HK1232895B - Anti-c-met antibody and anti-c-met antibody-cytotoxic drug conjugate and pharmaceutical use thereof - Google Patents

Description

Anti-c-Met antibodies and anti-c-Met antibody-cytotoxic drug conjugates and their pharmaceutical uses

Technical Field

The present invention discloses an anti-c-Met antibody or antigen-binding fragment thereof, a chimeric antibody or humanized antibody comprising the CDR region of the anti-c-Met antibody, an anti-c-Met antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutical composition comprising a human c-Met antibody or antigen-binding fragment thereof and an antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as well as their use as anticancer drugs. In particular, the present invention relates to the use of a humanized anti-c-Met antibody and an anti-c-Met antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating a c-Met-mediated disease or condition.

Background Art

Recent molecular biology and tumor pharmacology studies have demonstrated that cellular signaling pathways involving protein tyrosine kinases (PTKs) play a crucial role in tumor formation and progression, with over 50% of proto-oncogenes and oncogene products possessing tyrosine kinase activity. The c-Met proto-oncogene belongs to the Ron subfamily of the PTK family, and the encoded c-Met protein is a high-affinity receptor for hepatocyte growth factor/scatter factor (HGF/SF). The HGF/c-Met signaling pathway is closely linked to angiogenesis and tumor growth, with sustained activation being a key driver of tissue cell carcinogenesis and cancer cell hyperproliferation. Inhibiting this pathway has emerged as a novel approach for targeted tumor therapy.

The c-Met proto-oncogene is located on the long arm of human chromosome 7 (7q31). It is over 120 kb in size and encodes a c-Met protein precursor with a molecular weight of approximately 150 kDa. This protein undergoes partial glycosylation to produce a 170 kDa glycoprotein, which is further cleaved into an α subunit (50 kDa) and a β subunit (140 kDa), linked by disulfide bonds to form the mature c-Met protein receptor. This heterodimer consists of two chains: the β chain has an extracellular region, a transmembrane region (also known as a membrane extension), and an intracellular region (containing the intracellular tyrosine kinase binding site). The α chain has only an extracellular portion, but it is heavily glycosylated and attached to the β chain via disulfide bonds. The extracellular regions of both subunits serve as the recognition sites for their respective ligands, while the intracellular regions possess tyrosine kinase activity.

There are three mechanisms of c-Met activation: HGF-dependent, HGF-independent, and other membrane-dependent pathways, such as CD44, a receptor on the hyaluronic acid membrane, adhesins, and RON signaling. The most common mechanism is HGF-dependent. HGF binds to c-Met at the N-terminus, promoting dimerization and autophosphorylation at Tyr1234 and Tyr1235 on the β chain. Phosphorylation of Tyr1349 and Tyr1356 near the C-terminus creates multiple binding sites for adaptor proteins. These adaptor proteins induce downstream signaling mediated by PI3K/Akt, Ras/MAPK, c-Src, and STAT3/5, triggering diverse cellular responses, including cell survival and motility (closely linked to the PI3K/Akt pathway), tumor metastasis, and cell proliferation (primarily mediated by Ras/MAPK). In addition, c-Met cross-talks with other membrane receptors, and it is now known that this cross-talk can promote tumor formation and metastasis. Since c-Met is the intersection of many pathways that lead to tumor formation and metastasis, targeting c-Met can relatively easily achieve simultaneous interference with many pathways, making c-Met a promising target for anti-tumor and anti-metastasis therapy.

Antibody drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds, leveraging the antibody's specificity for tumor cell-specific or highly expressed antigens and the high efficacy of the cytotoxin while avoiding toxic side effects on normal cells. This means that compared to traditional chemotherapy drugs, ADCs can precisely bind to tumor cells while minimizing the effects on normal cells.

ADC drugs are composed of three parts: an antibody (target), a linker, and a toxin. Among them, a good target (antibody part) determines the specificity of the ADC drug, which includes not only specific target binding but also effective internalization.

Currently, there are three main types of inhibitors targeting c-Met kinase: biological antagonists of HGF and c-Met, antibodies against HGF and c-Met, and small molecule c-Met inhibitors. Existing clinical results indicate that antibodies directly targeting HGF and c-Met, or small molecules inhibiting c-Met, are less than ideal. ADCs targeting c-Met may be the most effective approach for treating tumors. Currently, no clinical studies have been conducted on c-Met ADCs.

The present invention is the first to introduce an anti-c-Met antibody ADC drug that not only retains the antibody-dependent cell proliferation inhibitory effect of the anti-c-Met antibodies of the present invention, but also enhances the efficacy of a potential cytotoxic drug. Furthermore, because the toxin is released in tumor cells, the drug's toxicity and side effects do not increase in tandem with the increased efficacy. The present invention provides humanized antibodies and chimeric antibodies that specifically bind to human c-Met, characterized by high affinity, high efficacy, endocytosis, good stability, and lack of c-Met agonist activity. Building on these excellent properties, the present invention also provides an antibody-cytotoxic drug conjugate that specifically binds to human c-Met, or a pharmaceutically acceptable salt or solvate thereof, that not only retains the antibody-dependent cell proliferation inhibitory effect of the anti-c-Met antibodies of the present invention, but also enhances the efficacy and broad-spectrum therapeutic potential of the potential cytotoxic drug. Furthermore, because the toxin is released in tumor cells (endocytosis of the anti-c-Met antibodies of the present invention), the drug's toxicity and side effects do not increase in tandem with the increased efficacy.

Summary of the Invention

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to a c-Met receptor, comprising at least one CDR region sequence or a mutant sequence thereof selected from the following:

Antibody heavy chain variable region HCDR region sequence: SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and

Antibody light chain variable region LCDR region sequence: SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the antibody heavy chain variable region comprises at least one HCDR region sequence or a mutant sequence thereof selected from the following: SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the antibody light chain variable region comprises at least one LCDR region sequence or a mutant sequence thereof selected from the following: SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 6 (HCDR1), SEQ ID NO: 7 (HCDR2) and SEQ ID NO: 8 (HCDR3), or a mutant sequence thereof, and a light chain variable region sequence of SEQ ID NO: 9 (LCDR1), SEQ ID NO: 10 (LCDR2) and SEQ ID NO: 11 (LCDR3) or a mutant sequence thereof.

In a preferred embodiment of the present invention, a c-Met antibody or antigen-binding fragment thereof as described above is provided, wherein the CDR region mutation sequence is 1-3 amino acid mutations in the CDR region that optimize the antibody activity, and the HCDR2 region mutation sequence is preferably SEQ ID NO: 12.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the c-Met antibody or the antigen-binding fragment thereof is a murine antibody or a fragment thereof.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the sequence of the murine antibody heavy chain variable region is: SEQ ID NO: 4.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the sequence of the light chain variable region of the murine antibody is: SEQ ID NO: 5.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the heavy chain variable region of the murine antibody is SEQ ID NO: 4, and the light chain variable region is SEQ ID NO: 5.

In a preferred embodiment of the present invention, a murine antibody or fragment thereof as described above is provided, wherein the antibody heavy chain variable region further comprises a heavy chain FR region derived from murine IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

In a preferred embodiment of the present invention, a murine antibody or a fragment thereof as described above is provided, which further comprises a heavy chain constant region derived from murine IgG1, IgG2, IgG3 or IgG4, or a variant thereof.

In a preferred embodiment of the present invention, a murine antibody or fragment thereof as described above is provided, wherein the antibody light chain variable region further comprises a light chain FR region selected from murine κ or λ chain, or variants thereof.

In a preferred embodiment of the present invention, a murine antibody or fragment thereof as described above is provided, which further comprises a light chain constant region selected from murine κ or λ chain, or variants thereof.

In a preferred embodiment of the present invention, there is provided a c-Met antibody or an antigen-binding fragment thereof as described above, which is a chimeric antibody or a humanized antibody or a fragment thereof.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

In a preferred embodiment of the present invention, a c-Met antibody or antigen-binding fragment thereof as described above is provided, wherein the heavy chain FR region sequence on the heavy chain variable region of the humanized antibody is derived from a human germline heavy chain sequence, preferably the human germline heavy chain IGHV 3-33*01; and comprises the framework sequence of the FR1, FR2, FR3 and FR4 regions of the human germline heavy chain IGHV 3-33*01 or a mutant sequence thereof, preferably, the mutant sequence is a back mutation of 0-10 amino acids.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 13-15 or a variant thereof.

In a preferred embodiment of the present invention, a c-Met antibody or antigen-binding fragment thereof as described above is provided, wherein the light chain FR region sequence on the light chain variable region of the humanized antibody is selected from a human germline light chain sequence, preferably a human germline light chain IGKV085 or IGKV 4-1*01, comprising the framework sequence of FR1, FR2, FR3 and FR4 regions of the human germline light chain IGKV085 and IGKV 4-1*01 or a mutant sequence thereof, preferably, the mutant sequence is a back mutation of 0-10 amino acids.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the humanized antibody comprises a light chain variable region sequence selected from SEQ ID NOs: 16-18 or a variant thereof.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the humanized antibody comprises a heavy chain variable region selected from SEQ ID NOs: 13-15 and a light chain variable region selected from SEQ ID NOs: 16-18.

In a preferred embodiment of the present invention, there is provided a c-Met antibody or an antigen-binding fragment thereof as described above, comprising a combination of a heavy chain variable region sequence and a light chain variable region sequence selected from any one of (a) to (c):

(a) the heavy chain variable region sequence of SEQ ID NO: 13 and the light chain variable region sequence of SEQ ID NO: 16;

(b) the heavy chain variable region sequence of SEQ ID NO: 14 and the light chain variable region sequence of SEQ ID NO: 17; or

(c) The heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 18.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the heavy chain constant region of the humanized antibody comprises a constant region derived from human IgG1 or a variant thereof, human IgG2 or a variant thereof, human IgG3 or a variant thereof, or human IgG4 or a variant thereof, preferably comprises a constant region of human IgG1 or a variant thereof, human IgG2 or a variant thereof, or human IgG4 or a variant thereof, more preferably a constant region of human IgG2 or a variant thereof.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, which comprises a full-length heavy chain sequence selected from SEQ ID NOs: 23-25 or a sequence having at least 90% homology thereto.

In a preferred embodiment of the present invention, a c-Met antibody or antigen-binding fragment thereof as described above is provided, wherein the humanized antibody light chain variable region further comprises a light chain FR region optionally selected from human κ or λ chain or variants thereof.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the light chain constant region of the humanized antibody comprises a constant region selected from human κ or λ chain or variants thereof.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, which comprises a full-length light chain sequence selected from SEQ ID NOs: 26-28 or a sequence having at least 90% sequence homology thereto.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the humanized antibody comprises a full-length heavy chain sequence selected from SEQ ID NOs: 23-25 and a full-length light chain sequence selected from SEQ ID NOs: 26-28.

In a preferred embodiment of the present invention, there is provided a c-Met antibody or an antigen-binding fragment thereof as described above, wherein the humanized antibody is selected from a combination of any one of (a) to (c) full-length light chain sequence and full-length heavy chain sequence:

Ab-9: heavy chain sequence of SEQ ID NO: 23 and light chain sequence of SEQ ID NO: 26;

Ab-10: the heavy chain sequence of SEQ ID NO: 24 and the light chain sequence of SEQ ID NO: 27; or

Ab-11: heavy chain sequence of SEQ ID NO: 25 and light chain sequence of SEQ ID NO: 28.

In a preferred embodiment of the present invention, a c-Met antibody or an antigen-binding fragment thereof as described above is provided, wherein the antigen-binding fragment comprises Fab, Fv, sFv or F(ab') ₂ .

The present invention further provides a DNA molecule encoding the expression precursor product of the c-Met antibody or antigen-binding fragment thereof as described above.

The present invention further provides an expression vector containing the DNA molecule described above.

The present invention further provides a host cell transformed with the expression vector described above.

In a preferred embodiment of the present invention, a host cell as described above is provided, wherein the host cell is preferably a mammalian cell, more preferably a CHO cell.

The present invention further provides a pharmaceutical composition comprising the c-Met antibody or antigen-binding fragment thereof as described above, and one or more pharmaceutically acceptable excipients, diluents or carriers.

The present invention further provides a use of the c-Met antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, as described above, in the preparation of a medicament for treating a disease or condition mediated by c-Met, wherein the disease or condition is preferably cancer; more preferably a cancer expressing c-Met; most preferably gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer; most preferably gastric cancer and non-small cell lung cancer.

The present invention further provides a method for treating and preventing a disease or condition mediated by c-Met, comprising administering to a patient in need thereof a therapeutically effective amount of the c-Met antibody or antigen-binding fragment thereof as described above, or a pharmaceutical composition comprising the same; wherein the disease or condition is preferably cancer; more preferably a cancer expressing c-Met; most preferably gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer; and most preferably gastric cancer and non-small cell lung cancer.

The present invention further provides an antibody-cytotoxic drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvent thereof:

Ab-[(L ₂ )tL ₁ -D)]y (I)

in:

D is the drug module;

L ₁ , L ₂ are linker units;

t is 0 or 1, preferably 1;

y is 1-8, preferably 2-5; y can be a decimal;

Ab is an antibody or an antigen-binding fragment thereof that specifically binds to the c-Met receptor as described above.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate of formula (I) or a pharmaceutically acceptable salt or solvent thereof is provided, wherein -L ₂ - is a compound of the following formula (-L ₂ -):

in

X ₁ is selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl;

_X2 is selected from -alkyl-, -cycloalkyl- and -heterocyclyl-;

m is 0-5, preferably 1-3; S is a sulfur atom.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the drug moiety D is a cytotoxic agent selected from toxins, chemotherapeutic agents, antibiotics, radioisotopes and nucleolytic enzymes.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent thereof is provided, wherein D is a compound of the following general formula (D):

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ -R ⁷ are selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl groups;

R ⁸ -R ¹¹ are selected from hydrogen atoms, halogen, alkenyl, alkyl, alkoxy and cycloalkyl groups; preferably, at least one of R ⁸ -R ¹¹ is selected from halogen, alkenyl, alkyl and cycloalkyl groups, and the rest are hydrogen atoms;

or any two of R ⁸ -R ¹¹ form a cycloalkyl group, and the remaining two groups are selected from hydrogen atoms, alkyl groups and cycloalkyl groups;

R ¹² -R ¹³ are selected from hydrogen atom, alkyl group or halogen;

R ¹⁴ is selected from an aryl group or a heteroaryl group, wherein the aryl group or the heteroaryl group is optionally further substituted by a substituent selected from a hydrogen atom, a halogen, a hydroxyl group, an alkyl group, an alkoxy group and a cycloalkyl group;

R ¹⁵ is selected from halogen, alkenyl, alkyl, cycloalkyl and COOR ¹⁷ ;

R ¹⁶ is selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl;

R ¹⁷ is selected from a hydrogen atom, an alkyl group and an alkoxy group.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein _L2 comprises a linker selected from Val-Cit, MC, PAB and MC-PAB, preferably MC.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein D is a maytansinoid; preferably selected from DM1, DM3 and DM4, more preferably DM1.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof is provided, wherein _L2 is selected from N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), and N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB); preferably SPP or SMCC.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate of formula (I) or a pharmaceutically acceptable salt or solvate thereof is provided, wherein D is a camptothecin alkaloid; preferably selected from CPT, 10-hydroxy-CPT, CPT-11 (irinotecan), SN-38 and topotecan, and more preferably SN-38.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the linker _L2 comprises a structure selected from Val-Cit, MC, PAB or MC-PAB, preferably MC or MC-vc-PAB.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent thereof is provided, which comprises a conjugate drug of general formula (II) or a pharmaceutically acceptable salt or solvent thereof:

in:

R ² -R ¹⁶ are as defined in the general formula (D);

Ab, t, y, L ₁ and L ₂ are as defined in the general formula (I).

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent thereof is provided, which comprises a conjugate drug of general formula (III) or a pharmaceutically acceptable salt or solvent thereof:

in:

R ² -R ¹⁶ are as defined in the general formula (D);

Ab, t, y, L ₁ and L ₂ are as defined in the general formula (I);

n is 3-6, preferably 5.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent thereof is provided, which comprises a conjugate drug of general formula (IV) or a pharmaceutically acceptable salt or solvent thereof:

in:

R ² -R ¹⁶ are as defined in the general formula (D);

Ab, y are as defined in the general formula (I);

n is as defined in formula (III);

X ¹ , X ² , and m are as defined in the general formula L2.

