodiments, the pharmaceutical composition may comprise formulation materials for altering, maintaining, or preserving properties of the composition such as pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability. In certain embodiments, suitable formulation materials include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate (such as phosphate buffered saline), or a suitable organic acid); fillers (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring agents, flavoring agents, and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; counterions for salting (such as sodium); solvents (such as glycerol, propylene glycol, or polyethylene glycol); diluents; excipients, and / or pharmaceutical adjuvants. In particular, the pharmaceutical composition may comprise any suitable carrier, formulation, or ingredient, etc., listed below, or a combination thereof: “Remington: The Science And Practice Of Pharmacy” Mack Publishing Co., Easton, PA, 19th edition, (1995) (hereinafter referred to as, Remington’95), or “Remington: The Science And Practice Of Pharmacy”, Pharmaceutical Press, Easton, PA, 22nd edition, (2013) (hereinafter referred to as, Remington 2013), the contents of which are incorporated herein by reference in their entirety. The various materials listed herein, alone or in combination, may be incorporated into or used in conjunction with the materials described in Remington’95 or Remington 2013. Any suitable techniques, carriers, and excipients may be used, including those understood in the art, for example, as described in Remington’95 or Remington 2013.

[0347] In certain embodiments, the pharmaceutical composition comprises suitable excipients, some non-limiting examples of which include anti-adhesives (e.g., magnesium stearate), binders, fillers, monosaccharides, disaccharides, other carbohydrates (e.g., glucose, mannose or dextrin), sugar alcohols (e.g., mannitol or sorbitol), coatings (e.g., cellulose, hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose, synthetic polymers, shellac, gelatin, zein, enteric or other polysaccharides), starches (e.g., potato, corn or wheat starch), silica, pigments, disintegrants, flavors, lubricants, preservatives, sorbents, sweeteners, carriers, suspending agents, surfactants and / or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates (e.g., polysorbate 20, polysorbate 80), triton, tromethamine, lecithin, cholesterol, tyloxapal), stability enhancers (e.g., sucrose or sorbitol) and tonicity enhancers (e.g., alkali metal halides, sodium chloride or potassium chloride, mannitol, sorbitol), and / or any excipients disclosed in Remington’95 or Remington 2013. The term "binder" as used herein refers to a compound or ingredient that helps to keep the pharmaceutical mixture combined. Suitable binders for the preparation of pharmaceutical formulations and commonly used in the preparation of pharmaceutical tablets, capsules and granules are known to those skilled in the art. For clarity, the term "binding agent" as used herein does not refer to the "binder" used in certain pharmaceutical formulations. However, in certain embodiments, the pharmaceutical composition may comprise a binding agent that specifically binds cMET, as well as a binder.

[0348] In some embodiments, the pharmaceutical composition comprises suitable pharmaceutically acceptable additives and / or carriers. Some non-limiting examples of suitable additives include suitable pH regulators, soothing agents, buffers, sulfur-containing reducing agents, antioxidants, etc. Some non-limiting examples of sulfur-containing reducing agents include those having a mercapto group (e.g., thiol), such as N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and C1 to C7 thioalkanoic acids. Some non-limiting examples of antioxidants include isoascorbic acid, dibutylhydroxytoluene, butylated hydroxyanisole, α-tocopherol, tocopheryl acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, tripentyl gallate and propyl gallate, and chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate. In addition, diluents, additives, and excipients may contain other commonly used components, such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate, and organic salts such as sodium citrate, potassium citrate, and sodium acetate.

[0349] The pharmaceutical composition used herein can be stable for a long time (e.g., about several months or years). In some embodiments, the pharmaceutical composition comprises one or more suitable preservatives. Some non-limiting examples of preservatives include benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, hydrogen peroxide, etc., and / or combinations thereof. Preservatives can include quaternary ammonium compounds, such as benzalkonium chloride, benzoxonium chloride, benzethonium chloride, cetrimide, sepazonium chloride, cetylpyridinium chloride, or domiphen bromide (BRADOSOL (registered trademark)). Preservatives can include alkyl mercury salts of thiosalicylic acid, such as thimerosal, phenylmercuric nitrate, phenylmercuric acetate, or phenylmercuric borate. Preservatives can include parabens, such as methylparaben or propylparaben. Preservatives can include alcohols, such as chlorobutanol, benzyl alcohol, or phenethyl alcohol. Preservatives can include biguanide derivatives, such as chlorhexidine or polyhexamethylene biguanide. Preservatives can include sodium perborate, imidazolidinyl urea, and / or sorbic acid. Preservatives can include stabilized oxy-chlorine complexes, such as those known and commercially available under the trade name PURITE (registered trademark). Preservatives can include polyethylene glycol-polyamine condensation resins, such as those known and commercially available under the trade name POLYQUART (registered trademark) from Henkel KGaA. Preservatives can include stabilized hydrogen peroxide. The preservative can be benzalkonium chloride. In some embodiments, the pharmaceutical composition is preservative-free.

[0350] In some embodiments, the composition, pharmaceutical composition, or binder-drug conjugate is substantially free of contaminants (e.g., blood cells, platelets, polypeptides, minerals, blood-borne compounds or chemicals, viruses, bacteria, other pathogens, toxins, etc.). In some embodiments, the composition, pharmaceutical composition, or binder-drug conjugate is substantially free of serum and serum contaminants (e.g., serum proteins, serum lipids, serum carbohydrates, serum antigens, etc.). In some embodiments, the composition, pharmaceutical composition, or binder-drug conjugate is substantially free of pathogens (e.g., viruses, parasites, or bacteria). In some embodiments, the composition, pharmaceutical composition, or binder-drug conjugate is substantially free of endotoxins. In some embodiments, the composition, pharmaceutical composition, or binder-drug conjugate is sterile. In certain embodiments, the composition or pharmaceutical composition comprises a binder-drug conjugate that specifically binds to the extracellular domain of cMET, and a suitable diluent (e.g., phosphate buffered saline).

[0351] The pharmaceutical compositions described herein can be configured for administration to a subject in any suitable form and / or amount depending on the treatment for which they are used. For example, a pharmaceutical composition configured for parenteral administration (e.g., by injection or infusion) can be in the form of a suspension, solution, or emulsion in an oily or aqueous carrier, and it can contain formulating agents, excipients, additives, and / or diluents such as aqueous or non-aqueous solvents, co-solvents, suspending agents, preservatives, stabilizers, and / or dispersing agents. In some embodiments, a pharmaceutical composition suitable for parenteral administration can contain one or more excipients. In some embodiments, the pharmaceutical composition is lyophilized into a dry powder form. In some embodiments, the pharmaceutical composition is lyophilized into a dry powder form that is suitable for reconstitution with a suitable pharmaceutical solvent (e.g., water, saline, isotonic buffer solution (e.g., PBS), etc.). In certain embodiments, the reconstituted form of the lyophilized pharmaceutical composition is suitable for parenteral administration (e.g., intravenous administration) to a mammal.

[0352] In certain embodiments, the pharmaceutical composition is configured for oral administration and can be formulated into tablets, microtablets, minitablets, pellets, powder granules, capsules (e.g., capsules filled with microtablets, pellets, powders or granules), emulsions or solutions. The pharmaceutical composition configured for oral administration may comprise a suitable coating to delay or sustain the release of the active ingredient (e.g., binder), some non-limiting examples of which include enteric coatings such as fatty acids, waxes, shellac, plastics, methyl acrylate-methacrylic acid copolymers, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, cellulose acetate trimellitate, sodium alginate, zein, vegetable fibers, and the like, and combinations thereof.