In a preferred embodiment of the present invention, an antibody-cytotoxic drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent thereof is provided, which includes a conjugate drug of general formula (V) or a pharmaceutically acceptable salt or solvent thereof:

in:

Ab, D, and y are as defined in the general formula (I);

n is as defined in formula (III);

X ¹ , X ² , and m are as defined in the general formula L2.

The antibody-cytotoxic drug conjugates or pharmaceutically acceptable salts or solvent compounds thereof of the present invention include but are not limited to:

Ab-9, Ab-10, and Ab-11 are c-Met antibodies as described above, and y is 1-8, preferably 2-5.

Wherein, the range of y is 1-8, preferably 1-4.

The present invention further provides a method for preparing an antibody-cytotoxic drug conjugate represented by general formula (V), comprising:

The compound of formula (Ab-L2) reacts with the compound of formula (L1-D) in an organic solvent to obtain a compound of formula (V); the organic solvent is preferably acetonitrile or ethanol;

in:

Ab is an antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor as described above;

X ₁ is selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl;

_X2 is selected from alkyl, cycloalkyl and heterocyclyl;

X is 0-5, preferably 1-3;

m is 0-5, preferably 1-3.

In a preferred embodiment of the present invention, there is provided an antibody-cytotoxic drug conjugate as described above or a pharmaceutically acceptable salt or solvent compound thereof, which has in vitro or in vivo cell killing activity.

The present invention further provides a pharmaceutical composition comprising the c-Met antibody or antigen-binding fragment thereof, or antibody-cytotoxic drug conjugate or pharmaceutically acceptable salt or solvent compound thereof and a pharmaceutically acceptable excipient, diluent or carrier as described above.

The present invention further provides a use of the c-Met antibody or antigen-binding fragment thereof, or antibody-cytotoxic drug conjugate or pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same, as described above, in the preparation of a medicament for treating a disease or condition mediated by c-Met, wherein the disease or condition is preferably cancer; more preferably a cancer expressing c-Met; most preferably gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer; and most preferably gastric cancer, pancreatic cancer, non-small cell lung cancer, and renal cancer.

The present invention further provides a method for treating and preventing a disease or condition mediated by c-Met, comprising administering to a patient in need thereof a therapeutically effective amount of the c-Met antibody or antigen-binding fragment thereof, or antibody-cytotoxic drug conjugate or pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same; wherein the disease or condition is preferably cancer; more preferably, a cancer expressing c-Met; most preferably, gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer; and most preferably, gastric cancer, pancreatic cancer, non-small cell lung cancer, and renal cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the tumor inhibition effects of the anti-c-Met antibodies and ADC molecules of the present invention. The results demonstrate that the novel ADC molecules, through the incorporated toxin, can achieve complete tumor inhibition, a trait not achieved by the antibody alone. The results also demonstrate that the ADC molecules of the present invention do not affect T _1/2 due to the toxin conjugation, and that the ADC drugs of the present invention exhibit no toxicity in mice.

Detailed Description of the Invention

1. Terminology

In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

The terms "c-Met" or "c-Met polypeptide" or "c-Met receptor" refer to a receptor tyrosine kinase that binds human growth factor (HGF). Unless otherwise specified herein, references to murine c-Met (m-c-Met) or monkey c-Met (cyno-c-Met) refer to human c-Met (h-c-Met). The human, murine, and cynomolgus monkey c-Met proteins used herein are encoded by nucleotide or polypeptide sequences provided by GenBank, such as the human polypeptide encoded by the nucleotide sequence provided in GenBank Accession No. NM_000245, or the human protein or its extracellular domain encoded by the polypeptide sequence provided in GenBank Accession No. NP_000236. The original single-chain precursor protein is post-translationally cleaved to produce α and β subunits, which are linked by disulfide bonds to form the mature receptor. The receptor tyrosine kinase c-Met participates in cellular processes including, for example, migration, invasion, and morphogenesis associated with embryonic tissue regeneration.

The term "c-Met-related disorder or condition" refers to any disease, disorder, or condition that results from the adverse expression or lack of expression, adverse regulation or lack of regulation, or deleterious activity or lack of activity of c-Met, or that can be regulated, treated, or cured by modulating c-Met expression or activity. For example, activation of the HGF/c-Met pathway can be expected in a large proportion of cancer patients, or in patients whose disease is driven by alterations associated with the c-Met pathway. For example, upregulation is attributed to various mechanisms, such as overexpression of HGF and/or c-Met, or constitutive activation through c-Met mutations. c-Met-related disorders or conditions include, but are not limited to, proliferative diseases and disorders and inflammatory diseases and disorders. Proliferative diseases include, but are not limited to, cancers including, for example, gastric cancer, esophageal cancer, renal cancer including papillary renal cell carcinoma, lung cancer, glioma, head and neck cancer, epithelial cancer, skin cancer, leukemia, lymphoma, myeloma, brain cancer, pancreatic cancer, colorectal cancer, gastrointestinal cancer, intestinal cancer, genital cancer, urinary cancer, melanoma, prostate cancer, and other tumors known to those skilled in the art. Inflammatory diseases include, but are not limited to, bacterial infections including infections caused by Listeria bacteria.

The antibodies described herein refer to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of the immunoglobulin heavy chains differ, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class, Ig can be further divided into different subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3), or conform to the numbering rules of Kabat and Chothia (HCDR1).

The term "murine antibody," as used herein, refers to anti-human c-Met monoclonal antibodies produced in mice according to the knowledge and skill in the art. These antibodies are prepared by injecting a test subject with a c-Met antigen, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional properties. In a preferred embodiment of the present invention, the murine c-Met antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variants thereof.

The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody. This can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies is first established. The variable region gene is then cloned from the mouse hybridoma cells and then cloned into the constant region gene of the human antibody for recombinant expression.

The term "humanized antibody," also known as CDR-grafted antibody humanization, refers to antibodies produced by transplanting mouse CDR sequences into human antibody variable region frameworks, i.e., different types of human germline antibody framework sequences. This can overcome the strong variable antibody response induced by chimeric antibodies due to the large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase ) and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. In a preferred embodiment of the present invention, the CDR sequences of the c-Met humanized antibody mouse are selected from SEQ ID NOs: 6, 7, 8, 9, 10, and 11. The human antibody variable region framework is designed and selected, wherein the light chain FR region sequence on the antibody light chain variable region is selected from a human germline light chain sequence, preferably the human germline light chain IGKV085 or IGKV 4-1*01, and comprises the FR1, FR2, FR3, and FR4 regions of the human germline light chain IGKV085 and IGKV4-1*01; wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from a human germline heavy chain sequence, preferably the human germline heavy chain IGHV 3-33*01, and comprises the FR1, FR2, FR3, and FR4 regions of the human germline heavy chain IGHV 3-33*01. To avoid a decrease in immunogenicity and a decrease in activity, the human antibody variable region can be subjected to minimal back mutations to maintain activity.

A variety of methods are available in the art for producing humanized antibodies. For example, humanized antibodies can be produced by obtaining the HCVR and LCVR sequences of an anti-c-Met-specific antibody (e.g., a murine antibody or an antibody produced by a hybridoma) and grafting them onto a selected human antibody framework coding sequence. Optionally, the CDR regions can be optimized by random mutagenesis or position-specific mutagenesis to replace one or more amino acids in the CDRs with different amino acids before grafting the CDR regions into the framework regions. Alternatively, the CDR regions can be optimized after insertion into the human framework regions using methods available to those skilled in the art. Preferably, a "humanized antibody" has CDRs derived from or from a parent antibody (i.e., a non-human antibody, preferably a mouse monoclonal antibody), and to the extent that they exist, the framework and constant regions (or a major or substantial portion thereof, i.e., at least about 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%) are identical in sequence to human germline immunoglobulin regions (see, e.g., the International, ImMunoGeneTics, Database) or recombinant or mutated forms thereof, whether or not the antibody is produced in human cells. Preferably, at least 2, 3, 4, 5 or 6 CDRs of the humanized antibody are optimized from the CDRs of the non-human parent antibody from which the humanized antibody is derived to produce desired properties, such as improved specificity, affinity or neutralization, which can be identified by screening assays, such as ELISA assays. Preferably, the optimized CDRs in the antibodies of the invention comprise at least one amino acid substitution when compared to the CDRs present in the parent antibody. Certain amino acid substitutions in the CDRs of the humanized antibodies of the invention (see Example 6 herein) compared to the CDRs of the parent antibody reduce the potential for antibody instability (e.g., removal of Asn residues in the CDRs) or reduce the immunogenicity of the antibody when administered to human subjects (e.g., as predicted by IMMUNOFILTER™ Technology).

After the CDR coding sequences are grafted onto the selected human framework coding sequences, the resulting DNA sequences encoding the humanized variable heavy and variable light chain sequences are then expressed to generate a humanized antibody that binds c-Met. The humanized HCVR and LCVR can be expressed as part of the entire anti-sclerostin antibody molecule, i.e., as a fusion protein with human constant domain sequences. However, the HCVR and LCVR sequences can also be expressed in the absence of constant sequences to generate a humanized anti-c-Met scFv.

References further describing methods for humanizing mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

The "antigen-binding fragment" referred to in the present invention refers to Fab fragments, Fab' fragments, F(ab') ₂ fragments, and Fv fragments (scFv) that bind to human c-Met, each with antigen-binding activity; these fragments comprise one or more CDR regions selected from SEQ ID NO:3 to SEQ ID NO:8 of the antibodies described herein. An Fv fragment is the smallest antibody fragment that contains the heavy and light chain variable regions of an antibody, but lacks the constant region, and possesses a complete antigen-binding site. Generally, an Fv antibody also includes a polypeptide linker between the VH and VL domains, enabling the formation of the required structure for antigen binding. Alternatively, two antibody variable regions can be linked into a single polypeptide chain using various linkers, resulting in a single-chain antibody or single-chain Fv (scFv). scFv can also be combined with other antibodies, such as anti-EGFR antibodies, to create bispecific antibodies. The term "binds to c-Met" herein refers to the ability to interact with human c-Met. The term "antigen-binding site" herein refers to a discrete, three-dimensional site on an antigen that is recognized by an antibody or antigen-binding fragment of the present invention. The term "ADCC," or antibody-dependent cell-mediated cytotoxicity, as used herein, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc region of an antibody. Modification of the Fc region of IgG can reduce or eliminate the antibody's ADCC effector function. Such modifications include mutations in the heavy chain constant region of the antibody, such as N297A, L234A, or L235A in IgG1; and F235E or L234A/E235A mutations in IgG2/4 chimeras and IgG4.

The fusion protein described in the present invention is a protein product co-expressed by two genes obtained through DNA recombination. The recombinant c-Met extracellular domain Fc fusion protein is a fusion protein co-expressed by DNA recombination of the c-Met extracellular domain and the human antibody Fc fragment. The c-Met extracellular domain refers to the portion of the c-Met protein expressed outside the cell membrane.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy chain (SEQ ID NO: 4) and light chain (SEQ ID NO: 5) can be cloned and recombined into the expression vector pEE6.4 (Lonza Biologics). The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred existing technology, mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human c-Met. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid containing the secreted antibody can be purified using conventional techniques. For example, it can be passed through an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, for example, at -70°C, or lyophilized.

The antibodies of the present invention are monoclonal antibodies. Monoclonal antibodies or mAbs described herein are antibodies derived from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clones. Monoclonal antibodies or antigen-binding fragments can be recombinantly obtained using hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR-grafting), or other existing technologies.

"Administering" and "treating" as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contacting of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contacting an agent with a cell, as well as contacting an agent with a fluid, wherein the fluid is in contact with the cell.

"Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present invention, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other health care professional to assess the severity or progression of the symptoms. While an embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in every patient, it should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

"Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th ed.)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity.

As used throughout the specification and claims, the term "consisting essentially of" or variations thereof means that all recited elements or groups of elements are included, and optionally, other elements of similar or different properties from the recited elements that do not significantly alter the basic or novel properties of a given administration regimen, method, or composition. As a non-limiting example, a binding compound consisting essentially of a recited amino acid sequence may further include one or more amino acids that do not significantly affect the properties of the binding compound.

An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or signs of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"Exogenous" refers to substances that are produced outside the body of an organism, cell, or human body, depending on the context. "Endogenous" refers to substances that are produced inside the body of a cell, organism, or human body, depending on the context.

"Homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally speaking, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence may but need not be present.

A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically/pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

The term "carrier," as used in the context of the present invention, refers to a system that can alter the drug's entry and distribution within the body, control its release rate, and deliver it to targeted organs. Drug carrier release and targeting systems can reduce drug degradation and loss, mitigate side effects, and improve bioavailability. For example, polymeric surfactants, which can serve as carriers, can self-assemble due to their unique amphiphilic structure, forming various aggregates, preferred examples of which include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers. The term "diluent," also known as filler, is primarily used to increase the weight and volume of a tablet. The addition of a diluent not only ensures a certain volume, but also reduces dosage variation of the main ingredient and improves the drug's compression moldability. When the drug in a tablet contains an oily component, an absorbent is added to absorb the oil and maintain a "dry" state, facilitating tablet production.

The term "pharmaceutically acceptable salt" refers to a salt of the ligand-cytotoxic drug conjugate of the present invention. Such salts are safe and effective when used in mammals and have the desired biological activity. The antibody-antibody drug conjugate compound of the present invention contains at least one amino group and can therefore form salts with acids.

The term "solvate" refers to a pharmaceutically acceptable solvent compound formed by a ligand-drug conjugate compound of the present invention and one or more solvent molecules.

The term "ligand" refers to a macromolecular compound that recognizes and binds to an antigen or receptor associated with a target cell. Ligands function to deliver drugs to the target cell population bound to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that bind to cells.

Therapeutic agents are molecules or atoms that are administered separately, simultaneously or sequentially with a binding moiety such as an antibody or antibody fragment, or a subfragment thereof, and can be used to treat a disease. Examples of therapeutic agents include, but are not limited to, antibodies, antibody fragments, conjugates, drugs, cytotoxic agents, pro-apoptotic agents, toxins, nucleases (including DNA enzymes and RNA enzymes), hormones, immunomodulators, chelating agents, boron compounds, photosensitizers or dyes, radioisotopes or radionuclides, oligonucleotides, interfering RNA, peptides, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins or peptides, or combinations thereof.

A conjugate is an antibody component or other targeting moiety coupled to a therapeutic agent as described above. As used herein, the terms "conjugate" and "immunoconjugate" are used interchangeably.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction.

"Toxin" refers to any substance capable of having a deleterious effect on the growth or proliferation of cells.

"Chemotherapeutic agents" refer to chemical compounds that can be used to treat cancer. This definition also includes anti-hormonal agents that act to modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or whole-body treatments. They can themselves be hormones.

Auristatins are fully synthetic drugs with relatively easy chemical modifications to optimize their physical properties and druggability. Auristatin derivatives used for antibody conjugation primarily include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). The former is a synthetic pentapeptide derived from the natural tubulin polymerase inhibitor dolastatin-10, with a 2-amino-1-phenylpropyl-1-ol residue added to the C-terminus. MMAE exhibits less than one nanomolar inhibitory activity against various human tumor cell lines. To reduce MMAE's inherent cytotoxic activity, MMAF incorporates a phenylalanine residue at the C-terminus of dolastatin-10. This addition of a carboxyl group significantly reduces MMAE's cell membrane permeability and thus significantly reduces its biological activity against cells. However, its inhibitory activity is significantly enhanced after antibody conjugation (US Pat. No. 7,750,116).

The term "tubulin inhibitors" refers to a class of compounds that inhibit or promote tubulin polymerization, thereby interfering with the mitotic process of cells and exerting anti-tumor effects. Non-limiting examples include maytansines, calicheamicins, taxanes, vincristine, colchicine, and dolestatins/auristatins, preferably selected from the group consisting of maytansines and dolestatins/auristatins; and more preferably selected from the group consisting of compounds represented by the general formula _D1 or _D2M .

CPT is the abbreviation for camptothecin and is used in this application to refer to camptothecin itself or an analog or derivative of camptothecin. The structures of camptothecin and some of its analogs with the numbers shown and rings labeled with letters A-E are provided in the following formulas.

CPT:R1＝R2＝R3＝H

10-Hydroxy-CPT: R1＝OH；R2＝R3＝H

Irinotecan (CPT-11): R2 = ethyl; R3 = H

SN-38: R1＝OH; R2＝ethyl; R3＝H

Topotecan: R1＝OH; R2＝H; R3＝CH-N(CH ₃ ) ₂

The term "intracellular metabolite" refers to a compound produced by a metabolic process or reaction within a cell that is directed against an antibody-drug conjugate (ADC). The metabolic process or reaction can be an enzymatic process, such as proteolytic cleavage of the peptide linker of the ADC, or hydrolysis of functional groups such as hydrazones, esters, or amides. Intracellular metabolites include, but are not limited to, antibodies and free drugs that undergo intracellular cleavage after entry, diffusion, uptake, or transport into a cell.

The terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction within a cell on an antibody-drug conjugate (ADC) whereby the covalent attachment, i.e., the linker, between the drug moiety (D) and the antibody (Ab) is broken, resulting in dissociation of the free drug from the antibody within the cell. The cleaved moiety of the ADC is thus an intracellular metabolite.

The term "bioavailability" refers to the systemic availability (i.e., blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term that measures both the time (rate) and the total amount (extent) of drug that reaches the general circulation from an administered dosage form.

The term "cytotoxic activity" refers to the cell-killing, cytostatic, or growth-inhibitory effect of an antibody-drug conjugate or an intracellular metabolite of an antibody-drug conjugate. Cytotoxic activity can be expressed as an IC50 value, which is the concentration (molar or mass) per unit volume at which half of the cells survive.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 2,2-diethylhexyl, and various branched chain isomers thereof. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen, or S(O) _m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spirocyclic, fused, and bridged heterocyclyls.

The heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring that is connected to the parent structure is a heterocyclyl, non-limiting examples of which include: and the like.

The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, most preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

The aryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5-membered or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein the definition of alkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

The term "bond" refers to a covalent bond represented by "—".

The term "hydroxy" refers to an -OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to _-NH2 .

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

The term "oxo" refers to =0.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and that the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

"Linker" refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches the antibody to the drug moiety. In various embodiments, linkers include divalent groups such as alkyldiyl, arylene, heteroarylene groups, such as -(CR2)nO(CR2)n-, repeating alkyloxy units (e.g., polyethyleneoxy, PEG, polymethyleneoxy), and alkylamino (e.g., polyethyleneamino, Jeffamine™) moieties; and diacids and amides, including succinate, succinamide, diglycolate, malonate, and hexanamide.

abbreviation

Connector assembly

MC＝6-maleimidocaproyl

Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker)

Citrulline = 2-amino-5-ureidopentanic acid

PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker component)

Me-Val-Cit = N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to protect it from cleavage by cathepsin B)

MC(PEG)6-OH = Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines)

SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate

SPDP = N-succinimidyl 3-(2-pyridyldithio) propionate

SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

IT = Iminothiolane

Cytotoxic drugs:

MMAE = Monomethyl auristatin E (MW 718)

MMAF = a variant of auristatin E (MMAE) with a phenylalanine (MW 731.5) at the C-terminus of the drug

MMAF-DMAEA = MMAF (MW 801.5) with DMAEA (dimethylaminoethylamine) amide-linked to the C-terminal phenylalanine

MMAF-TEG = MMAF esterified with tetraethylene glycol to phenylalanine

MMAF-NtBu = N-tert-butyl group attached as amide to the C-terminus of MMAF

DM1 = N(2')-deacetyl-N(2')-(3-mercapto-1-oxypropyl)-maytansine

DM3 = N(2')-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine

DM4 = N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine

The invention also provides antibody-cytotoxic drug conjugates or pharmaceutically acceptable salts or solvates thereof (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising any anti-c-Met antibody of the invention or other c-Met antibody having endocytosis activity (e.g., LY-2875358) conjugated to one or more cytotoxic agents, such as chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes (i.e., radioconjugates).

In certain embodiments, the antibody-cytotoxic drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprises an anti-c-Met antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents that can be used to generate the antibody-cytotoxic drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, are described herein (above). Enzymatically active toxins and fragments thereof may also be used, and these enzymatically active toxins and fragments thereof are described in the specification.

In certain embodiments, the antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof comprises an anti-c-Met antibody and one or more small molecule toxins, including but not limited to small molecule drugs such as camptothecin derivatives, calicheamicin, maytansinoids, dolastatin, auristatin, trichothecene and CC1065, and fragments of these drugs having cytotoxic activity.

Exemplary linkers _L include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). A variety of linkers are known in the art, and some are described below.

The linker can be a "cleavable linker" that facilitates release of the drug in the cell. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

In some embodiments, the linker component can be a "stretcher unit" that connects the antibody to another linker component or a drug moiety. An exemplary stretcher unit is shown below (wherein the wavy line indicates the site of covalent attachment to the antibody):

In some embodiments, the linker unit can be an amino acid unit. In one such embodiment, the amino acid unit allows for protease cleavage of the linker, thereby facilitating release of the drug from the antibody-cytotoxic drug conjugate or its pharmaceutically acceptable salt or solvate after exposure to intracellular proteases (such as lysosomal enzymes). See, for example, Doronina et al. (2003) Nat. Biotechnol. 21: 778-784. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include: valine-citrulline (VC or val-cit); alanine-phenylalanine (AF or ala-phe); phenylalanine-lysine (FK or phe-lys); or N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid unit can comprise naturally occurring amino acid residues, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid unit can be designed and optimized with respect to their selectivity for enzymatic cleavage by specific enzymes (e.g., tumor-associated proteases, cathepsins B, C and D, or plasma proteases).

In some embodiments, the linker component can be a "spacer" unit that connects the antibody (either directly or through a Stretcher unit and/or an Amino Acid unit) to the Drug Module. The Spacer unit can be "self-immolative" or "non-self-immolative." A "non-self-immolative" Spacer unit refers to a Spacer unit in which part or all of the Spacer unit remains bound to the Drug Module after enzymatic (proteolytic) cleavage of the ADC. Examples of non-self-immolative Spacer units include, but are not limited to, glycine Spacer units and glycine-glycine Spacer units. Other combinations of peptide spacers that are susceptible to sequence-specific enzymatic cleavage are also contemplated. For example, enzymatic cleavage of an ADC containing a glycine-glycine Spacer unit by tumor cell-associated proteases will result in the release of the glycine-glycine-Drug Module from the remainder of the ADC. In one such embodiment, the glycine-glycine-Drug Module is then subjected to a separate hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine Spacer unit from the Drug Module.

"Self-immolative" spacer units allow for release of the drug moiety without a separate hydrolysis step. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is formed between the benzyl alcohol and the cytotoxic agent. See, e.g., Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103. In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB).

Exemplary linkers in the present invention are as follows:

Linkers, including stretchers, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1.

Exemplary Drug Modules

Maytansine and maytansine alkaloids

In some embodiments, antibody-cytotoxic drug conjugates or their pharmaceutically acceptable salts or solvent compounds include antibodies of the present invention coupled to one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that play a role by inhibiting tubulin multimerization. Maytansine was initially isolated from the East African shrub Maytenus serrata (US Pat. No. 3,896,111). It was subsequently discovered that some microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (US Pat. No. 4,151,042).

Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relatively easy to prepare by fermentation or chemical modification or derivatization of fermentation products; (ii) easily derivatized with functional groups suitable for conjugation to antibodies via non-disulfide linkers; (iii) stable in plasma; and (iv) effective against a variety of tumor cell lines.

Maytansine compounds suitable for use as maytansinoid drug moieties are well known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973). Maytansinol and maytansinol analogs can also be synthesized according to known methods.

Exemplary embodiments of maytansinoid drug moieties include: DM1; DM3; and DM4, as disclosed herein.

Auristatin and dolastatin

In some embodiments, the antibody-cytotoxic drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprises an antibody of the invention conjugated to a dolastatin or dolastatin peptide analog or derivative (e.g., auristatin) (U.S. Patent Nos. 5,635,483; 5,780,588). Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug moiety may be attached to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF, as disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, March 28, 2004, the disclosure of which is expressly incorporated herein by reference in its entirety. The peptide drug moiety can be selected from the following general formulas _DE and _DF :

The wavy lines _DE and _DF indicate the covalent attachment sites of antibodies or antibody-linkers, and each position is independent;

^R2 is selected from H and C1-C8 hydrocarbon group;

^R3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

^R4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

^R5 is selected from H and methyl;

or R ⁴ and R ⁵ together form a carbocyclic ring and have the general formula -(CRaRb)n-, wherein ^Ra and R ^b are independently selected from H, C1-C8 hydrocarbon group and C3-C8 carbocyclic ring, and n is selected from 2, 3, 4, 5 and 6;

^R6 is selected from H and C1-C8 hydrocarbon group;

R ⁷ is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

Each R ⁸ is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O-(C1-C8 alkyl);

R ⁹ is selected from H and C1-C8 hydrocarbon groups;

R ¹⁰ is selected from aryl or C3-C8 heterocycle;

Z is O, S, NH or NR ¹² , wherein R ¹² is a C1-C8 hydrocarbon group;

R ¹¹ is selected from H, C1-C20 hydrocarbon group, aryl group, C3-C8 heterocycle, -(R ¹³ O)mR ¹⁴ and -(R ¹³ O)m-CH(R ¹⁵ ) ₂ ;

m is an integer selected from 1-1000;

^R13 is a C2-C8 hydrocarbon group;

R ¹⁴ is H or a C1-C8 hydrocarbon group;

R ¹⁵ is independently H, COOH, -(CH ₂ )nN(R16) ₂ , -(CH ₂ )n-SO ₃ H or -(CH ₂ )n-SO ₃ -C1-C8 hydrocarbon group at each occurrence;

Each occurrence of R ¹⁶ is independently H, C1-C8 hydrocarbon group or -(CH ₂ )n-COOH;

R ¹⁸ is selected from -C(R8) ₂ -C(R8) ₂ -aryl, -C(R8) ₂ -C(R8) ₂ -(C3-C8 heterocycle) and -C(R8) ₂ -C(R8) ₂ -(C3-C8 carbocycle); and

n is an integer selected from 0 to 6.

An exemplary auristatin of formula D _E , embodiment is MMAE, wherein the wavy line indicates the linker (L) covalently attached to the antibody-drug conjugate:

An exemplary auristatin of formula DF, an embodiment is MMAF, wherein the wavy line indicates a linker (L) covalently attached to the antibody-drug conjugate (see US 2005/0238649 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124):

Other drug moieties include MMAF derivatives selected from the following, where the wavy line indicates a linker (L) covalently attached to the antibody-drug conjugate:

In one aspect, a hydrophilic group can be attached to the drug moiety at R ¹¹ , including but not limited to triethylene glycol ester (TEG), as described above. Without being limited to any particular theory, the hydrophilic group facilitates internalization and non-agglomeration of the drug moiety. Exemplary embodiments of ADCs of Formula I comprising auristatin/dolastatin or derivatives thereof are described in US 2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124, which are expressly incorporated herein by reference. Exemplary embodiments of ADCs of Formula I comprising MMAE or MMAF and various linkers have the following structures and abbreviations (wherein "Ab" is an antibody; p is 1 to about 8; "Val-Cit" is a valine-citrulline dipeptide; and "S" is a sulfur atom):

Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schroder and K. Lübke, "The Peptides", Volume 1, pp 76-136, 1965, Academic Press). Auristatin/dolastatin drug moieties can be prepared according to the methods described in the following references: US2005-0238649A1; U.S. Patent No. 5635483; U.S. Patent No. 5780588; Pettit et al. (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13: 243-277; Pettit, G. R. et al., Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15: 859-863; and Doronina (2003) Nat. Biotechnol. 21(7): 778-784.

Specifically, auristatin/dolastatin drug moieties of formula DF, such as MMAF and its derivatives, can be prepared using the methods described in US 2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. Auristatin/dolastatin drug moieties of formula DE, such as MMAE and its derivatives, can be prepared using the methods described in Doronina et al. (2003) Nat. Biotech. 21: 778-784. Drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE can be conveniently synthesized by conventional methods, such as those described in Doronina et al. (2003) Nat. Biotech. 21: 778-784 and U.S. Patent Application Publication No. US 2005/0238649 A1, and then conjugated to the antibody of interest.

Drug payload

Drug loading is represented by y, i.e., the average number of drug modules per antibody in the molecule of Formula I. The range of drug loading can be 1-20 drug modules (D) per antibody. The ADC of Formula I includes a collection of antibodies coupled with a certain range (1-20) of drug modules. The average number of drug modules per antibody in the ADC preparation from the coupling reaction can be characterized by conventional means, such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of ADC in terms of y can also be measured. In some cases, separation, purification, and characterization of homogeneous ADCs having a certain value of p from ADCs with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis.

For some antibody-drug conjugates, y may be limited by the number of attachment sites on the antibody. For example, if the attachment is a cysteine thiol, as in the exemplary embodiments above, the antibody may have only one or several cysteine thiol groups, or may have only one or several thiol groups with sufficient reactivity to attach the linker. In certain embodiments, higher drug loadings, such as y>5, may cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. In certain embodiments, the drug loading of the ADC of the present invention ranges from 1 to about 8; about 2 to about 6; about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7. In fact, it has been shown that for some ADCs, the optimal ratio of each antibody drug module can be less than 8, and can be about 2 to about 5. See US 2005-0238649 Al (incorporated herein by reference in its entirety).

In certain embodiments, the drug module less than the theoretical maximum is coupled to the antibody in the coupling reaction. The antibody may include, for example, a lysine residue that does not react with the drug-linker intermediate or linker reagent, as discussed below. Only the most reactive lysine groups can react with amine-reactive linker reagents. Generally speaking, antibodies do not include many free and reactive cysteine thiol groups to which drug modules can be connected; in fact, most cysteine thiol groups in antibodies exist in the form of disulfide bridges. In certain embodiments, antibodies can be reduced to produce reactive cysteine thiol groups with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions. In certain embodiments, the antibody is placed in denaturing conditions to expose reactive nucleophilic groups, such as lysine or cysteine.

The loading of the ADC (drug/antibody ratio, DAR) can be controlled in various ways, for example, by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) partial or limited reducing conditions for cysteine thiol modification, (iv) engineering the amino acid sequence of the antibody by recombinant techniques such that the number and position of cysteine residues are altered in order to control the number and/or position of linker-drug attachments (such as thioMabs or thioFabs prepared as described herein and in WO2006/034488 (incorporated herein by reference in its entirety)).

It should be understood that if more than one nucleophilic group reacts with the drug-linker intermediate or with the linker reagent and the subsequent drug moiety reagent, the resulting product is a mixture of ADC compounds having a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay specific for the antibody and specific for the drug. The various ADC molecules in the mixture can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography. In certain embodiments, homogeneous ADCs with a single loading value can be separated from the conjugated mixture by electrophoresis or chromatography.

Certain methods for preparing antibody-cytotoxic drug conjugates or pharmaceutically acceptable salts or solvent compounds thereof

ADCs of Formula I can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting the nucleophilic group of the antibody with a bivalent linker reagent via a covalent bond to form Ab-L, which is then reacted with the drug moiety D; and (2) reacting the nucleophilic group of the drug moiety with a bivalent linker reagent via a covalent bond to form D-L, which is then reacted with the nucleophilic group of the antibody. Exemplary methods for preparing ADCs of Formula I via the latter route are described in US 2005-0238649 A1, which is expressly incorporated herein by reference.

Nucleophilic groups of antibodies include, but are not limited to: (i) N-terminal amine groups; (ii) side chain amine groups, such as lysine; (iii) side chain thiol groups, such as cysteine; and (iv) hydroxyl or amino groups of sugars in glycosylated antibodies. Amines, thiols, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker modules to form covalent bonds, and linker reagents include: (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyls, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. Antibodies can be fully or partially reduced by treatment with reducing agents such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), thereby making them reactive for coupling with linker reagents. Each cysteine bridge will theoretically form two reactive thiol nucleophiles. Alternatively, sulfhydryl groups can be introduced into antibodies through modification of lysine residues, for example, by reacting lysine residues with 2-iminothiolane (Traut's reagent), resulting in conversion of an amine to a thiol.