[0353] In some embodiments, the pharmaceutical compositions described herein can be configured for topical administration and may comprise one or more of a binder and / or lubricant, a polyalkylene glycol, gelatin, cocoa butter or other suitable wax or fat. In some embodiments, the pharmaceutical compositions described herein are incorporated into a topical formulation comprising a topical carrier that is generally suitable for topical drug administration and comprising any suitable materials known to those skilled in the art. In certain embodiments, the topical formulation of the pharmaceutical composition is formulated for administering the binder from a topical patch.

[0354] In certain embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art based on, for example, the intended route of administration, delivery form and desired dose (see, e.g., Remington’95 or Remington 2013, supra). In certain embodiments, such compositions may affect the physical state, stability, in vivo release rate and in vivo clearance rate of the antibody-drug conjugate of the present invention. The pharmaceutical composition can be prepared by any suitable means, including, for example, by conventional mixing, dissolving, granulating, preparing dragees, milling, emulsifying, encapsulating, entrapping or tableting processes (e.g., see the methods described in Remington’95 or Remington 2013).

[0355] Second Medical UseIn some embodiments, provided herein are compositions or pharmaceutical compositions that act as a medicament for treating cancer or a neoplastic disorder in a subject, wherein the composition or pharmaceutical composition comprises a binder-drug conjugate as described herein. In some embodiments, provided herein are compositions or pharmaceutical compositions comprising a binder-drug conjugate as described herein for treating cancer or a neoplastic disorder.

[0356] Treatment Method

[0357] In some embodiments, the compositions, pharmaceutical compositions or binder-drug conjugates described herein are used to treat a subject having or suspected of having a neoplastic disorder or cancer. In certain embodiments, the binder-drug conjugate or pharmaceutical composition described herein is used to treat a neoplastic disorder or cancer in a subject, wherein the binder-drug conjugate specifically binds to the extracellular domain of human cMET. In some embodiments, provided herein is a method of treating a subject having or suspected of having a neoplastic disorder or cancer. In certain embodiments, the method of treating a subject having or suspected of having a neoplastic disorder or cancer comprises administering to the subject a therapeutically effective amount of the composition, pharmaceutical composition or binder-drug conjugate described herein. In certain embodiments, the method comprises contacting a cell (e.g., one or more cells) of the subject with a therapeutically effective amount of the composition, pharmaceutical composition or binder-drug conjugate described herein. In certain embodiments, the method comprises contacting a cancer cell or tumor cell of the subject with a therapeutically effective amount of the composition, pharmaceutical composition or binder-drug conjugate described herein. In certain embodiments, the method comprises contacting a cell (e.g., one or more cells) of the subject with a therapeutically effective amount of a binder-drug conjugate that specifically binds to the extracellular portion of human cMET or a variant thereof. In certain embodiments, the method comprises contacting a cancer cell or tumor cell with a therapeutically effective amount of a binder-drug conjugate that specifically binds to the extracellular portion of human cMET or a variant thereof, wherein the cell expresses cMET on its cell surface. The cells of the subject are typically cells that express the extracellular portion of cMET. The cells contacted with the binder-drug conjugate can be within the subject (e.g., in vivo) or outside the subject (e.g., in vitro or ex vivo).

[0358] In certain embodiments, the binder-drug conjugate blocks, inhibits, ameliorates, alleviates, or represses the growth, viability, or metastasis of cancer or cancer cells. In certain embodiments, the binder-drug conjugate induces the death, necrosis, or apoptosis of cancer or cancer cells. In certain embodiments, contacting the cells of a subject with a binder-drug conjugate disclosed herein induces or promotes cell death, necrosis, or apoptosis. In certain embodiments, contacting the cells of a subject with a binder-drug conjugate disclosed herein induces or promotes cell death through ADCC, ADCP, or complement-dependent cellular cytotoxicity (CDCC). In certain embodiments, contacting the cells of a subject with a binder-drug conjugate disclosed herein reduces, inhibits, or decreases cell mitosis. In certain embodiments, contacting the cancer or cancer cells of a subject with a binder-drug conjugate disclosed herein reduces, inhibits, or decreases the metastasis of the cancer or cancer cells.

[0359] Subject

[0360] The term "subject" refers to a mammal. Any suitable mammal can be treated by the methods or compositions described herein. Some non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., adult, adolescent, child, infant, or mammalian in utero). The mammal can be male or female.

[0361] In some embodiments, a subject is in need of the treatment or composition described herein. In certain embodiments, the subject has or is suspected of having a neoplastic disorder or cancer. In some embodiments, a subject in need of the treatment or composition described herein has or is suspected of having a neoplastic disorder or cancer. In certain embodiments, a binder-drug conjugate or composition described herein is used to treat a subject having or suspected of having a neoplastic disorder or cancer.

[0362] Cancer Type

[0363] The compositions, pharmaceutical compositions or linker-drug conjugates disclosed herein can be used to treat neoplastic disorders or cancers, some non-limiting examples of which include: carcinoma, sarcoma, neuro neoplasia, lymphoma, myeloma, leukemia, melanoma, mesothelioma, solid or soft tissue tumors, and secondary cancers (e.g., originating from a primary site). Some non-limiting examples of carcinoma include: respiratory system carcinoma, gastrointestinal system carcinoma, urogenital system carcinoma, testicular cancer, prostate cancer, endocrine system carcinoma, basal cell carcinoma of the skin, carcinoma of unknown primary, cholangiocarcinoma, ductal carcinoma in situ (DCIS), Merkel cell carcinoma, lung cancer, thymoma and thymic carcinoma, midline tract carcinoma, small cell lung cancer, thyroid cancer, hepatocellular carcinoma, squamous cell carcinoma, squamous cell carcinoma of the head and neck, breast cancer, epithelial carcinoma, adrenocortical carcinoma, ovarian surface epithelial carcinoma, etc., and also include: uterine cancer, cervical cancer, colon cancer, pancreatic cancer, kidney cancer, esophageal cancer, gastric cancer and ovarian cancer. Some non-limiting examples of sarcoma include: Ewing sarcoma, lymphosarcoma, liposarcoma, osteosarcoma, breast sarcoma, soft tissue sarcoma, Kaposi sarcoma, rhabdomyosarcoma, uterine sarcoma, chondrosarcoma, leiomyosarcoma, fibrosarcoma, etc. Some non-limiting examples of neuro neoplasia include: glioma, glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma, etc.Some non-limiting examples of lymphomas, myelomas, and leukemias include: acute and chronic lymphocytic leukemia, granulocytic leukemia, multiple myeloma, poorly differentiated acute leukemia (e.g., erythroleukemia and acute megakaryoblastic leukemia), acute promyeloid leukemia (APML), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) (which includes B-lineage ALL and T-lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), Waldenstrom’s macroglobulinemia (WM), non-Hodgkin lymphoma and variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin disease, and Reed-Sternberg disease. Some non-limiting examples of soft tissue or solid tissue tumors include: visceral tumors, seminoma, hepatoma, and additional tumors of the following: breast, liver, lung, pancreas, uterus, ovary, testis, head, neck, eye, brain, mouth, pharynx, vocal cords, ear, nose, esophagus, stomach, intestine, colon, adrenal gland, kidney, bone, bladder, urethra, epithelial carcinoma, lung, muscle, skin, foot, hand, and soft tissue. In some embodiments, the neoplastic disorders or cancers treatable by the pharmaceutical compositions or binder-drug conjugates disclosed herein are selected from: bladder cancer, breast cancer, colorectal cancer, cervical cancer, gastric cancer, liver cancer, hepatocellular carcinoma, hypopharyngeal cancer, lung cancer, adenocarcinoma, ovarian cancer, and kidney cancer.In some embodiments, the neoplastic disorders or cancers treatable by the pharmaceutical compositions or binder-drug conjugates disclosed herein are selected from: pancreatic cancer (e.g., pancreatic adenocarcinoma, exocrine pancreatic cancer, or neuroendocrine pancreatic cancer), colorectal cancer (e.g., colorectal adenocarcinoma), small intestine malignancies, cholangiocarcinoma, non-small cell lung cancer (NSCLC), thyroid cancer, esophageal cancer or esophagogastric junction (EGJ) cancer, gastric adenocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, female genital tract malignancies, breast cancer, small cell lung cancer, ovarian surface epithelial carcinoma, retroperitoneal or peritoneal sarcoma, prostate adenocarcinoma, neuroendocrine tumors, gastrointestinal stromal tumors, glioblastoma, or non-epithelial ovarian cancer. In some embodiments, the neoplastic disorder or cancer treatable by the pharmaceutical compositions or binder-drug conjugates disclosed herein is breast cancer, some non-limiting examples of which include: ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) (e.g., breast ductal carcinoma, breast medullary carcinoma, breast mucinous carcinoma, breast papillary carcinoma, and breast cribriform carcinoma), invasive lobular carcinoma (ILC), inflammatory breast cancer, lobular carcinoma in situ (LCIS), male breast cancer, breast cancer molecular subtypes (e.g., luminal B breast cancer or hormone receptor-positive breast cancer, triple-negative breast cancer, HER2-enriched breast cancer, and normal-like breast cancer), Paget’s disease of the nipple, phyllodes tumor of the breast, and metastatic breast cancer. In some embodiments, the neoplastic disorder or cancer treatable by the pharmaceutical compositions or binder-drug conjugates disclosed herein is triple-negative breast cancer.

[0364] In some embodiments, the efficacy of the treatments described herein can be determined or predicted in part by the amount of cMET expressed by a cancer or neoplasia. Many cancer and tumor types are known to express cMET, some non-limiting examples of which include certain bladder cancers, breast cancers, colorectal cancers, gastric cancers, hepatocellular carcinomas, HNSCC, hypopharyngeal cancers, lung cancers, adenocarcinomas, ovarian cancers, and kidney cancers (see, e.g., Ariyawutyakorn et al. (2016) Journal of Cancer 7(6):633-649), and the amount of cMET expressed by many cancer types is known (see, e.g., Arguello et al. (2013) Annual Meeting of Association for Molecular Pathology (AMP) Abstract No. 294319). In addition, the cMET expression of tumor cells or cancer cells can be rapidly determined using a suitable anti-cMET binding agent (e.g., an antibody) using a suitable method (e.g., whole cell ELISA, FAC, any suitable immunoassay, etc.). Thus, in some embodiments, a method of treating a subject having or suspected of having cancer comprises administering to the subject a therapeutically effective amount of a binder-drug conjugate described herein or a pharmaceutical composition comprising a binder-drug conjugate described herein, wherein the cancer expresses a detectable level of cMET. In certain embodiments, a cancer expressing a detectable level of cMET can be a cancer known or reported to express cMET, or a cancer suspected of expressing cMET (e.g., by having a genotype or phenotype similar to another cancer known to express cMET). In some embodiments, a cancer expressing cMET or suspected of expressing cMET is a cancer that expresses an RNA transcript encoding cMET or a portion thereof. In some embodiments, a cancer expressing cMET or suspected of expressing cMET is a cancer that expresses cMET on its cell surface.

[0365] Route of Administration

[0366] Any suitable method for administering a composition, pharmaceutical composition, or binder-drug conjugate to a subject can be used. The exact formulation and route of administration of the composition used in accordance with the methods of the invention described herein can be selected by a medical professional (e.g., a physician) based on the circumstances of the patient. (See, e.g., Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, which is incorporated herein by reference in its entirety). Any suitable route of administration can be used to administer the pharmaceutical compositions or binder-drug conjugates described herein. Some non-limiting examples of routes of administration include topical or local (e.g., transdermal or through the skin, (e.g., on the skin or epidermis), intraocular or ocular, intranasal, transmucosal, in the ear, intratympanic (e.g., behind the ear drum)), enteral (e.g., delivery through the gastrointestinal tract, e.g., oral (e.g., as tablets, capsules, granules, liquids, emulsions, lozenges, or combinations thereof), sublingual, through a gastrostomy tube, rectal, etc.), by parenteral administration (e.g., parenterally, e.g., intravenous, intraarterial, intramuscular, intraperitoneal, intradermal, subcutaneous, intracavitary, intracranial, intraarticular, into the joint space, intracardiac (into the heart), intracavernous injection, intralesional (into a skin lesion), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intrauterine, intravaginal, intravesical infusion, intravitreal), etc., or combinations thereof.

[0367] In some embodiments, the compositions herein are provided to a subject. Sometimes the compositions provided to a subject are provided to the subject for self-administration or for administration to the subject by another person (e.g., a non-medical professional). For example, the compositions described herein can be provided in accordance with instructions (e.g., a prescription) written by a medical practitioner that permit the provision of the compositions or treatment described herein to a patient. In another example, a composition can be provided to a subject, where the subject self-administers the composition, e.g., orally, intravenously, or via an inhaler.

[0368] Alternatively, the compositions used in accordance with the methods of the invention can be administered in a local rather than a systemic manner, e.g., by direct application to the skin, mucosa, or target treatment area, including the use of depot or sustained release formulations.

[0369] In some embodiments, a pharmaceutical composition comprising a binder-drug conjugate can be administered alone (e.g., as a single active ingredient (AI) or, for example, as a single active pharmaceutical ingredient (API)). In other embodiments, a pharmaceutical composition comprising a binder-drug conjugate can be administered in combination with one or more additional AIs / APIs, e.g., as two separate compositions or as a single composition in which one or more of the additional AIs / APIs are mixed or formulated with the binder-drug conjugate in the pharmaceutical composition.

[0370] In certain embodiments, a cMET binder-drug conjugate is delivered to a cell (e.g., a mammalian cell). Any suitable method can be used to deliver the cMET binder-drug conjugate to a cell. In certain embodiments, delivering the cMET binder-drug conjugate to a cell comprises contacting a mammalian cell, in vitro or in vivo, with a composition comprising the cMET binder-drug conjugate under conditions that permit the binder-drug conjugate to bind to the cell.

[0371] Dose and Therapeutically Effective Amount

[0372] In some embodiments, the amount of the binder-drug conjugate in the composition is a therapeutically effective amount. In some embodiments, a therapeutically effective amount of the binder-drug conjugate is administered to a subject. In some embodiments, a therapeutically effective amount of the binder-drug conjugate in the composition is the amount required to achieve an effective therapeutic outcome. In certain embodiments, the amount of the binder-drug conjugate in a composition (e.g., a pharmaceutical composition) is an amount sufficient to prevent, treat, reduce the severity of, delay the onset of, and / or alleviate the symptoms of a neoplastic disorder or cancer contemplated herein.