The antibody-drug conjugates of the present invention can also be generated by the reaction between an electrophilic group (such as an aldehyde or ketone carbonyl) on the antibody and a nucleophilic group on a linker reagent or drug. Useful nucleophilic groups on linker reagents include, but are not limited to, hydrazides, oximes, amino groups, hydrazine groups, thiosemicarbazone groups, hydrazine carboxylates, and arylhydrazides. In one embodiment, the sugars of the glycosylated antibody can be oxidized with, for example, a periodate oxidant to form an aldehyde or ketone group that can react with the amine group of the linker reagent or drug moiety. The resulting imine Schiff base can form a stable linkage, or can be reduced with, for example, a borohydride reagent to form a stable amine linkage. In one embodiment, the reaction of the carbohydrate portion of the glycosylated antibody with galactose oxidase or sodium metaperiodate can generate carbonyl groups (aldehyde and ketone groups) in the antibody, which can react with suitable groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate to form an aldehyde at the first amino acid (Geoghegan and Stroh, (1992) Bioconjugate Chem. 3: 138-146; US5362852). Such an aldehyde can react with a drug moiety or linker nucleophile.

Nucleophilic groups on the drug moiety include, but are not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups, which are capable of reacting with electrophilic groups on the linker moiety to form a covalent bond, and linker reagents include: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups.

The compounds of the present invention specifically encompass, but are not limited to, ADCs prepared with the following cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., Pierce Biotechnology, Inc., Rockford, IL., U.S.A., see 2003-2004 Applications Handbook and Catalog, pages 467-498).

Antibody-cytotoxic drug conjugates comprising an antibody and a cytotoxic agent, or pharmaceutically acceptable salts or solvates thereof, can also be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for coupling radionucleotides to antibodies. See WO94/11026.

Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be prepared, for example, by recombinant techniques or peptide synthesis. The recombinant DNA molecule can comprise regions encoding the antibody and cytotoxic portions of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

In yet another embodiment, the antibody can be coupled to a "receptor" (such as streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient, followed by the use of a clearing agent to clear the unbound conjugate from the circulation, and then the "ligand" (e.g., avidin) coupled to a cytotoxic agent (e.g., radionucleotide) is administered. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

DETAILED DESCRIPTION

The present invention is further described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

Experimental methods in the Examples or Test Examples of the present invention, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the raw material or product manufacturers. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Contemporary Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

Example

Example 1. Antigen and Antibody Cloning and Expression

The antibodies (light and heavy chains) used in the present invention were constructed using the well-known overlap extension PCR method. The DNA fragments generated by overlap extension PCR were inserted into the expression vector pEE6.4 (Lonza Biologics) using the HindIII/BstBI restriction sites and expressed in 293F cells (Invitrogen, Cat# R790-07). The resulting recombinant proteins were used for immunization or screening. The c-Met gene template was obtained from origene (Cat. No. RC217003). The cloned and expressed DNA sequences are as follows.

Human c-Met extracellular domain (ECD) and mouse Fc domain fusion protein (human c-Met ECD-mFc) DNA sequence:

atgaaggcccccgctgtgcttgcacctggcatcctcgtgctcctgtttaccttggtgcagaggagcaatggggagtgtaaagaggcactagcaaagtccgagatgaatgtgaatatgaagtatcagcttcccaacttcaccgcggaaacacccatccagaatgtcattctacatg agcatcacattttccttggtgccactaactacatttatgttttaaatgaggaagaccttcagaaggttgctgagtacaagactgggcctgtgctggaacacccagattgtttcccatgtcaggactgcagcagcaaagccaatttatcaggaggtgtttggaaagataacatcaa catggctctagttgtcgacacctactatgatgatcaactcattagctgtggcagcgtcaacagagggacctgccagcgacatgtctttccccacaatcatactgctgacatacagtcggaggttcactgcatattctccccacagatagaagagcccagccagtgtcctgactgt gtggtgagcgccctgggagccaaagtcctttcatctgtaaaggaccggttcatcaacttctttgtaggcaatacccataaattcttcttatttcccagatcatccattgcattcgatatcagtgagaaggctaaaggaaacgaaagatggttttatgtttttgacggaccagtcct acattgatgttttacctgagttcagagattcttaccccattaagtatgtccatgcctttgaaagcaacaattttatttacttcttgacggtccaaagggaaactctagatgctcagacttttcacacaagaataatcaggttctgttccataaactctggattgcattcctacat ggaaatgcctctggagtgtattctcacagaaaagagaaaaaagagatccacaaagaaggaagtgtttaatatacttcaggctgcgtatgtcagcaagcctggggcccagcttgctagacaaataggagccagcctgaatgatgacattcttttcggggtgttcgcacaaagcaag ccagattctgccgaaccaatggatcgatctgccatgtgtgcattccctatcaaatatgtcaacgacttcttcaacaagatcgtcaacaaaaacaatgtgagatgtctccagcatttttacggacccaatcatgagcactgctttaataggacacttctgagaaattcatcaggct gtgaagcgcgccgtgatgaatatcgaacagagtttaccacagctttgcagcgcgttgacttattcatgggtcaattcagcgaagtcctcttaacatctatatccaccttcattaaaggagacctcaccatagctaatcttgggacatcagagggtcgcttcatgcaggttgtggt ttctcgatcaggaccatcaacccctcatgtgaattttctcctggactcccatccagtgtctccagaagtgattgtggagcatacattaaaccaaaatggctacacactggttatcactgggaagaagatcacgaagatcccattgaatggcttgggctgcagacatttccagtcc tgcagtcaatgcctctctgccccaccctttgttcagtgtggctggtgccacgacaaatgtgtgcgatcggaggaatgcctgagcgggacatggactcaacagatctgtctgcctgcaatctacaaggttttcccaaatagtgcaccccttgaaggagggacaaggctgaccatat gtggctgggactttggatttcggaggaataataaatttgatttaaagaaaactagagttctccttggaaatgagagctgcaccttgactttaagtgagagcacgatgaatacattgaaatgcacagttggtcctgccatgaataagcatttcaatatgtccataattatttcaaa tggccacgggacaacacaatacagtacattctcctatgtggatcctgtaataacaagtatttcgccgaaatacggtcctatggctggtggcactttacttactttaactggaaattacctaaacagtgggaattctagacacatttcaattggtggaaaaacatgtactttaaaa agtgtgtcaaacagtattcttgaatgttataccccagcccaaaccatttcaactgagtttgctgttaaattgaaaattgacttagccaaccgagagacaagcatcttcagttaccgtgaagatcccattgtctatgaaattcatccaaccaaatcttttattagtggtggggagca caataacaggtgttgggaaaaacctgaattcagttagtgtcccgagaatggtcataaatgtgcatgaagcaggaaggaactttacagtggcatgtcaacatcgctctaattcagagataatctgttgtaccactccttccctgcaacagctgaatctgcaactccccctgaaaac caaagcctttttcatgttagatgggatcctttccaaatactttgatctcatttatgtacataatcctgtgtttaagccttttgaaaagccagtgatgatctcaatgggcaatgaaaatgtactggaaattaagggaaatgatattgaccctgaagcagttaaaggtgaagtgtta aaagttggaaataagagctgtgagaatatacacttacattctgaagccgttttatgcacggtccccaatgacctgctgaaattgaacagcgagctaaatatagagtggaagcaagcaatttcttcaaccgtccttggaaaagtaatagttcaaccagatcagaatttcaca(SEQ IDNO:1)

Human c-Met extracellular Sema region and Flag-His tag (Human c-Met Sema-Flis) DNA sequence:

atgaaggcccccgctgtgcttgcacctggcatcctcgtgctcctgtttaccttggtgcagaggagcaatggggagtgtaaagaggcactagcaaagtccgagatgaatgt gaatatgaagtatcagcttcccaacttcaccgcggaaacacccatccagaatgtcattctacatgagcatcacattttccttggtgccactaactacatttatgttttaa atgaggaagaccttcagaaggttgctgagtacaagactgggcctgtgctggaacacccagaattgtttcccatgtcaggactgcagcagcaaagccaatttatcaggaggt gtttggaaagataacatcaacatggctctagttgtcgacacctactatgatgatcaactcattagctgtggcagcgtcaacagagggacctgccagcgacatgtctttcc ccacaatcatactgctgacatacagtcggaggttcactgcatattctccccacagatagaagagcccagccagtgtcctgactgtgtggtgagcgccctgggagccaaag tcctttcatctgtaaaaggaccggttcatcaacttctttgtaggcaataccataaattcttcttatttcccagatcatccattgcattcgatatcagtgagaaggctaaag gaaacgaaagatggttttatgtttttgacggaccagtcctacattgatgttttacctgagttcagagattcttaccccattaagtatgtccatgcctttgaaagcaacaa ttttatttacttcttgacggtccaaagggaaactctagatgctcagacttttcacacaagaataatcaggttctgttccataaactctggattgcattcctacatggaaat gcctctggagtgtattctcacagaaaagagaaaaaagagatccacaaagaaggaagtgtttaatatacttcaggctgcgtatgtcagcaagcctggggcccagcttgcta gacaaataggagccagcctgaatgatgacattcttttcggggtgttcgcacaaagcaagccagatctgccgaaccaatggatcgatctgccatgtgtgcattccctatc aaatatgtcaacgacttcttcaacaagatcgtcaacaaaaacaatgtgagatgtctccagcatttttacggacccaatcatgagcactgctttaataggacacttctgag aaattcatcaggctgtgaagcgcgccgtgatgaatatcgaacagagtttaccacagctttgcagcgcgttgacttattcatgggtcaattcagcgaagtcctcttaacat ctatatccaccttcattaaaggagacctcaccatagctaatcttgggacatcagagggtcgcttcatgcaggttgtggtttctcgatcaggaccatcaacccctcatgtg aattttctcctggactcccatccagtgtctccagaagtgattgtggagcatacattaaaccaaaatggctacacactggttatcactgggaagaagatcacgaagatccc attgaatggcttgggctgcagacatttccagtcctgcagtcaatgcctctctgccccaccctttgttcagtgtggctggtgccacgacaaatgtgtgcgatcggaggaat gcctgagcgggacatggactcaacagatctgtctgcctgcaatctacaaggactacaaggacgacgacgacaagcatgtccaccatcatcaccatcactgattcgaa(SEQ ID NO:2)

Human c-Met ECD his tag (Human c-Met ECD-His) recombinant protein DNA sequence:

atgaaggcccccgctgtgcttgcacctggcatcctcgtgctcctgtttaccttggtgcagaggagcaatggggagtgtaaagaggcactagcaaagtccgagatgaatgtgaatatgaagtatcagcttcccaacttcaccgcggaaacacccatccagaatgtcattctacatga gcatcacattttccttggtgccactaactacatttatgttttaaatgaggaagaccttcagaaggttgctgagtacaagactgggcctgtgctggaacacccagattgtttcccatgtcaggactgcagcagcaaagccaatttatcaggaggtgtttggaaagataacatcaacat ggctctagttgtcgacacctactatgatgatcaactcattagctgtggcagcgtcaacagagggacctgccagcgacatgtctttccccacaatcatactgctgacatacagtcggaggttcactgcatattctccccacagatagaagagcccagccagtgtcctgactgtgtgg tgagcgccctgggagccaaagtcctttcatctgtaaaaggaccggttcatcaacttctttgtaggcaataccataaattcttcttatttcccagatcatccattgcattcgatatcagtgagaaggctaaaggaaacgaaagatggttttatgtttttgacggaccagtcctacattg atgttttacctgagttcagagattcttaccccattaagtatgtccatgcctttgaaagcaacaattttatttacttcttgacggtccaaagggaaactctagatgctcagacttttcacacaagaataatcaggttctgttccataaactctggattgcattcctacatggaaatg cctctggagtgtattctcacagaaaagagaaaaaagagatccacaaagaaggaagtgtttaatatacttcaggctgcgtatgtcagcaagcctggggcccagcttgctagacaaataggagccagcctgaatgatgacattcttttcggggtgttcgcacaaaagcaagccagattct gccgaaccaatggatcgatctgccatgtgtgcattccctatcaaatatgtcaacgacttcttcaacaagatcgtcaacaaaaacaatgtgagatgtctccagcatttttacggacccaatcatgagcactgctttaataggacacttctgagaaattcatcaggctgtgaagcgcgc cgtgatgaatatcgaacagagtttaccacagctttgcagcgcgttgacttattcatgggtcaattcagcgaagtcctcttaacatctatatccaccttcattaaaggagacctcaccatagctaatcttgggacatcagagggtcgcttcatgcaggttgtggtttctcgatcagga ccatcaacccctcatgtgaattttctcctggactcccatccagtgtctccagaagtgattgtggagcatacattaaaccaaaatggctacacactggttatcactgggaagaagatcacgaagatcccattgaatggcttgggctgcagacatttccagtcctgcagtcaatgcct ctctgccccaccctttgttcagtgtggctggtgccacgacaaatgtgtgcgatcggaggaatgcctgagcgggacatggactcaacagatctgtctgcctgcaatctacaaggttttcccaaatagtgcaccccttgaaggagggacaaggctgaccatatgtggctgggactttgg atttcggaggaataaatttgatttaaagaaaactagagttctccttggaaatgagagctgcaccttgactttaagtgagagcacgatgaatacattgaaatgcacagttggtcctgccatgaataagcatttcaatatgtccataattatttcaaatggccacgggacaacaca atacagtacattctcctatgtggatcctgtaataacaagtatttcgccgaaatacggtcctatggctggtggcactttacttactttaactggaaattacctaaacagtgggaattctagacacatttcaattggtggaaaaacatgtactttaaaaaagtgtgtcaaacagtattct tgaatgttataccccagcccaaaccatttcaactgagtttgctgttaaattgaaaattgacttagccaaccgagagacaagcatcttcagttaccgtgaagatcccattgtctatgaaattcatccaaccaaatcttttattagtggtgggagcacaataacaggtgttgggaaaa acctgaattcagttagtgtcccgagaatggtcataaatgtgcatgaagcaggaaggaactttacagtggcatgtcaacatcgctctaattcagagataatctgttgtaccactccttccctgcaacagctgaatctgcaactccccctgaaaaccaaagcctttttcatgttagatg ggatcctttccaaatactttgatctcatttatgtacataatcctgtgtttaagccttttgaaaagccagtgatgatctcaatgggcaatgaaaatgtactggaaattaagggaaatgatattgaccctgaagcagttaaaggtgaagtgttaaaagttggaaataagagctgtgtgaga atatacacttacattctgaagccgttttatgcacggtccccaatgacctgctgaaattgaacagcgagctaaatatagagtggaagcaagcaatttcttcaaccgtccttggaaaagtaatagttcaaccagatcagaatttcacacaccatcatcaccatcactgattcgaa(SEQ ID NO:3)

Example 2. Antigen-antibody binding assay (ELISA)

This experiment uses the enzyme-linked immunosorbent assay (ELISA) method to test the affinity of anti-c-Met antibodies (including hybridoma supernatants or recombinantly expressed monoclonal antibodies, etc.) to c-Met antigen in vitro.

The experimental steps were as follows: dilute the antigen (humanc-Met-His, Example 1) to 2 μg/ml in coating solution (PBS) (Hyclone, Cat No.: SH30256.01B), add 100 μl/well to a 96-well ELISA plate (Costar 9018, Cat No.: 03113024), and incubate at 4°C overnight. The next day, the 96-well ELISA plate coated with the antigen was returned to room temperature and washed three times with washing solution (PBS + 0.05% Tween 20 (Sigma, Cat No.: P1379). Then, 200 μl/well blocking solution (PBS + 1% BSA (Roche, Cat No.: 738328) was added and incubated at 37°C for 1 hour. Washed three times with washing solution. Anti-c-Met antibody to be tested was added to the 96-well ELISA plate and incubated at room temperature for 1 hour. Washed three times with washing solution. Secondary antibody (Goat anti-Mouse IgG (H + L) (HRP) (Thermo, Cat No.: 31432) diluted 10000 times with blocking solution was added 100 μl/well to the 96-well ELISA plate and incubated at room temperature for 1 hour. Washed three times with washing solution. 100 μl/well color development solution TMB (eBioscience) was added. REF: 00-4201-56) to a 96-well microtiter plate. Add 100 μl/well stop solution (2N H ₂ SO ₄ ) to the 96-well microtiter plate. Read the plate using a plate reader at 450 nm.

Example 3. Production of mouse monoclonal antibody against human c-Met by cell line

The present invention generates a mouse-derived anti-human c-Met monoclonal cell line through mouse immunization, spleen cell fusion, and hybridoma screening. This method is well known in the art. Specifically, the recombinantly expressed antigen (human c-Met ECD-mFc, human c-Met Sema-flis, see Example 1) is diluted to 1 mg/ml with PBS (Hyclone, Cat No.: SH30256.01B), emulsified with Freund's adjuvant (complete Freund's adjuvant for the first immunization, incomplete Freund's adjuvant for the booster immunization), and then subcutaneously inoculated into Balb/C mice (5 mice per group), with each mouse receiving 100 μg of antigen. Booster immunizations are administered two weeks apart. Starting from the first booster immunization, mouse sera are collected within 7 to 10 days after each booster immunization, and the titer is determined by ELISA (see Example 2 for the method).

Mice with serum titers higher than 1:10 ⁵ after immunization were selected for cell fusion. Mouse B cells and myeloma cells (SP2/0, ATCC number: CRL-1581 ^™ ) were aseptically prepared and counted. The two cells were mixed at a ratio of B cells:SP2/0 = 1:4 and centrifuged (1500 rpm for 7 min). The supernatant was discarded and 1 ml of 50% polyethylene glycol (Supplier: SIGMA, Catalogue #RNBB306) was added. The mixture was then terminated with 1 ml of serum-free RPMI1640 (Supplier: GIBCO, Catalogue #C22400). The mixture was centrifuged for 10 min, the supernatant was discarded, and the pellet was resuspended in RPMI1640 containing hybridoma growth factor (Supplier: Roche, Catalogue #1363735001), serum (Supplier: GIBCO, Catalogue #C20270), and HAT (Supplier: Invitrogen, Catalogue #21060-017). 10 cells were added to each well. ^Five B cells were plated in a standard format, with 100 μl per well, and cultured in a 37°C cell culture incubator. After 3 days, 100 μl of RPMI-1640 supplemented with hybridoma growth factor, serum, and HT (Supplier: Invitrogen, Catalogue #11067-030) was added to each well. After 2 to 4 days, the medium was completely replaced with 150 μl of RPMI-1640 supplemented with hybridoma growth factor, serum, and HT per well. The next day, positive clones were detected by ELISA (see Example 2 for the method). Results are shown in Table 1.

Table 1. Hybridoma fusion detection of human c-Met immunized mice

Clone number Test results (OD450) Negative control 0.07 Ab-1 1.48 Ab-2 1.38 Ab-3 1.29 Ab-4 1.6 Ab-5 1.64 Ab-6 1.75 Ab-7 1.58 Ab-8 1.24

Example 4 Inhibitory Effect of Anti-Human c-Met Monoclonal Antibody on Proliferation of Gastric Cancer Cells MKN45

The clones were selected and further cultured to obtain monoclonal clones. After binding activity was verified by ELISA, the culture supernatants of the monoclonal clones were selected for cell viability testing. The experimental principle is that the anti-human c-Met antibodies of the present invention can inhibit the phosphorylation of c-Met on the surface of human gastric cancer cells (MKN45), thereby inhibiting the proliferation of MNK45 cells.

Human gastric cancer cells (MKN45, JCRB, JCRB0254, P11) were plated at 1× ^10⁵ cells/mL in 96-well cell culture plates (Costar, #3799) at 50 μl/well in RPMI 1640 medium (GIBCO, cat#11835) supplemented with 10% fetal bovine serum (FBS) (GIBCO-10099141). Anti-human c-Met antibody (50 μl/well) was then added and cultured in a 37°C incubator (SANYO, instrument number TINC035) for 5 days. Cell proliferation was assessed using the CellTiter-Luminescent Cell Viability Assay (Promega, G7573) according to the kit's instructions. Plates were read using a plate reader (PerkinElmer, TREA001-RDA-IBA100). The cell proliferation percentage was calculated using the following formula: Cell proliferation rate % = (1-cell count in the experimental group/cell count in the untreated group) x 100%. The results are shown in Table 2.

Table 2. Cellular activity of anti-human c-Met monoclonal antibodies

Clone number MKN45 inhibition rate (%) Blank control 0.01 Ab-1 59.2 Ab-2 58.4 Ab-3 59.6 Ab-4 54.9 Ab-5 77.4 Ab-6 70.8 Ab-7 56.4 Ab-8 53.8

Example 5. Anti-c-Met antibody sequence cloning

The active single cell strain Ab-5 obtained in Example 4 was subjected to cDNA sequence cloning, and then recombinantly expressed monoclonal antibodies were tested for various activities. The present invention uses reverse transcription PCR to amplify the heavy and light chain variable regions of the antibody gene, which are then linked to a vector for sequencing to obtain the monoclonal antibody light and heavy chain sequences. First, an RNA purification kit (Qiagen, Cat. No. 74134, see instructions for this step) is used to extract total cellular RNA from the active single cell strain in Example 4. Then, a single-stranded cDNA is prepared using Invitrogen's Cat. No. 18080-051 cDNA synthesis kit, i.e., oligo-dT primer-cDNA reverse transcription. Using this as a template, the antibody light and heavy chain variable region sequences are synthesized using PCR. The PCR products are cloned into the TA vector pMD-18T and then sent for sequencing. The resulting antibody light and heavy chain sequences are respectively cloned into expression vectors (see Example 1), recombinant monoclonal antibodies are expressed, and after verifying activity (see Examples 2 and 4), humanization is performed.

The sequence of the mouse hybridoma monoclonal antibody Ab-5 of the present invention is:

Heavy chain variable region:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLPNYGVHWVRQSPGKGLEWLGVIWSGGSTNYAAAFVSRLRISKDNKSQVFFEMNSLQADDTAVYYCARNHDNPYNYAMDYWGQGTTVTVSS(SEQ ID NO:4)

Light chain variable region:

DIVLTQSPGSLAVYLGQRATISCRANKSVSTSTYNYLHWYQQKPGQPPKLLIYLASNLASGVPARFSGSGSGTDFTLNIHPLEEEDAATYYCQHSRDLPPTFGAGTKLELKR(SEQ ID NO:5)

The amino acid residues of the VH/VL CDRs of the anti-human c-Met antibody were determined and annotated using the Kabat numbering system.

The murine CDR sequences of the present invention are shown in Table 3:

Table 3. CDR sequences of mouse anti-sclerostin antibodies

Antibody Ab-5 Heavy chain CDR1 NYGVH (SEQ ID NO: 6) Heavy chain CDR2 VIWSGGSTNYAAAFVS (SEQ ID NO: 7) Heavy chain CDR3 NHDNPYNYAMDY (SEQ ID NO: 8) Light chain CDR1 RANKSVSTSTYNYLH (SEQ ID NO: 9) Light chain CDR2 LASNLAS (SEQ ID NO: 10) Light chain CDR3 QHSRDLPPT (SEQ ID NO: 11)

Example 6. Humanization of anti-c-Met antibody

After comparing the light and heavy chain sequences of the murine anti-c-Met monoclonal antibody obtained in Example 5 for homology in an antibody database, a humanized antibody model was constructed. Based on the model, back mutations were selected to identify the optimal humanized anti-c-Met monoclonal antibody as the preferred molecule of the present invention. This method begins by searching a database of published mouse Fab crystal structure models (e.g., the PDB database) for crystal structures with similar homology to the resulting murine candidate molecule. High-resolution (e.g., high-resolution) Fab crystal structures were selected to construct a mouse Fab model. The light and heavy chain sequences of the murine antibody of the present invention were aligned with those in the model, retaining sequences consistent with the murine antibody sequence in the model to obtain a structural model of the murine antibody of the present invention. Inconsistent amino acids were identified as potential back mutation sites. The murine antibody structural model was run using Swiss-pdb viewer software for energy optimization (minimization). Back mutations were performed at various amino acid sites in the model, excluding the CDRs. The resulting mutant (humanized) antibodies were compared to the pre-humanized antibodies for activity testing. Humanized antibodies with good activity were retained. Subsequently, the CDR regions were optimized, including avoiding glycosylation, deamidation, and oxidation sites. The optimized CDR regions of the humanized anti-c-Met antibody are shown in Table 4:

Table 4. CDR sequences of optimized anti-c-Met antibodies

The humanized light and heavy chain variable regions are shown below:

1. Heavy chain variable region:

Ab-9

QVTLKESGPVLVKPTETLTLTCTVSGFSLPNYGVHWVRQPPGKALEWLAVIWSGGSTNYAAAFVSRLRISKDTSKSQVVFTMNNMDPVDTATYYCARNHDNPYNYAMDYWGQGTTVTVSS(SEQ ID NO:13)

Ab-10

QVQLVESGGGVVQPGRSLRLSCAASGFSLSNYGVHWVRQAPGKGLEWLAVIWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYNYAMDYWGQGTTVTVSS(SEQ ID NO:14)

Ab-11

QVQLVESGGGVVQPGRSLRLSCAASGFTLPNYGVHWVRQAPGKGLEWLAVIWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYNYAMDYWGQGTTVTVSS(SEQ ID NO:15)

2. Light chain variable region:

Ab-9

DIVLTQSPASLAVSPGQRATITCRANKSVSTSTYNYLHWYQQKPGQPPKLLIYLASNLASGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRDLPPTFGQGTKLEIKR(SEQ ID NO:16)

Ab-10

DIVLTQSPDSLAVSLGERATINCRADKSVSTSTYNYLHWYQQKPGQPPKLLIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPPTFGQGTKLEIKR(SEQ ID NO:17)

Ab-11

DIVLTQSPDSLAVSLGERATINCRANKSVSTSTYNYLHWYQQKPGQPPKLLIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPPTFGQGTKLEIKR(SEQ ID NO:18)

The humanized light and heavy chains and the IgG Fc segment are recombined to obtain the humanized anti-c-Met monoclonal antibody of the present invention. The Fc sequence used is selected from the following sequences:

Heavy chain constant region:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:20)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:21)

Light chain constant region:

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:22)

The above antibodies were cloned, expressed, and purified by gene cloning and recombinant expression methods. After ELISA (Example 2) and in vitro binding activity assay (Example 7), the humanized antibodies Ab-9, Ab-10, and Ab-11 with the best activity retention were finally selected. The sequences are as follows:

Ab-9 humanized antibody:

Heavy chain:

QVTLKESGPVLVKPTETLTLTCTVSGFSLPNYGVHWVRQPPGKALEWLAVIWSGGSTNYAAAFVSRLRISKDTSKSQVVFTMNNMDPVDTATYYCARNHDNPYNYAMDYWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:23)

Light chain:

DIVLTQSPASLAVSPGQRATITCRANKSVSTSTYNYLHWYQQKPGQPPKLLIYLASNLASGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRDLPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:26)

Ab-10 humanized antibody:

Heavy chain:

QVQLVESGGGVVQPGRSLRLSCAASGFSLSNYGVHWVRQAPGKGLEWLAVIWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYNYAMDYWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:24)

Light chain:

DIVLTQSPDSLAVSLGERATINCRADKSVSTSTYNYLHWYQQKPGQPPKLLIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:27)

Ab-11 humanized antibody:

Heavy chain:

QVQLVESGGGVVQPGRSLRLSCAASGFTLPNYGVHWVRQAPGKGLEWLAVIWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYNYAMDYWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:25)

Light chain:

DIVLTQSPDSLAVSLGERATINCRANKSVSTSTYNYLHWYQQKPGQPPKLLIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:28)

Example 7. In vitro binding activity assay of anti-c-Met humanized antibodies

In addition to testing the in vitro activity of the humanized antibody of the present invention by ELISA (Example 2), its binding to the c-Met high-expressing cell line MKN45 and its affinity to the c-Met antigen (BIACore assay) were also tested. The results are shown in Tables 5 and 6.

The binding activity of the anti-c-Met humanized compound of the present invention to the c-Met high-expressing cell line MKN45 was detected by FACS method.

MKN45 cells (JCRB, Cat No.: JCRB0254) were resuspended in RPMI 1640 medium (GIBCO, Cat No.: 11835-030) supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063) to a concentration of 10,000 cells/mL. Two mL of the resuspended MKN45 cells were plated at 150,000 cells/well into a 96-well microtiter plate (Corning, Cat No.: 3799). Eight concentrations of c-Met antibody (five-fold serial dilutions starting from 20 μg/mL) were added to the corresponding wells in a final volume of 100 μL. The plates were incubated at 4°C for 1 hour. FACS buffer (phosphate-buffered saline (PBS) with 2.5% (v/v) fetal bovine serum (FBS) (Hyclone, Cat: SH30256.01B)) was added, centrifuged at 1300 rpm at 4°C for 4 minutes, and the supernatant was discarded. Repeat three times. 100 μl of secondary antibody solution (fluorescently labeled goat anti-mouse secondary antibody: 1:200 dilution, Biolegend, Cat#405307; fluorescently labeled anti-human secondary antibody: 1:30 dilution, Biolegend, Cat#409304) was added to each well and incubated at 4°C for 1 hour. FACS buffer was added, centrifuged at 1300 rpm at 4°C for 4 minutes, and the supernatant was discarded. Repeat three times. 200 μl of FACS buffer was added, the cells were resuspended, and the samples were prepared for analysis using a flow cytometer (BD FACS Array).

The present invention adopts surface plasmon resonance (SPR) technology to detect the affinity between c-Met antibody and c-Met antigen Sema-His.

Anti-mouse IgG (GE Life Sciences catalog #BR-1008-38) or anti-human IgG (GE Life Sciences catalog #BR-1008-39) antibodies were diluted to 30 μg/ml and 50 μg/ml, respectively, with pH 5.0 sodium acetate solution (GE Healthcare, Cat #BR-1003-51) and immobilized to the test and control channels of a CM5 chip (GE Life Sciences catalog #BR-1000-12) using an amino coupling kit (GE Life Sciences, Cat #BR100050). The coupling level was set at 15,000 RU. c-Met antibody was diluted to 1.5 μg/ml in PBS (Hyclone, Cat#SH30256.01B) + 0.05% P20 (GE Life Sciences, Cat#BR-1000-54) running buffer. Sema-his antigen was diluted to 200 nM in running buffer and then diluted 1:2 in the same buffer to 0.78 nM. The diluted antibody was flowed through the experimental channel at a flow rate of 30 μl/min for one minute, and the antigen was flowed through the experimental and control channels at the same flow rate for 3 minutes. After 10 minutes of dissociation, the flow rate was adjusted to 10 μl/min, and regeneration buffer was flowed through the experimental and control channels for 3 minutes. Data were double-subtracted and fitted using BiaEvaluation 4.1 using a 1:1 (Langmuir) model.

Table 5. Binding activity of humanized anti-c-Met antibodies

Humanized antibodies Ab-9 Ab-10 Ab-11 0.13 0.39 0.2

Table 6. MKN45 binding activity and antigen affinity of humanized anti-c-Met antibodies

Humanized antibodies MKN45/FCAS binding activity (nM) and antigen affinity Biacore (nM) Ab-9 1.6 4 Ab-10 1.23 8

The above results show that the binding activity of the humanized antibody of the present invention to the antigen is 0.13-8 nM, and the test results vary depending on the detection method. The results show that the humanized anti-c-Met antibody retains the binding activity of the antibody before humanization.

Example 8. In vitro function and cell activity evaluation of anti-c-Met humanized antibodies

To test the function of the antibody of the present invention, the antibody in Example 7 was evaluated using the binding assay of blocking c-Met ligand (hepatocyte growth factor HGF) and c-Met, and the cell proliferation inhibition assay (Example 4).

The binding of HGF to c-Met results in tyrosine phosphorylation of c-Met molecules and activation of the c-Met signaling pathway. This experiment used ELISA to detect the activity of the anti-c-Met antibody of the present invention in blocking HGF binding to the receptor c-Met protein, ie, IC ₅₀ .

c-Met ECD-mFc (Example 1) was diluted in PBS (Hyclone, Cat#SH30256.01B) to a final concentration of 2 μg/ml and coated in a 96-well ELISA plate (Costar, Cat#2592) overnight at room temperature. The plates were washed three times with PBST (PBS + 0.05% Tween 20 (Simga, Cat#P1379)) using a plate washer (Suppler: BioTex; Model: ELX405; S/N: 251504). 300 μl of blocking buffer (PBS + 1% BSA (Roche, Cat#738328)) was added to the 96-well plate and incubated at 37°C for 60 minutes. After washing three times with PBST, 50 μl of the antibody diluted in blocking buffer was added to the 96-well plates and incubated at 37°C for 90 minutes. Add 50 μl of human HGF (Sino Biological, #10463-HNAS) diluted in blocking buffer to a final concentration of 20 ng/ml to a 96-well plate containing c-Met antibody and incubate at room temperature for 120 minutes. After washing three times with PBST, add 100 μl of biotinylated anti-HGF antibody (R&D, Cat#BAF294) diluted in blocking buffer to a final concentration of 100 ng/ml to the 96-well plate and incubate for 90 minutes. After washing with PBST, add 100 μl of horseradish peroxidase (eBioscience, #18-4100-51) diluted in blocking buffer to the 96-well plate and incubate for 30 minutes. After washing with PBST, 100 μl of substrate (ebioscience, cat# 00-4201-56) was added and incubated at room temperature for 10 minutes. 100 μl of stop solution (2N H ₂ SO ₄ ) was added and the plate was read using a 450 nm microplate reader (Supplier: Moleculer Devices; Model: MNR0643; Equipment ID: TMRP001). Data were analyzed using SoftMax Pro v5. The results are shown in Table 7.