[0373] "Therapeutically effective amount" means an amount sufficient to achieve an effective therapeutic outcome and / or an amount sufficient to prevent, treat, reduce the severity of, delay the onset of, and / or alleviate the symptoms of a neoplastic disorder or cancer. In certain embodiments, "therapeutically effective amount" means an amount sufficient to terminate the growth of a tumor or cancer and / or slow its growth. In certain embodiments, "therapeutically effective amount" means an amount sufficient to inhibit the replication of and / or induce the death of one or more tumor cells or cancer cells. Determination of a therapeutically effective amount is well within the ability of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0374] In certain embodiments, a therapeutically effective amount is an amount that is high enough to provide an effective therapeutic effect and is low enough to minimize undesirable adverse reactions. Thus, in certain embodiments, the therapeutically effective amount of the binder-drug conjugate can vary with the subject and generally depends on the subject's age, weight, general health, the severity of the condition being treated, and the particular combination of drugs being administered. Accordingly, in some embodiments, the therapeutically effective amount is determined empirically. Thus, the therapeutically effective amount of the binder-drug conjugate for treating a subject can be determined by one of ordinary skill in the art based on, for example, the effective amounts found in animal or clinical studies, the experience of the physician, and the recommended dosage ranges or dosing guidelines.

[0375] In certain embodiments, the binder-drug conjugate (e.g., the binder-drug conjugate in a pharmaceutical composition) is administered in a suitable therapeutically effective amount or dose (e.g., administered in a suitable volume and concentration, which sometimes depends in part on the particular route of administration). In certain embodiments, the therapeutically effective amount of the binder-drug conjugate is selected from one or more doses of about 0.01 mg / kg (e.g., per kilogram of subject body weight) to 500 mg / kg, 0.1 mg / kg to 500 mg / kg, 0.1 mg / kg to 400 mg / kg, 0.01 mg / kg to 300 mg / kg, 0.1 mg / kg to 300 mg / kg, 0.1 mg / kg to 200 mg / kg, 0.1 mg / kg to 150 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 75 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 25 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 1 mg / kg, intervening amounts, and combinations thereof. In some aspects, the therapeutically effective amount of the binder-drug conjugate includes one or more doses of about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, and 0.1 mg / kg, intervening amounts, and combinations thereof. In some embodiments, the therapeutically effective amount of the binder-drug conjugate is from about 0.1 mg / kg to 100 mg / kg, or from about 1 mg / kg to about 50 mg / kg.

[0376] In some embodiments, administering a therapeutically effective amount of a binder-drug conjugate or a pharmaceutical composition comprising a binder-drug conjugate comprises administering a suitable dose at a frequency or interval as needed to obtain an effective therapeutic outcome. In some embodiments, administering a therapeutically effective amount of a binder-drug conjugate or a pharmaceutical composition comprising a binder-drug conjugate comprises administering a suitable dose hourly, every two hours, every 4 hours, every 6 hours, three times a day, twice a day, once a day, six times a week, five times a week, four times a week, three times a week, twice a week, weekly, in combination therewith, and / or at regular or irregular intervals, and / or simply at a frequency or interval as needed or as recommended by a medical professional. In certain embodiments, an effective therapeutic outcome can be determined by monitoring the number, size, viability, growth, mitosis, or metastasis of cancer, tumor growth, or cancer cells in a subject. Thus, in certain embodiments, a decrease or reduction in the number, viability, size, growth, mitosis, or metastasis of tumor cells or cancer cells in a subject is considered an effective therapeutic outcome.

[0377] Kit

[0378] If desired, a pharmaceutical composition comprising an amount or dose of a binder-drug conjugate can be provided in a kit, package, or dispensing device, which can comprise one or more doses of the binder. In some embodiments, the kit comprises a package and / or a dispensing device. Some non-limiting examples of packages include metal, glass, or plastic containers, or blister packs, which contain the binder-drug conjugate or composition described herein. In certain embodiments, the kit comprises a dispensing device, such as a syringe or an inhaler. The package and / or dispenser device can be accompanied by instructions for administration. The package or dispenser can also be accompanied by a container-related notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects approval by the agency of the form of the drug for human or veterinary administration. Such a notice can be, for example, the label approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert.

[0379] In some embodiments, the kit or package contains an amount of a binder-drug conjugate sufficient to treat a patient for a period of 1 day to 1 year, 1 day to 180 days, 1 day to 120 days, 1 day to 90 days, 1 day to 60 days, 1 day to 30 days, 1 to 24 hours, 1 to 12 hours, 1 to 4 hours, or an amount of time therebetween.

[0380] The kit optionally includes a product label and / or one or more package inserts that provide a description of the components therein or instructions for the use of the components in vitro, in vivo, or ex vivo. Exemplary instructions include instructions for diagnostic methods, treatment regimens, or therapeutic regimens. In certain embodiments, the kit includes packaging material, which refers to the physical structure that houses the components of the kit. The packaging material can maintain the sterility of the components and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.). The product label or insert includes "print matter", e.g., paper or cardboard, or is separate or affixed to the component, kit, or packaging material (e.g., box), or is attached to an ampoule, tube, or vial containing the components of the kit. The label or insert can additionally include a computer-readable medium, a compact disc (e.g., CD- or DVD-ROM / RAM, DVD), an MP3, a magnetic tape, or an electronic storage medium (e.g., RAM and ROM) or a hybrid of these (e.g., magnetic / optical storage medium), a FLASH medium, or a memory card. The product label or insert can contain labeling information for one or more of the components, dosages, clinical pharmacology of the active ingredient (including mechanism of action, pharmacokinetics (PK), and pharmacodynamics (PD)). The product label or insert can contain information indicating manufacturer information, lot number, manufacturer location, date, information about the specified disorders, ailments, diseases, or symptoms for which the components of the kit can be used. The product label or insert can contain instructions for a clinician or for an object for using one or more of the components of the kit in a method, treatment regimen, or therapeutic regimen. The instructions can include dosage, frequency, or duration, and instructions for performing any method, treatment regimen, or therapeutic regimen described herein. Thus, the kit of the present invention can additionally include markings or instructions for performing any method and use of the present invention described herein. The product label or insert can contain information about potential adverse side effects and / or warnings.

[0381] Examples

[0382] Example 1 - Antibody Production

[0383] To induce an antibody response against cMET, mice were immunized with cMET-Fc or cMET peptides as described in Figure 1 and 2 . In some embodiments, peptides from strategic regions were selected for immunization. Figure 3An example of a structural loop on MET is shown, which inspired the design of peptide 3. Spleens were obtained from immunized mice, and splenocytes were fused with appropriate fusion partners using standard protocols to generate hybridomas. Hybridoma clones were isolated and tested for their ability to bind to MET and / or induce cMET internalization on human cancer cell lines, as measured by flow cytometry ( Figure 4 ). The selected hybridoma antibodies were selected based on their ability to induce MET degradation ( Figure 5 ) or their inability to induce ERK phosphorylation ( Figure 6 ). The lead hybridoma F6B1P3D12 was deposited on March 20, 2019, at the Patent Depository of the American Type Culture Collection (10801 University Boulevard Manassas, Virginia 20110-2209 USA). The deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and its Regulations (Budapest Treaty). The ATCC number has not been assigned.

[0384] Additional assays were performed to select ideal anti-cMET antibody candidates. For example, the species cross-reactivity of anti-cMET antibodies was tested by determining their ability to bind to human cMET, monkey cMET (e.g., Macaca fascicularis, i.e., cynomolgus monkey), rat cMET, and mouse cMET, as measured by ELISA (Figure 7 and Table 11). In vivo half-life and other pharmacokinetic characteristics were also evaluated (data not shown). The potency and specificity of antibody-drug conjugates (ADCs) against high, medium, and negative cMET-expressing cell lines were also determined using anti-cMET antibodies conjugated to MMAF ( Figure 9 and 15 , Tables 11 and 12). The in vivo potency of the ADC was tested using the MKN45 xenograft model.

[0385] [Table 11]

[0386]

[0387] Summary of the characteristics of the selected humanized monoclonal binders in Example 2

[0388] Generate humanized and isotype-switched monoclonal binders that contain the heavy-chain CDRs and light-chain CDRs of the murine monoclonal antibody P3D12. Sixteen different heavy-chain (HC) and light-chain (LC) combinations were tested for solubility in PBS, binding to human cMET, binding to rat cMET, binding affinity to human and rat cMET as determined by surface plasmon resonance (SPR), presence of agonist activity as reported by Meso Scale Discovery plates (MSD), and cMET degradation. The results are summarized in Table 12 below.

[0389] [Table 12]

[0390]

[0391] The inventors selected an IgG2 isotype monoclonal humanized antibody containing the humanized light-chain variable region of SEQ ID NO:47 and the humanized heavy-chain variable region of SEQ ID NO:108 as a representative humanized anti-cMET antibody (referred to as hD12) and used this antibody in the following examples.

[0392] Example 3 - Binding Assay

[0393] A representative humanized anti-cMET antibody hD12 containing the humanized heavy-chain of SEQ ID NO:108 and the humanized light-chain sequence of SEQ ID NO:47 was randomly conjugated to five representative payloads, namely payloads of Chemical Formulas II, IV, VI, VII, and XI, each of which contains a pyrrolobenzodiazepine toxin and a linker. In this example, the linker of the payload was randomly linked to the hD12 antibody using disulfide bond chemistry. Briefly, the hD12 antibody was first reduced with glutathione (GSH), unreacted GSH was removed, and a linker containing a reactive maleimide group was reacted with one or more free sulfhydryl groups (i.e., thiol groups) on the antibody. Using this method, one or more payloads were covalently linked to the hD12 antibody at random positions occupied by cysteine residues. Therefore, this method is called random conjugation.

[0394] Binding of the hD12 antibody-drug conjugates obtained by ELISA analysis (i.e., hD12-II (antibody hD12 conjugated to the payload of Chemical Formula II); hD12-IV (antibody hD12 conjugated to the payload of Chemical Formula IV); hD12-VI (antibody hD12 conjugated to the payload of Chemical Formula VI); hD12-VII (antibody hD12 conjugated to the payload of Chemical Formula VII); and hD12-XI (antibody hD12 conjugated to the payload of Chemical Formula XI)) to cMET bound to the plate. Denosumab conjugated to each of the five payloads was used as a negative control because the monoclonal antibody denosumab specifically binds to RANK ligand (RANKL) and does not bind to cMET. The results of the ELISA binding study are shown in Figure 17 Figures A and 17B. There was no significant difference in the binding of the five different hD12 drug conjugates to cMET. The isotype control conjugate of denosumab did not bind to cMET as expected.

[0395] Materials: High-binding 384-well plates (Thermo Fisher #: 8755), blocking buffer (SkyTek Lab #AAA500), recombinant human c-Met-10X His (1.04 mg / ml, in-house, Lot #140924TA), anti-human κ light chain HRP-conjugated (1 mg / ml, Brthyl #AP80-219P), 1×KPL wash buffer in water (20×, 200 ml, KPL #50-63-01), TMB (100 ml, KPL #53-00-00), and stop solution (Cell Signaling #7002L).

[0396] Example 4 - Cytotoxicity Assay

[0397] The cytotoxicity of the five hD12 antibody-drug conjugates of Example 3 against cells expressing different levels of surface cMET was tested. Denosumab conjugated to each of the five representative payloads was used as a negative control (data not shown). The denosumab conjugate had little or no effect on cell killing in cell lines expressing cMET.

[0398] The results of the cytotoxicity assays are shown in FIGS. 18A to 18E and FIGS. 19A to 19F. The cell lines tested were SNU-1 (ATCC, does not express cMET, FIG. 18A), SNU-16 (ATCC, moderately expresses cMET, FIG. 18B), SNU-620 (KCLB, highly expresses cMET, FIG. 18C), MKN-45 (DSMZ, highly expresses cMET, FIG. 18D), H441 (ATCC, moderately expresses cMET, FIG. 19A), H1373 (ATCC, moderately expresses cMET, FIG. 19B), H1975 (ATCC, moderately expresses cMET, FIG. 19C), SNU-5 (ATCC, highly expresses cMET, FIG. 19D), and H1573 (ATCC, moderately expresses cMET, FIG. 19E). In some of the cell lines with moderate and high cMET expression, hD12-II and hD12-VII showed slightly higher potency.

[0399] Example 5 - Xenograft Studies

[0400] Two in vivo xenograft studies were conducted to evaluate the potency of the five hD12 antibody-drug conjugates of Example 3 (i.e., hD12-VI, hD12-II, hD12-VII, and hD12-XI). Note that the terms hD12-vc-XI, hD12-vc-VI, hD12-vc-II, hD12-vc-VII, and hD12-vc-IV shown in FIGS. 20 to 22 are used synonymously with the terms hD12-XI, hD12-VI, hD12-II, hD12-VII, and hD12-IV, respectively. Also, as shown in the figures, denosumab-* (e.g., denosumab-II) is used synonymously with the term denosumab-vc-* (e.g., denosumab-vc-II). The "vc" designation has no significant meaning. The term "denosumab-II" refers to the monoclonal antibody "denosumab" conjugated to the payload of Chemical Formula II.

[0401] A first H1975 in vivo xenograft study was established in groups of ten mice (Nu / nu: (Charles River)). Each mouse was inoculated with H1975 cells and subsequently treated with one of the designated antibody-drug conjugates or with PBS. The antibody-drug conjugate was administered as a single dose by i.v. tail vein injection on Day 1. Two different doses (0.5 mg / kg and 0.125 mg / kg) of each antibody-drug conjugate were tested. Denosumab-II was used as a negative control. Tumor volume and weight were measured three times per week. The results of the H1975 in vivo xenograft study are shown in FIGS. 20A to 20G.

[0402] All animals tolerated the antibody-drug conjugates well. No significant weight loss was observed in any group (e.g., see Figure 20B). hD12-VII showed the highest potency at both dose concentrations of all the tested ADCs (Figures 20A and 20F). In the low-dose group (0.125 mg / kg), the efficacy of hD12-II and hD12-VI (Figures 20E and 20D) was slightly lower than that of hD12-VII. In the low-dose group, hD12-3315 and hD12-XI had the lowest efficacy (Figures 20G and 20C). The isotype control denosumab-II showed some efficacy at the 0.5 mg / kg dose. Since H1975 has the RANK-RANKL signaling pathway, denosumab can show tumor growth inhibitory effects (Journal of Thoracic Oncol., 2014, 9(3)345-54). In summary, all five hD12 drug conjugates showed significant potency against H1975 xenografts. hD12-VII, hD12-II, and hD12-VI exhibited the highest therapeutic potency, with VII slightly superior to the other two.