Table 7. In vitro function and cell activity of humanized anti-c-Met antibodies of the present invention

The above results indicate that the humanized antibody of the present invention not only retains the antigen-binding activity, but also can prevent the binding of the antigen to the ligand, and simultaneously exhibits the activity of inhibiting the growth of tumor cells.

Example 9 Evaluation of the agonist activity of the humanized anti-c-Met antibody of the present invention

Anti-c-Met antibodies block HGF/c-Met binding but may also activate c-Met signaling, i.e., exhibit agonist activity. However, anti-c-Met agonist activity is not required by the present invention. To test whether the antibodies of the present invention have agonist activity, three assays were performed: c-Met phosphorylation, proliferation of human renal clear cell carcinoma skin metastasis cells (caki-1), and migration of human lung cancer H441 cells.

HGF binding to c-Met activates tyrosine phosphorylation of c-Met and the c-Met signaling pathway. Therefore, HGF activation of c-Met was used as a positive control in agonist experiments. The human lung cancer cell line A459 was used to assess the potency of HGF-induced phosphorylation of c-Met at tyrosine residue 1349.

A549 cells were suspended in Ham's F12K + 2mM glutamine (Invitrogen, #21127-022) + 10% (v/v) fetal bovine serum (FBS) (GIBCO, #10099141). 0.2 mL of the cell suspension was added to a 96-well plate (Corning, #3599) at a cell density of 60,000 cells/well. The cells were incubated at 37°C, 5% CO₂ for ₂₄ hours. After 24 hours, the medium in the 96-well plates was discarded and 100 μL of reduced-serum medium (Ham's F12K + 2mM glutamine + 0.5% FBS) was added. The cells were starved for 6 hours at ₃₇ °C, 5% CO₂. Antibodies were diluted in the reduced-serum medium (final concentration: 20 μg/mL). The positive control HGF was used at a concentration of 200 ng/mL. The cells were incubated at 37°C for 15 minutes. After discarding the culture medium, 50 μl of cell lysis buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA), 50 mM NaF, 1% (v/v) TRITON-X 100, protease inhibitors (Roche cat#05892791001), phosphatase inhibitor cocktail II (Sigma#P5726), and phosphatase inhibitor cocktail III (Sigma#P0044) were added. After cell lysis, c-Met tyrosine phosphorylation was detected by ELISA. A c-Met capture antibody (CST, cat#3148s) was diluted 1:1000 in PBS and added to a 96-well ELISA plate (Costar, cat#9018), 100 μl per well, and incubated overnight at 4°C. After washing three times with TBS-T, 300 μl of blocking buffer (TBS-T plus 2% (w/v) BSA) was added and incubated for 1 hour. After washing three times with TBS-T, 75 μL of cell blocking buffer was added, followed by 25 μL of cell lysate, and incubation at 4°C overnight. The cells were washed three times with TBS-T and 100 μL of pY1349c-Met antibody (cell signaling, #3133) diluted 1:1000 in blocking buffer was added to each well. After incubation at room temperature for 2 hours, the cells were washed four times with TBST and HRP-conjugated goat anti-rabbit polyclonal antibody (cell signaling, cat#7074) diluted 1:12000 in blocking buffer was added to each well for 1 hour at room temperature. The cells were washed five times with TBS-T, and 100 μL of TMB (ebioscience #TMB,004201) was added to each well. 100 μL of stop solution (2N H ₂ SO ₄ ) was then added, and the plate was read using a 450 nm microplate reader (Supplier: Moleculer Devices; Model: MNR0643; Equip ID: TMRP001). Data analysis was performed using SoftMaxPro v5. The results are shown in Table 8.

Human clear cell renal carcinoma (Caki-1) cells express the hepatocyte growth factor receptor (c-Met). HGF can bind to c-Met and stimulate Caki-1 cell proliferation. Therefore, the humanized anti-c-Met antibodies of the present invention can be expressed in parallel with HGF in Caki-1 cells to evaluate the agonist activity of the anti-c-Met antibodies.

Caki-1 (Shanghai Institute of Science, TCHu135, P12) cells were plated at 1000 cells/well in a 96-well cell culture plate (Costar, #3799) in McCoy's 5A (Invitrogen, #16600) plus 10% fetal bovine serum (FBS) (GIBCO-10099141) at 37°C for 24 hours. The cells were then starved for 24 hours in McCoy's 5A plus 0.5% FBS. After starvation, cells were treated with serially diluted anti-c-Met antibodies (maximum concentration: 20 μg/ml) and a positive control for 5 days. Cell proliferation was then assessed using a cell proliferation assay kit (CellTiter-Luminescent Cell Viability Assay (Promega, G7573). Plates were read using a PerkinElmer plate reader (Manufacturer: PerkinElmer, Instrument No.: TREA001-RDA-IBA100). The percentage of cell proliferation was calculated as follows: % cell proliferation = number of cells in the experimental group / number of cells in the untreated group × 100%. The results showed that none of the antibodies of the present invention had any effect on the proliferation of Caki-1 cells. See Table 8.

If the anti-c-Met antibody has agonist activity, it can affect the migration ability of cells. The present invention uses the H441 cell line expressing c-Met to evaluate the ability of the c-Met antibody of the present invention to affect cell migration.

H441 cells (ATCC, Cat No.: HTB-174) were resuspended in RPMI 1640 medium (GIBCO, Cat No.: 11835-030) with 10% (v/v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063) to a concentration of 500,000 cells/ml. The resuspended H441 cells were added to a 12-well culture plate (Costar, Cat No.: 3513) at 1 ml/well and cultured at 37°C, 5% _CO2 for 3 days. The cells were washed twice with phosphate-buffered saline (PBS) and added with RPMI 1640 medium containing low concentration fetal bovine serum (0.5% FBS) and cultured at 37°C, 5% CO2 _for 16 hours. The bottom of each well was scratched with a 5ml pipette tip and washed once with culture medium containing low-concentration fetal bovine serum. 1ml of low-concentration serum culture medium RPMI 1640 was added. The scratched area was randomly selected and photographed under a 4x inverted microscope, and the culture plate was marked. This time was marked as time zero. Cells were treated with 10μg/ml c-Met antibody or HGF control (200ng/ml) for 16 hours (37°C, 5% _CO2 ). The marked scratched area was photographed under a 4x inverted microscope, and this time was marked as the post-migration time. The percentage of affected cell migration was calculated as the migration distance relative to time zero divided by the migration distance of the culture medium group relative to time zero, multiplied by 100. The results are shown in Table 8.

Table 8. Evaluation of agonist activity of humanized anti-c-Met antibodies of the present invention

Humanized antibodies Activated c-Met phosphorylation (%)* Used to stimulate Caki-1 proliferation H441 migration rate (%)# Ab-9 29.9 none 53 Ab-10 33.7 none 34

*: Antibody concentration was 20 μg/ml, and the activation of c-Met phosphorylation by control HGF (200 ng/ml) was set as 100%. #: Antibody concentration was 20 μg/ml, and the effect of HGF (200 ng/ml) on H441 mobility was set as 100%.

The above results indicate that the agonist activity of the humanized antibody of the present invention is relatively weak (c-Met phosphorylation, H441 migration assay results) or non-existent (20 μg/ml antibody failed to stimulate Caki-1 proliferation).

Example 10 In vivo efficacy evaluation of anti-c-Met antibodies

In order to evaluate the anti-tumor activity of the antibody of the present invention, a human gastric cancer MKN45 cell model was subcutaneously transplanted into BALB/c nude mice for detection.

MKN45 cells were cultured in monolayers using RPMI-1640 medium (10% fetal bovine serum) at 37°C and 5% _CO₂ . Cells were counted and harvested during the logarithmic growth phase. Cells were resuspended in PBS to an appropriate concentration and inoculated subcutaneously in the right flank of female BALB/c nude mice (10 weeks old, weighing 22-28 g, purchased from Shanghai Slake Laboratory Animal Co., Ltd., Animal Certification Number: 2007000548777, housed in SPF-grade housing). ^When the average tumor volume reached 114 ^mm⁻ , mice were weighed and tumor volume was measured. Groups were then divided and dosing began. The control group received PBS, while the antibody-treated group received the antibody of the present invention at 5 mg/kg twice weekly. Tumor volume and body weight were measured twice weekly, and the experiment was terminated on day 25. Tumor size was calculated using the formula: Tumor volume ( ^mm⁻ ) = 0.5 × (longest diameter of tumor × shortest diameter of tumor²). The formula for calculating the tumor inhibition rate was: tumor inhibition rate = (V0-VT)/V0×100%, where _V0 and _VT were the tumor volumes at the beginning and end of the experiment, respectively.

The results showed that antibodies Ab-9 and Ab-10 of the present invention had tumor inhibition rates of 56% and 64%, respectively. The body weight of the mice did not change significantly during the experiment (22-24 g). These results demonstrate that the humanized anti-c-Met antibodies of the present invention have tumor growth inhibition activity in vivo.

Example 11 Endocytosis of anti-c-Met antibodies

The antibodies of the present invention bind to human c-Met and exhibit excellent in vitro activity and in vivo tumor suppression activity. Furthermore, the antibodies have no or very weak agonist activity. To test whether the antibodies of the present invention can be internalized into cells along with human c-Met after binding to human c-Met, evaluation was performed using c-Met-expressing human gastric cancer cells MKN45 (JCRB, Cat No.: JCRB0254).

MKN45 cells were resuspended in RPMI 1640 medium (GIBCO, Cat No.: 11835-030) supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063) to a concentration of 10,000 cells/mL. Two mL of the resuspended MKN45 cells were plated at a density of 250,000 cells/well into a 96-well microtiter plate. 10 μg/mL c-Met antibody was added to the corresponding wells in a final volume of 100 μL. The cells were incubated at 4°C for 1 hour. Add FACS buffer (phosphate buffered saline with 2.5% fetal bovine serum (Hyclone, Cat: SH30256.01B), centrifuge at 4°C, 1300 rpm, for 4 minutes, discard the supernatant, and repeat three times. Add 100 μl of secondary antibody solution (fluorescently labeled goat anti-mouse secondary antibody: 1:200 dilution, Biolegend, Cat#405307; fluorescently labeled anti-human secondary antibody: 1:30 dilution, Biolegend, Cat#409304) to each well and incubate at 4°C for 1 hour. Add FACS buffer, centrifuge at 4°C, 1300 rpm, for 4 minutes, discard the supernatant, and repeat three times. Add complete cell culture medium (RPMI 1640 medium with 10% fetal bovine serum) and incubate at 37°C, 5% _CO2 for 0 hours, 0.5 hours, 1 hour, 2 hours, and 4 hours. Add 5 μl of 7-AAD (Biolegend, Cat: 420403) to 100 μl per well. Incubate in FACS buffer at 4°C for 30 minutes. Add FACS buffer, centrifuge at 4°C at 1300 rpm for 4 minutes, and discard the supernatant. Repeat three times. Add 200 μl of stripping buffer (0.05 M glycine, pH 3.0; 0.1 M sodium chloride, 1:1 (v/v)) to each well, resuspend the cells, and incubate at room temperature for 7 minutes. Centrifuge at room temperature at 1300 rpm for 4 minutes and discard the supernatant. Add 200 μl of neutralizing wash buffer (0.15 M Tris, pH 7.4) to each well, resuspend the cells, and centrifuge at room temperature at 1300 rpm for 4 minutes. Discard the supernatant. Add 200 μl of FACS buffer, resuspend the cells, and prepare the samples for analysis using a flow cytometer (BD FACSCalibur). Results are shown in Table 9.

c-Met antibody internalization percentage = (fluorescence intensity value at each time point - average fluorescence intensity value at zero point) / average fluorescence intensity value at zero point.

Table 9. Evaluation of cellular endocytosis performance of humanized anti-c-Met antibodies of the present invention (endocytosis percentage %)

Humanized antibodies 0h 0.5h 1h 2h 4h hIgG (control)* 0 -0.9 -4.4 -4.9 3.6 Ab-9 0 26 32 32 31 Ab-10 0 twenty four 38 53 59

*: Control group -4.9% -3.6% are experimental errors (background values). All are considered to have no endocytosis

The results in Table 9 show that the antibodies of the present invention, in addition to having no agonist activity, also have good endocytosis. After binding to target cells, the antibodies and receptors are rapidly internalized into the target cells, with endocytosis reaching maximum within 2-4 hours.

Example 12 Analysis of biophysical stability characteristics of anti-c-Met antibodies

In order to evaluate the biophysical stability of the antibodies of the present invention, such as the presence of glycosylation and deamination sites, and their stability, the anti-c-Met antibodies of the present invention were comprehensively evaluated using mass spectrometry (LC-MS) analysis.

Glycosylation analysis was performed by direct LC-MS analysis of light and heavy chain molecular weights. Deamination was analyzed by LC-MS under conditions of prolonged treatment (over 3 months) at 4°C or accelerated treatment (21 days at 40°C). After treatment under various conditions, samples were removed and diluted to 2 mg/ml with Tris-HCl (pH 7.2). A final concentration of 10 mM TCEP and 6 M urea (AMRESCO, Cat#0378) were added and incubated _{at 37} °C for 20 min. Sulfhydryl groups were protected by the addition of IAA (Sigma-Aldrich, Cat#I1149) at a final concentration of 20 mM and incubated at room temperature in the dark for 15 min. The sample was diluted with Tris-HCl (pH 7.2) to adjust the pH. Protease (Sigma-Aldrich, Cat#T6567) was added at a protein:enzyme ratio of 10:1 (weight to weight). After incubation at 37°C for 25 min, the reaction was terminated with formic acid (Fluca, Cat#94318) at a final concentration of 0.1%. The reaction was centrifuged and analyzed by LC-MS.

BiopharmaLynx was used to analyze the presence of deamidation. The mass spectrometric data were analyzed by identifying the native peptide and modified products containing the deamidation site, extracting the precursor ions to generate an EIC (Extracted Ion Chromatogram), integrating the peak areas, and calculating the proportions of deamidated and oxidized products. The results are shown in Table 10.

Table 10. Physical stability evaluation of humanized anti-c-Met antibodies of the present invention

*: All heavy chains are glycosylated, and the molecular weights are consistent with expectations. #: Deaminated molecule ratio (%). 0.66-1.0% is within the detection background range.

The above results show that the antibody of the present invention is stable and has good physical properties.

Example 13: Anti-c-Met Antibody Ab-10 Conjugated to Toxin MC-MMAF

The anti-c-Met antibodies of the present invention possess properties such as receptor binding blocking activity, lack of agonist activity, target cell endocytosis activity, and physical stability. These properties make the antibodies of the present invention particularly suitable for conjugation with toxins to form ADC drugs for the treatment of c-Met-expressing cancers. The conjugation process is shown in the figure below:

In the first step, S-(3-carbonylpropyl)thioacetate (0.7 mg, 5.3 μmol) was dissolved in 0.9 mL of acetonitrile and set aside. The pre-prepared S-(3-carbonylpropyl)thioacetate in acetonitrile was added to an acetic acid/sodium acetate buffer (10.35 mg/mL, 9.0 mL, 0.97 mmol) solution of Ab-10 mAb at pH 4.3. Then, 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 μmol) was added dropwise. The mixture was shaken at 25°C for 2 hours. After completion of the reaction, the product 1b was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS, pH 6.5) to obtain a solution. This solution was concentrated to approximately 10 mg/mL and then directly used in the next step.

In the second step, 0.35 mL of 2.0 M hydroxylamine hydrochloride solution was added to solution 1b (11.0 mL). After shaking at 25°C for 30 minutes, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5) to obtain the title product Ab-10 monoclonal antibody-propanethiol 1c solution (concentration 6.17 mg/ml, 14.7 mL).