[0403] A second in vivo H1373 xenograft study was conducted to further evaluate the potency of the five hD12 antibody-drug conjugates of Example 3 (i.e., hD12-VI, hD12-II, hD12-VII, and hD12-XI). The in vivo H1373 xenograft study was established with ten mice per group (Nu / nu: (Charles River)). Each mouse was inoculated with H1373 cells and subsequently treated with one of the designated antibody-drug conjugates or with PBS. The antibody-drug conjugates were administered as a single dose by i.v. tail vein injection on Day 7. Two different doses (0.5 mg / kg and 0.125 mg / kg) of each antibody-drug conjugate were tested. Denosumab-II was used as a negative control. Tumor volume and weight were measured three times per week. The results of the in vivo H1373 xenograft study are shown in Figures 21A-21E and Figure 22 in.

[0404] All animals tolerated the antibody-drug conjugates well. No significant weight loss was observed in any group (e.g., see Figure 22 ). As seen in the first H1975 xenograft model, hD12-VII (Figure 21D) showed slightly better potency in the low-dose group (0.125 mg / kg) compared to hD12-II and hD12-VI (Figures 21C and 21B). As previously observed, hD12-IV was the least effective drug (Figure 21E).

[0405] Example 6 - PK Study in Mice

[0406] Over a 72-hour period, the circulating half-lives of the hD12 drug conjugates of Example 3 (i.e., hD12-VI, hD12-II, hD12-VII, and hD12-XI) were evaluated in 5 groups of 3 mice each. Each group of mice received a single i.v. injection of 1 mg / kg of one of hD12-II, hD12-IV, hD12-VI, hD12-XI, or denosumab-II. Blood was drawn at 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, and 72 hours. Serum samples were prepared and the amount of each of the specified antibody-drug conjugates was analyzed ( Figure 23 ). Serum antibodies were captured with anti-Fc specific antibodies and detected with goat anti-human IgG (H+L)-HRP.

[0407] Example 7 - Site-Specific Conjugation of Payload to hD12

[0408] The coding region of hD12 was mutated at multiple sites to introduce cysteine residues into the heavy chain constant region of the IgG2 antibody to obtain hD12 variant antibodies hD12-T289C (T at position 289 mutated to cysteine), hD12-V442C (V at position 442 mutated to cysteine), hD12-V282C (V at position 282 mutated to cysteine), hD12-S119C (S at position 119 mutated to cysteine). The mutation sites are in the constant region of hD12 and are defined according to the EU numbering system as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969. The payload of Formula II was site-specifically conjugated to each mutated cysteine residue site using maleimide chemistry. The quality and extent of conjugation were evaluated by determining the total recovery, aggregate content, monomer content, and drug-antibody ratio (DAR). Optimal conjugation was observed for the hD12 conjugates hD12-T289C-II, hD12-V442C-II, and hD12-V282C-II. As described in Example 3, the relative binding affinities of these three site-specific conjugates to cMET were compared to randomly conjugated hD12-II using cMET binding ELISA. The results of the cMET binding assay are shown in Figure 24 A and 24B. All site-specific compounds successfully bound to human cMET with similar affinities. The site-specific conjugates bound with similar or slightly better affinities than randomly conjugated hD12-II.

[0409] Example 8 - Cytotoxicity assessment of site - specific conjugates against cMET - expressing cell lines. The inventors determined the cytotoxic potency of site - specifically conjugated II conjugates (i.e., hD12 - T289C - II, hD12 - V442C - II, and hD12 - V282C - II) against the following eight cMET - expressing cell lines compared to randomly conjugated hD12 - II as a benchmark: SNU - 16 (Figure 25B), SNU - 620 (Figure 25C), MKN - 45 (Figure 25D), NCI - N87 (Figure 25E), SNU - 5( Figure 26 A), H1373 (Figure 27A), H1573 (Figure 27B), H1975 (Figure 27C), and the cMET - negative cell line SNU - 1 (Figure 25A). The cytotoxicity results are summarized in Figures 25F, 26B, and 27E. The doses administered are indicated in Figures 25G, 26C, and 27D.

[0410] Against the MKN - 45, SNU - 620, SNU - 5, SNU - 16, NCI - N87, and H1975 cell lines, all site - specifically conjugated antibodies showed cytotoxicity comparable to that of the randomly conjugated hD12 - II control. Due to complex killing curves significantly different from the S - shaped curve, the EC50 values for the site - specific conjugates could not be determined for the H1373 and H1573 cell lines. The complex shape of the killing curve was more evident in the case of the site - specific conjugates and was reproducible in repeated experiments. Overall, the cytotoxic potency of the site - specific hD12 conjugates appears to be superior to that of the randomly conjugated hD12 - II.

[0411] Example 9 - In vivo xenograft analysis of site - specific conjugates

[0412] The site - specific conjugates hD12 - T289C - II, hD12 - V442C - II, and hD12 - V282C - II, as well as randomly conjugated hD12 - II, were evaluated in an H1975 (moderate cMET expression) xenograft tumor model. Denosumab - II was used as a negative control. Each mouse was inoculated with H1975 cells (day 0) and subsequently treated with one of the designated antibody - drug conjugates or with PBS. The antibody - drug conjugates were administered as a single dose by i.v. tail vein injection on day 14 after tumor cell inoculation. Two different doses (0.5 mg / kg and 0.125 mg / kg) of each antibody - drug conjugate were tested. Ten mice were tested for each treatment. Tumor volume and weight were measured three times a week. The results of the H1975 in vivo xenograft study are shown in Figures 28A to 28C.

[0413] All high-dose groups of the site-specific ADCs, as well as the randomly conjugated II (0.5 mg / kg), showed high potency in the H1975 model. Complete tumor regression was observed (Figures 28A to 28C). The low-dose groups (0.125 mg / kg) showed tumor regression in the case of hD12-II (random) and hD12-T289C-II, with hD12-V422C-II being the most effective. hD12-V282C-II was the least effective. Denosumab control showed slight potency at high doses, which was also seen in a previous H1975 model run in the case of randomly conjugated ADCs. All test articles administered were well tolerated, and no significant weight loss was observed( Figure 29 ).

[0414] Example 10 - Pharmacokinetics in Rats for Site-Specific hD12 Conjugates (Total Antibody Assay)

[0415] The circulating half-lives of the site-specific hD12 conjugates hD12-V422C-II, hD12-V282C-II, and hD12-T289C-II were evaluated in rats for 21 days. Each group of rats (3 rats / group) received a single i.v. injection of 1 mg / kg of the designated antibody-drug conjugate( Figure 30 ). Blood samples were taken at 0.5 h, 2 h, 6 h, 24 h, 48 h, 72 h, 168 h, 312 h, and 480 h after injection. Antibody levels in serum samples were analyzed in a PK ELISA using a capture antibody (anti-Fc specific) and goat anti-human IgG (H+L)-HRP. The results are summarized in Figure 31 . Pharmacokinetic (PK) parameters of the site-specific conjugates were determined using WinNonlin software. All 3 site-specific conjugates showed concentration-time curves of similar shape. The calculated half-lives were 12 to 18 days. The variants could not be distinguished from each other based solely on total antibody pharmacokinetic data. Based on the results of this experiment, there were no significant pharmacokinetic differences among hD12-V422C-II, hD12-V282C-II, and hD12-T289C-II.

[0416] Example 11 - Non-Human Primate Tolerability

[0417] The tolerability of randomly conjugated hD12-II and the site-specific variants hD12-V282C-II and hD12-T289C-II was tested in non-human primates at up to 1 mg / kg. In general, all antibody-drug conjugates were well tolerated. No severe weight loss was observed by the end of the study (day 21).

[0418] Example 12 - PDX Models, Methods, and Results

[0419] Patient - derived xenograft (PDX) is a model of cancer in which tissue or cells from a patient's tumor are implanted into immunodeficient mice. PDX models are commonly used to create an environment similar to the natural growth of cancer for studying cancer progression and treatment. Multiple CRown Bio HuPRime (registered trademark) gastric, colorectal, and head and neck (H&N) PDX models were conducted to evaluate the potency of hD12 - T289C - II. PDX models with varying c - Met expression levels (from low to high) were selected.

[0420] Briefly, female BALB / c nude mice, 14 to 15 weeks old, were subcutaneously inoculated in the right flank with a fragment of primary human tumor (gastric cancer, colorectal cancer, or H&N cancer, sized 2 to 3 mm) for tumor development. When the tumor size reached an average volume of 200 mm 3 the mice were randomized and grouped (6 treatment groups). Each group consisted of 10 mice. Starting from day 0, the test article was administered as a single dose i.v. to the tumor - bearing mice. A second dose was administered if needed. Secukinumab - II, a non - targeting antibody conjugated to the payload of chemical formula II, was administered at a dose of 1 mg / kg. hD12 - T289C - II was administered at single doses of 1.0 mg / kg, 0.5 mg / kg, 0.25, and 0.125 mg / kg. The vehicle control group was administered a single i.v. dose of 1×PBS.

[0421] After random grouping, tumor size was measured two - dimensionally by calipers twice a week. Tumor volume (mm 3 ) was calculated by TV = 0.5a×b 2 where a and b are the major and minor diameters of the tumor surface. Body weight was measured and updated along with its tumor measurements.

[0422] Then, TGI% was calculated using tumor size according to the following formula: TGI% = ((mean(C) - mean(C0))-(mean(T) - mean(T0)) / (mean(C) - mean(C0))*100%, where T is the current group value; T0 is the initial value of the current group; C is the control group value; C0 is the initial value of the control group.

[0423] The results of the PDX study are shown in Figure 32In FIGS. 32 and 33. Each data point represents one PDX model group consisting of 10 mice inoculated with a single PDX tumor. The TGI% was calculated as described above. Mice were treated with a vehicle (PBS) or secukinumab-II as a negative control. Tumor volume (y-axis) was determined over time (i.e., days, x-axis) (FIG. 33C). Figure 32 The results in FIGS. 32 and 33 show that hD12-T289C-II effectively inhibited the growth of human tumor tissues derived from gastric cancer, colorectal cancer, or H&N cancer in a dose-dependent manner.

[0424] Example 13

[0425] A human subject has multiple metastatic cancers of 2 cm or greater in size, which are present in the liver and lungs. A biopsy was performed to determine whether the cancer cells express cMET on their cell surface. The presence of cell surface cMET expression was confirmed from the biopsy results.

[0426] The conjugate-drug conjugate described herein that specifically binds to the extracellular domain of human cMET was administered to the human subject. The conjugate optionally comprises a human κ and IgG2 heavy chain constant region, a light chain variable region of SEQ ID NO: 41, and a heavy chain variable region of SEQ ID NO: 98. The conjugate-drug conjugate was administered once daily at a dose of 15 mg / kg, intravenously, in a volume of 100 ml over a period of 1 hour for six weeks. The presence, size, and viability of the tumor were determined by subsequent biopsies and ultrasound. After two weeks of treatment, the size and number of tumors were significantly reduced. After six weeks of treatment, the subject was determined to be in remission.

[0427] Example 14

[0428] A human subject has colorectal adenocarcinoma with a solid tumor having a diameter of 2 cm. The conjugate-drug conjugate described herein was administered to the human subject. The conjugate of the conjugate-drug conjugate is a monoclonal humanized antibody of the IgG2 isotype comprising a light chain variable region of SEQ ID NO: 47 and a humanized heavy chain variable region of SEQ ID NO: 108. The drug of the conjugate-drug conjugate is a payload having the structure of Chemical Formula II. The conjugate was administered once daily at a dose of 1 mg / kg, intravenously, in a volume of 50 ml over a period of 30 minutes for six weeks. After two weeks of treatment, the tumor size was reduced by more than 50%. After six weeks of treatment, the subject was determined to be in remission.

[0429] Example 15 - cMET Sequence

[0430] SEQ ID NO: 109 (human cMET - UniProtKB - P08581 (MET_HUMAN)). * Residues E168 and N375 are shown in bold and underlined.

[0431]

[0432] SEQ ID NO:110 (Rat cMET - UniPRotKB - P97523 (MET_RAT))

[0433]

[0434] SEQ ID NO:111 (Mouse cMET - UniPRotKB - P16056 (MET_MOUSE))

[0435]

[0436]

[0437] SEQ ID NO:112 (Dog cMET)

[0438]

[0439]

[0440] SEQ ID NO:113 (Cynomolgus macaque (Macaca mulatta), Rhesus cMET - NCBI Reference Sequence: NP_001162100.1)

[0441]

[0442] Example 16 - Humanized Monoclonal Antibody hD12

[0443] SEQ ID NO:114 Heavy Chain Sequence of hD12

[0444]

[0445] SEQ ID:115 Light Chain Sequence of hD12

[0446]

[0447] SEQ ID NO:116 Heavy Chain Sequence of hD12 T289C (Residue C289 not underlined)

[0448]

[0449] The entire content of each patent, patent application, publication, or any other reference or document cited herein is hereby incorporated by reference. In case of conflict, the specification (including definitions) shall prevail.

[0450] The citation of any patent, patent application, publication or any other document does not admit that any of the foregoing is relevant prior art, nor does it constitute any admission as to the content or date of these publications or documents.

[0451] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0452] All features disclosed herein can be combined in any combination. Each feature disclosed in the specification can be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless otherwise expressly stated, the features disclosed (e.g., antibodies) are an instance of a class of equivalent or similar features.

[0453] Unless the context clearly dictates otherwise, all numerical values or ranges of values used herein include the integers within such ranges and the values or fractions of integers within the ranges. In addition, when a list of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%), the list includes all intermediate values and fractional values therebetween (e.g., 54%, 85.4%). Thus, by way of example, reference to 80% or higher identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., and 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so on.

[0454] References to integers greater than (more than) or less than a given integer include any numbers greater than or less than the recited number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc., down to the number 1; and reference to less than 10 includes 9, 8, 7, etc., down to the number 1.

[0455] Unless the context clearly indicates otherwise, all numerical values or ranges used herein include the integers and fractional values within such ranges, as well as the fractional values of the integers within such ranges. Thus, by way of illustration, reference to a numerical range, such as 1 to 10, includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so on. Thus, reference to a range of 1 to 50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so on.

[0456] Reference to a series of ranges includes ranges that combine the values at the boundaries of different ranges within the series. Thus, by way of illustration, reference to a series of ranges, such as, 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500 to 750, 750 to 1,000, 1,000 to 1,500, 1,500 to 2,000, 2,000 to 2,500, 2,500 to 3,000, 3,000 to 3,500, 3,500 to 4,000, 4,000 to 4,500, 4,500 to 5,000, 5,500 to 6,000, 6,000 to 7,000, 7,000 to 8,000 or 8,000 to 9,000, includes ranges such as 10 to 50, 50 to 100, 100 to 1,000, 1,000 to 3,000, 2,000 to 4,000, etc.