In the third step, the compound MC-MMAF (1.1 mg, 1.2 μmol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile and added to the Ab-10 monoclonal antibody-propanethiol solution 1c (6.17 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After filtration through a 0.2 μm filter under sterile conditions, the title product ADC-1 in PBS buffer (3.7 mg/mL, 4.7 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 148119.2 (M _Ab + 0D), 149278.1 (M _Ab + 1D), 150308.1 (M _Ab + 2D), 151314.1 (M _Ab + 3D). The average value of the toxin amount attached to each antibody molecule (DAR) was y = 1.7.

Example 14: Anti-c-Met Antibody Ab-10 Conjugated to Toxin MC-VC-PAB-MMAE

The compound MC-VC-PAB-MMAE (1.6 mg, 1.2 μmol, prepared using the method disclosed in PCT patent WO2004010957) was dissolved in 0.3 mL of acetonitrile and added to Ab-10 monoclonal antibody-propanethiol solution 1c (6.17 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After aseptic filtration through a 0.2 μm filter under sterile conditions, the title product ADC-2 in PBS buffer (3.6 mg/mL, 4.8 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 148118.4 (M _Ab + 0D), 149509.2 (M _Ab + 1D), 150903.1 (M _Ab + 2D), 152290.4 (M _Ab + 3D), 153680.7 (M _Ab + 4D). The average value of the toxin-bound amount (DAR) per antibody molecule was y = 1.8.

Example 15: Anti-c-Met Antibody Ab-10 Conjugated to Toxin MC-VC-PAB-MMAF

The compound MC-VC-PAB-MMAF (1.6 mg, 1.2 μmol, prepared using the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile and added to Ab-10 monoclonal antibody-propanethiol solution 1c (6.17 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After aseptic filtration through a 0.2 μm filter under sterile conditions, the title product ADC-3 in PBS buffer (3.5 mg/mL, 4.9 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 148119.1 (M _Ab + 0D), 149525.3 (M _Ab + 1D), 150930.7 (M _Ab + 2D), 152335.2 (M _Ab + 3D), 153739.8 (M _Ab + 4D). The average value of the toxin bound to each antibody molecule (DAR) was y = 1.6.

Example 16: Anti-c-Met Antibody Ab-10 Conjugated to Toxin MC-MMAE

The compound MC-MMAE (1.2 mg, 1.2 μmol, prepared by the method disclosed in patent application "US7/750/116B1") was dissolved in 0.3 mL of acetonitrile and added to Ab-10 monoclonal antibody-propanethiol solution 1c (6.17 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After aseptic filtration through a 0.2 μm filter under sterile conditions, the title product ADC-4 in PBS buffer (3.4 mg/mL, 5.0 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 148118.6 (M _Ab + 0D), 149104.3 (M _Ab + 1D), 150090.1 (M _Ab + 2D), 151075.8 (M _Ab + 3D). The average value of the toxin amount (DAR) bound to each antibody molecule was y = 1.6.

Example 17: Anti-c-Met Antibody Ab-9 Conjugated to Toxin MC-MMAE

In the first step, S-(3-carbonylpropyl)thioacetate (0.7 mg, 5.3 μmol) was dissolved in 0.9 mL of acetonitrile and set aside. This pre-prepared S-(3-carbonylpropyl)thioacetate in acetonitrile was added to an acetic acid/sodium acetate buffer (10.85 mg/mL, 9.0 mL, 0.976 mmol) solution of Ab-9 monoclonal antibody at pH 4.3. Then, 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 μmol) was added dropwise. The mixture was shaken at 25°C for 2 hours. After completion of the reaction, the product was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M PBS, pH 6.5) to obtain a solution of the title product 5b. This solution was concentrated to approximately 10 mg/mL and directly used in the next step.

In the second step, 0.35 mL of 2.0 M hydroxylamine hydrochloride solution was added to solution 5b (11.0 mL). After shaking at 25°C for 30 minutes, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5) to obtain the title product Ab-9 monoclonal antibody-propanethiol 5c solution (concentration 6.2 mg/ml, 15.0 mL).

In the third step, the compound MC-MMAE (1.1 mg, 1.2 μmol) was dissolved in 0.3 mL of acetonitrile and added to the Ab-9 monoclonal antibody-propanethiol solution 5c (6.2 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After filtration through a 0.2 μm filter under sterile conditions, the title product ADC-5 in PBS buffer (3.8 mg/mL, 4.6 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 150530.9 (M _Ab + 0D), 151915.7 (M _Ab + 1D), 153333.6 (M _Ab + 2D), 154763.4 (M _Ab + 3D), 156271.9 (M _Ab + 4D). The average value of the toxin bound to each antibody molecule (DAR) was y = 1.5.

Example 18: Anti-c-Met Antibody Ab-9 Conjugated to Toxin MC-MMAF

The compound MC-MMAF (1.1 mg, 1.2 μmol) was dissolved in 0.3 mL of acetonitrile and added to the Ab-9 monoclonal antibody-propanethiol solution 5c (6.2 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After aseptic filtration through a 0.2 μm filter under sterile conditions, the title product ADC-6 in PBS buffer (3.8 mg/mL, 4.6 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 150537.8 (M _Ab + 0D), 152087.9 (M _Ab + 1D), 153486.5 (M _Ab + 2D), 154911.7 (M _Ab + 3D), 156499.9 (M _Ab + 4D). The average value of the toxin-bound amount (DAR) per antibody molecule was y = 1.7.

Example 19: Anti-c-Met Antibody Ab-9 Conjugated to Toxin MC-VC-PAB-MMAF

The compound MC-VC-PAB-MMAF (1.6 mg, 1.2 μmol) was dissolved in 0.3 mL of acetonitrile and added to the Ab-9 monoclonal antibody-propanethiol solution 5c (6.2 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After aseptic filtration through a 0.2 μm filter under sterile conditions, the title product ADC-7 in PBS buffer (3.8 mg/mL, 4.6 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 150537.8 (M _Ab + 0D), 152087.9 (M _Ab + 1D), 153486.5 (M _Ab + 2D), 154911.7 (M _Ab + 3D), 156499.9 (M _Ab + 4D). The average value of the toxin-bound amount (DAR) per antibody molecule was y = 1.8.

Example 20: Anti-c-Met Antibody Ab-9 Conjugated to Toxin MC-VC-PAB-MMAE

The compound MC-VC-PAB-MMAE (1.6 mg, 1.2 μmol) was dissolved in 0.3 mL of acetonitrile and added to the Ab-9 monoclonal antibody-propanethiol solution 5c (6.2 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After aseptic filtration through a 0.2 μm filter, the title product ADC-8 in PBS buffer (3.8 mg/mL, 4.6 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 150508.6 (M _Ab + 0D), 151903.6 (M _Ab + 1D), 153314.5 (M _Ab + 2D), 154747.8 (M _Ab + 3D), 156039.5 ( _{M Ab} + 4D). The average value of the toxin bound to each antibody molecule (DAR) was y = 1.6.

Example 21: Anti-c-Met Antibody Ab-10 Conjugated to Toxin SMCC-DM1

first step

SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (1.65 mg, 4.94 μmol, purchased from Shanghai Hanhong Chemical Technology Co., Ltd., batch number BH-4857-111203) was dissolved in 0.9 mL of acetonitrile solution and set aside. The pre-prepared acetonitrile solution of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate was added to Ab-10 monoclonal antibody (10.15 mg/ml, 9.0 mL, 0.62 μmol) in PBS buffer (pH 6.5) and reacted at 25°C for 2 hours with shaking. After completion of the reaction, the title product 9b was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M PBS solution, pH 6.5) to obtain a solution. The solution was concentrated to approximately 10 mg/ml (8.3 mg/ml, 11 ml) and directly used for the next reaction.

Step 2

To the 9b solution (11.0 mL) was added 3.0 mg of L-DM1 (prepared using the known method described in the Journal of Medicinal Chemistry, 2006, 49, 4392-4408) in ethanol (3.0 mg L-DM1/1.1 mL ethanol). After shaking at 25°C for approximately 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (eluting phase: 0.05 M PBS, pH 6.5) to obtain the title product ADC-9 solution (6.3 mg/mL, 14 mL) and stored frozen at 4°C. Q-TOF LC/MS analysis revealed characteristic peaks: 148119.6 (M _Ab + 0D), 149078.1 (M _Ab + 1D), 149836.4 (M _Ab + 2D), 150593.7 (M _Ab + 3D), and 151552.5 (M _Ab + 4D). Average value: y = 2.3.

Example 22: Anti-c-Met Antibody Ab-9 Conjugated to Toxin SMCC-DM1

first step

SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (1.65 mg, 4.94 μmol) was dissolved in 0.9 mL of acetonitrile solution and set aside. The pre-prepared acetonitrile solution of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate was added to Ab-9 monoclonal antibody (10.15 mg/ml, 9.0 mL, 0.62 μmol) in PBS buffer (pH 6.5), and the mixture was shaken at 25°C for 2 hours. After completion of the reaction, the solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution, pH 6.5) to obtain the title product 10b solution, which was concentrated to approximately 10 mg/ml (8.3 mg/ml, 11 ml) and directly used for the next reaction.

Step 2

To solution 9b (11.0 mL), 3.0 mg of L-DM1 (3.0 mg L-DM1/1.1 ml ethanol) in ethanol was added. After shaking at 25°C for approximately 4.0 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (eluting phase: 0.05 M PBS solution, pH 6.5) to obtain the title product ADC-10 solution (concentration 6.3 mg/ml, 14 mL) and stored frozen at 4°C.

Q-TOF LC/MS: characteristic peaks: 150534.2 (M _Ab +0D), 151492.6 (M _Ab +1D), 152451.7 (M _Ab +2D), 153409.7 (M _Ab +3D), 154368.1 (M _Ab +4D).

Average value: y = 2.2.

Example 23: Anti-c-Met Antibody Ab-9 Conjugated to Toxin-SN-38

Compound MC-VC-PAB-SN-38 (1.3 mg, 1.2 μmol) was dissolved in 0.3 mL of acetonitrile and added to Ab-9 monoclonal antibody-propanethiol solution 5c (6.2 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After aseptic filtration through a 0.2 μm filter, the title product ADC-11 in PBS buffer (3.7 mg/mL, 4.5 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: characteristic peaks: 150537.1 (M _Ab +0D), 151786.6 (M _Ab +1D), 152948.6 (M _Ab +2D), 154161.7 (M _Ab +3D), 155365.9 (M _Ab +4D), 156477.8 (M _Ab +5D).

Average value: y = 2.6.

Example 24: Anti-c-Met Antibody Ab-10 Conjugated to Toxin

1. Preparation of toxin

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid

first step

(S)-tert-Butyl 2-amino-3-(2-fluorophenyl)propanoate

The raw material ((S)-2-amino-3-(2-fluorophenyl)propionic acid 12a (400 mg, 2.18 mmol, was prepared by the well-known method "Advanced Synthesis & Catalysis, 2012, 354 (17), 3327-3332” prepared) was dissolved in 10 Ml tert-butyl acetate, perchloric acid (300 mg (70%), 3.3 mmol) was added, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, 6 Ml of water was added, the liquid was separated, and the organic phase was washed with saturated sodium bicarbonate solution (5 Ml). The aqueous phase was adjusted to Ph = 8 with saturated sodium bicarbonate solution, extracted with dichloromethane (5 Ml × 3), and the organic phases were combined, washed with water (3 Ml) and saturated sodium chloride solution (5 Ml) in sequence, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude title product (S)-tert-butyl 2-amino-3-(2-fluorophenyl)propionic acid 12b (390 mg, yellow oil). The product was directly used for the next reaction without purification.

Step 2

(1S,3S,5S)-tert-Butyl 3-((1R,2R)-3-(((S)-1-(tert-butoxy)-3-(2-fluorophenyl)-1-carbonylpropyl-2-yl)amino)-1-methoxy-2-methyl-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylate

The starting material (2R,3R)-3-((1S,3S,5S)-2-(tert-butoxycarbonyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanoic acid 12e (100 mg, 0.334 mmol) was dissolved in a 6 mL mixture of dichloromethane and dimethylformamide (V/V = 5:1). The crude reactant (S)-tert-butyl 2-amino-3-(2-fluorophenyl)propanoic acid 12b (80 mg, 0.334 mmol) was added. N,N-Diisopropylethylamine (0.29 mL, 1.67 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (152.3 mg, 0.40 mmol) were then added. The reaction system was stirred at room temperature under an argon atmosphere for 1 hour. After the reaction, 10 mL of water was added with stirring. The layers were separated and the dichloromethane layer was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System B to afford the title product, (1S,3S,5S)-tert-butyl 3-((1R,2R)-3-(((S)-1-(tert-butoxy)-3-(2-fluorophenyl)-1-carbonylpropyl-2-yl)amino)-1-methoxy-2-methyl-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid 12c (173 mg, colorless liquid) in a yield of 99.5%.

MS m/z(ESI):521.2[M+1]

Step 3

(S)-tert-Butyl 2-((2R,3R)-3-((1S,3S,5S)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propanoic acid

The starting material (1S,3S,5S)-tert-butyl 3-((1R,2R)-3-(((S)-1-(tert-butoxy)-3-(2-fluorophenyl)-1-carbonylpropyl-2-yl)amino)-1-methoxy-2-methyl-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid 12c (173 mg, 0.33 mmol) was dissolved in 2 mL of dioxane. A 5.6 M solution of hydrogen chloride in dioxane (0.21 mL, 1.16 mmol) was added. Under an argon atmosphere, the mixture was stirred at room temperature for 1 hour and then placed in a refrigerator at 0°C for 12 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, diluted with 5 mL of dichloromethane, and then 10 mL of saturated sodium bicarbonate solution was added, followed by stirring for 10 minutes. The layers were separated, and the aqueous layer was extracted with dichloromethane (5 mL x 3). The combined dichloromethane layers were washed with a saturated sodium chloride solution (10 mL), and dried over anhydrous sodium sulfate. The residue was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude title product (S)-tert-butyl 2-((2R,3R)-3-((1S,3S,5S)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propanoic acid 12d (77 mg, yellow liquid). The product was directly used in the next reaction without purification.

MS m/z(ESI):421.2[M+1]

Step 4

(S)-tert-Butyl 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxo-4,7,10-triazatetradec-14-yl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propanoic acid

The crude (S)-tert-butyl 2-((2R,3R)-3-((1S,3S,5S)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propanoic acid 12d (77 mg, 0.183 mmol), (5S,8S,11S,12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propanoic acid 12d (77 mg, 0.183 mmol), 4,7,10-Triazatetradecane-14-carboxylic acid 12i (116.8 mg, 0.183 mmol, prepared using the method disclosed in patent application WO2013072813) was dissolved in a 6 mL mixture of dichloromethane and dimethylformamide (V/V = 5:1). N,N-diisopropylethylamine (0.16 mL, 0.915 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (84 mg, 0.22 mmol) were added. The reaction system was stirred at room temperature under an argon atmosphere for 1 hour. After completion of the reaction, 10 mL of water was added with stirring, and the layers were separated. The dichloromethane layer was washed with saturated sodium chloride solution (10 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with eluent System B to give the title product (S)-tert-butyl 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxa-4,7,10-triazatetradec-14-yl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propanoic acid 12e (190.5 mg, yellow viscous material) in a 100% yield.

MS m/z(ESI):1040.6[M+1]

Step 5

(S)-tert-Butyl 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid

The starting material (S)-tert-butyl 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxa-4,7,10-triazatetradec-14-yl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propanoic acid 12e (190.5 mg, 0.183 mmol) was dissolved in 1.5 mL of dichloromethane, and 2 mL of diethylamine was added. The reaction system was stirred at room temperature under an argon atmosphere for 3 hours. After the reaction, the reaction solution was concentrated under reduced pressure to give the crude title product (S)-tert-butyl 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid 12f (150 mg, yellow viscous substance). The product was directly used for the next reaction without purification.

MS m/z(ESI):818.5[M+1]

Step 6

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid

The crude (S)-tert-butyl ester 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid 12f (150 mg, 0.183 mmol) was dissolved in 1 mL of dioxane. 3 mL of a 5.6 M solution of hydrogen chloride in dioxane was added, and the mixture was stirred at room temperature under an argon atmosphere for 12 hours. After the reaction, the reaction solution was concentrated under reduced pressure and the solvent was spun with diethyl ether. The resulting residue was purified by high performance liquid chromatography to give 12 g (28 mg, white powder solid) of the title product, (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid, in a yield of 20%.