[0457] The foregoing may be modified without departing from the basic aspects of the technology. Although the technology has been described in considerable detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed herein, and such modifications and improvements are within the scope and spirit of the technology.

[0458] Positive language is used herein to describe multiple embodiments and aspects to generally disclose the invention. The invention also specifically includes embodiments in which specific subject matter (in whole or in part) (such as substances or materials, method steps and conditions, schemes or operations) is excluded. For example, in certain embodiments or aspects of the invention, materials and / or method steps are excluded. Thus, even though the invention is not generally described herein in terms of what it does not include, aspects that are not explicitly excluded from the invention are still disclosed herein.

[0459] The techniques illustratively described herein can suitably be practiced in the absence of any element not specifically disclosed herein. Thus, for example, in each instance herein, any one of the terms "comprising", "consisting essentially of", and "consisting of" can be replaced with any one of the other two terms. The terms and expressions that have been used are used as descriptive terms and not of limitation, and the use of such terms and expressions does not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the claimed technology. A noun that is not qualified by a quantity term can refer to one or more of the elements it modifies (e.g., "reagent" can mean one or more reagents), unless it is clear from the context that any one element or more than one element is being described. The term "about" as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and the term "about" used at the beginning of a string of values modifies each value (i.e., "about 1, 2, and 3" means about 1, about 2, and about 3). For example, a weight of "about 100 grams" can include weights from 90 grams to 110 grams. The term "substantially" as used herein refers to a value modifier meaning "at least 95%", "at least 96%", "at least 97%", "at least 98%", or "at least 99%" and can include 100%. For example, a composition substantially free of X can contain less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of X, and / or X can be absent or undetectable in the composition.

[0460] Accordingly, it is to be understood that although the inventive techniques have been specifically disclosed by way of some representative embodiments and optional features, modifications and variations of the concepts disclosed herein will be accessible to those skilled in the art, and such modifications and variations are considered to be within the scope of the technology.

Claims

1. An agent-drug conjugate for treating a subject having or suspected of having a neoplastic disorder expressing cMET or a cancer expressing cMET, comprising an agent and a payload, wherein the agent has a light chain variable region and a heavy chain variable region, the light chain variable region comprises the amino acid sequence SEQ ID NO:47, and the heavy chain variable region comprises the amino acid sequence SEQ ID NO:

108.

2. The conjugate - drug conjugate according to claim 1, wherein the payload is a pyrrolobenzodiazepine toxin, and the pyrrolobenzodiazepine toxin comprises a structure of Formula I: Wherein: Both Z1 and Z2 are N; Both Z3 and Z4 are C; Double dashed line represents a single bond or a double bond; n is from 1 to 10; R3 and R4 are each independently H or C 1-4 alkoxy; and R1 and R2 are each independently selected from H, C 1-5 alkyl, C 3-6 cycloalkyl, C 2-5 alkenyl and phenyl optionally substituted by R5, Wherein R5 is selected from -NH2, -NHR6 and a piperazinyl group substituted by R7 having the following structure: wherein R6 contains a linking group and R7 is H or C 1-5 alkyl; X1 is empty, a protecting group or comprises a linker; X2 is empty, a protecting group or comprises a linker; only one of X1, X2, R1 and R2 comprises a linker; and Y1 and Y2 are each independently empty, OH or SO3H; Provided that: (i) When X1 contains the linking group, Z1 Z3 is N-C, (ii) When X2 contains the linking group, Z2 Z4 is N-C, (iii) When X1 contains the protecting group, Z1 Z3 is N-C, and (iv) When X2 contains the protecting group, Z2 Z4 is N-C, Where empty indicates the absence of that moiety or the presence of one or more hydrogens to satisfy the required valency.

3. The agent-drug conjugate according to claim 2, wherein the linker comprises the structure of formula A: Wherein: The asterisk indicates the point of attachment to the pyrrolobenzodiazepine toxin; The wavy line indicates the point of attachment to the agent; m is from 1 to 20; q is from 0 to 10; and E is a linker.

4. The agent-drug conjugate according to claim 2, wherein the linker comprises the structure of formula B: Wherein: The asterisk indicates the point of attachment to the pyrrolobenzodiazepine toxin; the wavy line indicates the point of attachment to the binder; E is a linking group; v is from 0 to 10; and u is 0 or 1; where when u is 1, t is from 1 to 10.

5. The agent-drug conjugate according to claim 2, wherein the protecting group has the following structure (D): wherein the asterisk indicates the point of attachment to the pyrrolobenzodiazepine toxin; and w is from 1 to 5.

6. The agent-drug conjugate according to claim 1, wherein the payload comprises a structure selected from the following: Where m is 8; Where m is 8, p is 3, and X2 is a protecting group; Where m is 8; Where t is 8, and v is 1; and Where the wavy line indicates the point of attachment to the agent.

7. The agent-drug conjugate according to claim 6, wherein the protecting group of X2 has the following structure (D): Where the asterisk indicates the point of attachment to the payload; and w is from 1 to 5.

8. The agent-drug conjugate according to claim 1, wherein the agent is an antibody or an antigen-binding fragment thereof.

9. The agent-drug conjugate according to claim 8, wherein the agent specifically binds to human cMET, monkey cMET and rat cMET.

10. The agent-drug conjugate according to claim 9, wherein the agent specifically binds to the extracellular domain of a cMET variant.

11. Use of the agent-drug conjugate according to any one of claims 1 to 10 in the preparation of a medicament for treating a subject having or suspected of having a neoplastic disorder or cancer, wherein the neoplastic disorder or cancer is a neoplastic disorder expressing cMET or a cancer expressing cMET.

12. The use according to claim 11, wherein the neoplastic disorder or cancer comprises epithelial cancer, sarcoma, neuroblastoma, glioblastoma, myeloma, lymphoma or melanoma.

13. The use according to claim 11, wherein the neoplastic disorder or cancer comprises solid or soft tissue tumors.

14. The use according to claim 11, wherein the neoplastic disorder or cancer comprises bladder cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer, hepatocellular carcinoma, hypopharyngeal cancer, lung cancer, ovarian cancer or renal cancer.

15. The use according to claim 11, wherein the neoplastic disorder or cancer comprises liver cancer or adenocarcinoma.

16. The use according to claim 11, wherein the neoplastic disorder or cancer comprises pancreatic adenocarcinoma, colorectal adenocarcinoma, small intestinal malignancy, cholangiocarcinoma, non-small cell lung cancer (NSCLC), thyroid cancer, esophageal or esophagogastric junction (EGJ) cancer, gastric adenocarcinoma, hepatocellular carcinoma of the liver, head and neck squamous cell carcinoma, breast cancer, small cell lung cancer, ovarian surface epithelial carcinoma, retroperitoneal or peritoneal sarcoma, prostatic adenocarcinoma, neuroendocrine tumor, gastrointestinal stromal tumor, glioblastoma or non-epithelial ovarian cancer.

17. The use according to claim 11, wherein the neoplastic disorder or cancer comprises pancreatic neuroendocrine carcinoma, female genital tract malignancy or triple-negative breast cancer.

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