MS m/z(ESI):762.7[M+1]

¹ H NMR (400 MHz, CD ₃ OD): δ7.38-7.18(m,2H),7.13-7.01(m,2H),4.80-4.67(m,2H),4.30-4.15(m, 1H),4.13-4.01(m,1H),3.96-3.83(m,2H),3.75-3.60(m,2H),3.42-3.11(m,9H ),3.06-2.95(m,1H),2.70-2.58(m,4H),2.28-2.01(m,4H),1.88-1.70(m,3H) ,1.57-1.25(m,4H),1.22-0.95(m,18H),0.92-0.80(m,4H),0.78-0.65(m,1H).

2. Preparation of toxin intermediates

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid

The raw material (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl) 12 g (25 mg, 0.033 mmol) of propionic acid was dissolved in 3 mL of dichloromethane, and N,N-diisopropylethylamine (0.029 mL, 0.164 mmol) was added. The reaction system was placed under an argon atmosphere and an ice bath with a pre-prepared dichloromethane solution of 6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl chloride 4b (11.3 mg, 0.049 mmol) added dropwise, and the reaction was carried out at room temperature for 3 hours. After the reaction, 5 mL of water was added, stirred for 20 minutes, and the liquid was separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography to give the title product (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propanoic acid 12h (7 mg, yellow viscous material) in a yield of 22.4%.

MS m/z(ESI):955.4[M+1]

¹ H NMR (400MHz, CD ₃ OD): δ7.36-7.30(m,1H),7.29-7.21(m,1H),7.17-7.02(m,2H),6.83-6.79(m,2H),4.81-4.71(m,2H),4.69-4.55(m, 2H),4.25-4.15(m,1H),4.13-4.04(m,1H),3.96-3.85(m,2H),3.70-3.61(m,1H),3.55-3.46(m,3H),3.40-3.21(m,4H) ),3.18-3.10(m,2H),3.07-2.96(m,4H),2.67-2.56(m,2H),2.54-2.34(m,3H),2.29-2.17(m,2H),2.10-1.99(m,1H) ,1.89-1.57(m,7H),1.52-1.28(m,6H),1.21-1.11(m,4H),1.07-0.96(m,6H),0.95-0.81(m,12H),0.80-0.69(m,1H).

3. Preparation of Antibody-Toxin Conjugates

Compound 12h (1.2 mg, 1.2 μmol) was dissolved in 0.3 mL of acetonitrile and added to Ab-10 monoclonal antibody-propanethiol 1c solution (6.17 mg/mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After filtration through a 0.2 μm filter under sterile conditions, the title product ADC-12 in PBS buffer (3.3 mg/mL, 5.0 mL) was obtained and stored frozen at 4°C.

Q-TOF LC/MS: characteristic peaks: 148119.6 (M _Ab +0D), 149150.5 (M _Ab +1D), 150221.1 (M _Ab +2D), 151265.1 (M _Ab +3D), 152314.3 (M _Ab +4D).

Average value: y=1.6.

Referring to Examples 13-24, the ADC compounds of Examples 25-27 were prepared.

Anti-c-Met Antibody-Toxin Conjugate (ADC) Molecule Test Example

Test Example 1: Stability Evaluation of Anti-c-Met Antibody-Toxin Conjugate (ADC) Molecules

The toxin intermediates and toxins of the ADC molecules of the present invention or other c-Met antibody-toxin conjugates with endocytosis (e.g., LY-2875358-ADC) were evaluated for the stability of free toxins in PBS, human and monkey plasma.

The toxin intermediates and toxins of Example compounds ADC-1 and ADC-12 were diluted to 500 μg/mL with PBS or human or monkey plasma (Suzhou Xishan Zhongke Pharmaceutical Research and Development Co., Ltd., Animal Production License No.: SCXK(Su)2012-0009) and incubated at 37°C for 7 days. Samples were taken on days 0, 3, and 7 to determine the concentrations of free toxins and toxin intermediates. To 50 μL of drug-containing human or monkey plasma or PBS sample, 20 μL of internal control (camptothecin, Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., batch number 090107, 100 ng/mL) was added, followed by 150 μL of acetonitrile, vortex mixing for 3 minutes, and centrifugation at 15,000 rpm for 10 minutes. 80 μL of the supernatant was mixed with 80 μL of 0.2% formic acid, and 10 μL was injected. The standard curve analysis method is as follows: 50 μL of blank human, monkey plasma or PBS sample is added to 50 μL of the series working solution, 20 μL of the internal control (camptothecin, 100 ng/ml), 100 μL of acetonitrile, vortex mixing for 3 minutes, centrifugation at 15000 rpm for 10 minutes, 80 μL of the supernatant is mixed with 80 μL of 0.2% formic acid, and 10 μL is injected.

A Shimadzu LC-30AD ultra-high performance liquid chromatography system (Shimadzu Corporation, Japan) was used, and the UPLC-MS/MS mass spectrometer was an API4000 triple quadrupole tandem mass spectrometer (AB SCIEX, USA). Chromatographic conditions were set (chromatographic column: Waters XBridge ^™ BEH300 C18 (100 mm × 4.6 mm id, 3.5 mm), and the mobile phase was 0.2% formic acid-acetonitrile (gradient elution). The results are shown in Tables 11 and 12.

Table 11. Plasma stability evaluation of toxin intermediates and toxin drugs of ADC-1 of the present invention

*: Detection value is the percentage of free toxin in the sample. 0.01-0.19% is within the background range. ND: Not detectable.

Table 12. Plasma stability evaluation of toxin intermediates and toxin drugs of ADC-12 of the present invention

ND: Not detectable.

These results demonstrate that the toxin intermediates and toxins of ADC-1 and ADC-12 of the present invention are stable in various solvents (PBS, human plasma, monkey plasma, etc.). No degradation products, free toxins and toxin intermediates (toxin-linkers), were detected after incubation at 37°C for 0, 3, and 7 days.

Test Example 2: In vitro activity evaluation of anti-c-Met antibody-toxin conjugate (ADC) molecules

The in vitro activities of ADC-1 and ADC-12 of the present invention were evaluated by FACS (to detect binding to c-Met-positive cells) and endocytosis (methods are described in Example 11). The results are shown in Table 13.

Table 13. In vitro activity of ADC molecules of the present invention

Humanized antibodies MKN45/FCAS binding activity (nM) Endocytosis (%)* Ab-10 1.01 32.7 ADC-1 1.22 32.9 ADC-12 0.48 31.8

*: The data is the throughput ratio within 1 hour

The above results indicate that after the antibody of the present invention is coupled to the toxin, the binding activity and endocytosis activity of the antibody are retained.

Test Example 3: Anti-c-Met Antibody-Toxin Conjugate (ADC) Molecular Cytotoxicity Experiment

To evaluate the cytotoxicity of the ADC molecules of the present invention, a cell ATP toxicity assay was performed. ATP is an indicator of the metabolism of living cells, and measuring ATP can reflect the toxicity of the molecules to cells.

HepG2 cells (Cell Bank of the Chinese Academy of Sciences, Cat#TCHu 72) were cultured in EMEM complete medium supplemented with 10% FBS, and MKN45 cells were cultured in RPMI1640 complete medium supplemented with 10% FBS. During the experiment, 2-3 ml of trypsin was added for digestion for 2-3 minutes. After complete digestion, 10-15 ml of complete medium was added to elute the digested cells. The cells were centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. Subsequently, 10-20 ml of complete medium was added to resuspend the cells to prepare a single-cell suspension. The cell density was adjusted to 4×10 ⁴ cells/ml. 0.1 ml of the above cell suspension was added to each well of a 96-well cell culture plate and incubated in a 37°C, 5% _CO₂ incubator. After 24 hours, the medium was removed and 90 μl of EMEM medium containing 2% FBS or RPMI1640 medium containing 2% FBS was added to each well. The test samples (compounds of Example 13 and toxins) were diluted with PBS to various concentrations, and 10 μl was added to each well. The cells were incubated in a 37°C, 5% _CO₂ incubator for 72 hours. The assay was performed using the CellTiter-Luminescent Cell Viability Assay Kit (Promega, Cat# G7571) according to the manufacturer's instructions. Chemiluminescence was detected using a microplate reader (VICTOR 3, PerkinElmer), and data were analyzed using GraphPad Prism (version 5.0) software. The results are shown in Table 14.

Table 14. Cytotoxic effects of ADC molecules of the present invention and corresponding free cytotoxins

Test samples ADC-1 0.51 ND MMAF 0.85 4.88 ADC-12 0.59 ND 12g 79.4 400.8

ND: no activity detected; NA: not applicable

Discussion: These results demonstrate that ADC-1 and ADC-12 exhibit similar cytotoxicity against c-Met-positive MKN45 cells (IC ₅₀ values of 0.51 nM and 0.59 nM, respectively). However, the cytotoxicity of each toxin component on c-Met-positive MKN45 cells differed by a 93-fold difference (79.4/0.85).

Neither ADC-1 nor ADC-12 of the present invention had any cytotoxic effect on c-Met-negative HepG2 cells, demonstrating that the ADC compounds possess specific targeting effects. However, the cytotoxic effects of their respective toxin moieties on c-Met-negative HepG2 cells differed by 82 times (400.8/4.88).

These results demonstrate that ADC-1 and ADC-12 of the present invention have specific targeting effects, inhibiting the proliferation of c-Met-positive cells while lacking toxic effects on nonspecific (normal) cells. ADC-1 and ADC-12 differ in the toxicity of their respective free toxins toward targeted and non-targeted cells. The cytotoxicity of the toxin portion of ADC-12 toward c-Met-positive and negative HepG2 cells is 93 and 82 times weaker than that of the toxin portion of ADC-1. Therefore, if any free toxin is released during the molecule's journey to target cells, its nonspecific toxic effects are less pronounced than those of ADC-1. Consequently, toxic side effects are minimized and safety is improved.

Test Example 4: Inhibitory Effect of Anti-c-Met Antibody-Toxin Conjugate (ADC) Molecules on Tumor Cell Proliferation

The above results demonstrate that ADC-1 (Example 13) can specifically kill c-Met-expressing tumor target cells. To examine the inhibitory effect of this toxic effect on tumor cell proliferation, the molecules of the present invention were used to test various tumor cells. The inhibitory effects of the samples on cell proliferation were tested using the CCK assay, and the in vitro cellular activity of the ADC molecules of the present invention was evaluated based on the _IC50 value.

The cells used and the corresponding culture medium are shown in Table 15 below. Cell proliferation was detected using Cell Counting Kit (Dongren Chemical Technology Co., Ltd., Cat#CK04) (operated according to the instructions).

During the experiment, 2-3 ml of trypsin was added for 2-3 minutes. Once the cells were completely digested, 10-15 ml of complete culture medium was added to elute the digested cells. The cells were centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. Then, 10-20 ml of culture medium was added to resuspend the cells to prepare a single-cell suspension, and the cell density was adjusted to 4 × 10 ⁴ cells/ml. 0.1 ml of the above cell suspension was added to each well of a 96-well cell culture plate and incubated in a 37°C, 5% CO ₂ incubator. After 24 hours, the culture medium was removed, and 90 μl of culture medium containing 2% FBS was added to each well. The sample to be tested was diluted with PBS to various concentrations, and 10 μl was added to each well. The cells were incubated in a 37°C, 5% CO ₂ incubator for 72 hours. 10 μl of CCK8 was added to each well, and the cells were incubated in the incubator for another 2 hours. The OD450 value was measured using a microplate reader (VICTOR 3, PerkinElmer), and data were analyzed using GraphPad Prism (version 5.0) software. The results are shown in Table 16.

Table 15. Culture medium for cells used in this example

cell lines culture medium Manufacturer's Part Number MKN45 RPMI1640 + 10% FBS JCRB, JCRB0254 SNU5 IMDM + 10% FBS BxPC3 RPMI1640 + 10% FBS Chinese Academy of Sciences Cell Bank (Cat#TCHu 12) Caki-1 McCOY's 5A+10% FBS Chinese Academy of Sciences Cell Bank, Cat#TCHu135 NCI-H1993 RPMI1640 + 10% FBS PC9 DMEM + 10% FBS Shanghai Baili Biotechnology Co., Ltd. NCI-H596 DMEM + 10% FBS Shanghai Baili Biotechnology Co., Ltd.

Table 16. Inhibitory effects of the molecules of the present invention on proliferation of different cancer cells

The results in Table 16 demonstrate that the anti-c-Met antibodies of the present invention exhibit relatively good activity against gastric cancer cell lines MKN45 and SUN, but very weak or no activity against other tumor cells with low or no c-Met expression, such as lung cancer cells. ADC-1 of the present invention, however, possesses an additional toxin and exhibits excellent activity against c-Met-expressing tumor cells, including the gastric cancer cell lines MKN45 and SUN, and particularly against lung, pancreatic, and renal cell carcinoma cells, where anti-c-Met antibodies have no or very weak effects.

Test Example 5: In vivo efficacy evaluation of anti-c-Met antibody-toxin conjugate (ADC) molecules

1. Test Purpose

To better evaluate the anti-tumor efficacy of the anti-c-Met antibodies and ADC molecules of the present invention, a parallel comparative experiment was conducted using the method of Example 10 for Ab-10 and ADC-1. Unlike Example 10, this test case used a single administration, and the experiment was stopped after a tumor suppression effect was observed and a trend toward recovery was observed.

2. Antibodies to be tested

Ab-10 (5 mg/kg), stock solution (2.18 mg/ml) was made up to a final concentration of 0.5 mg/ml in PBS;

Ab-10 (10 mg/kg), stock solution (2.18 mg/ml) was made up to a final concentration of 1 mg/ml in PBS;

Ab-10 (30 mg/kg), stock solution (2.18 mg/ml) was made up to a final concentration of 3 mg/ml in PBS;

ADC-1 (2.5 mg/kg), the stock solution (10 mg/ml) was made up to a final concentration of 0.25 mg/ml with PBS;

ADC-1 (5 mg/kg), stock solution (10 mg/ml) was prepared with PBS to a final concentration of 0.5 mg/ml;

ADC-1 (10 mg/kg), the stock solution (10 mg/ml) was made up to a final concentration of 1 mg/ml with PBS;

All animals were administered with tail vein injection, with a volume of 0.2 ml per animal.

3. Test methods

MKN-45 cells (1×10 ⁶ /mouse) were subcutaneously inoculated in the right flank of nude mice. The tumors grew to an average volume of (150.19+8.44) mm ³ . The mice were randomly divided into groups for drug administration, with 8 mice in each group. The specific drug administration schedule is shown in Table 17.

The tumor volume and body weight were measured twice a week and the data were recorded.

Excel statistical software was used: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV/SQRT; and P value of inter-group difference was calculated as TTEST.

The formula for calculating tumor volume (V) is: V = 1/2 × L _length × L _short ²

Tumor inhibition rate = (V ₀ -V _T )/V _{0 *} 100%

V ₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively.

4. Test results

Table 17. Efficacy of the administered compounds on MKN-45 nude mouse transplanted tumors

**p<0.01*p<0.05

Conclusion: The antibodies and ADC compounds of the present invention have significant therapeutic effects on MKN-45 nude mouse transplanted tumors.

To evaluate the in vivo efficacy of ADC-12 of the present invention, ADC-1 and ADC-12 were compared in parallel using the same test method as above. The results are shown in Table 18 after a single administration of the same dose of 3 mg/kg.

Table 18. Inhibitory effects of ADC-1 and ADC-12 on tumors

Inhibition rate (%) 11 days 15 days 18 days 21 days ADC-1 42.8 44.7 35.4 27.1 ADC-12 44.6 54.5 50.5 50.4

The above results show that ADC-1 and ADC-12 had similar tumor inhibition rates on day 11, but after day 15, the efficacy of ADC-1 weakened (27.1% on day 21), while the tumor inhibition effect of ADC-12 remained at the level of day 11 (50.4%).

Test Example 6: Efficacy of ADC-12 on subcutaneous transplanted tumors of human lung cancer NCI-H1993 in nude mice

1. Purpose of the experiment

To evaluate and compare the efficacy of ADC-12 and Ab-10 antibody solutions on subcutaneous xenografts of human lung cancer NCI-H1993 in nude mice.

2. Drug preparation

ADC-12 was dissolved in water for injection into a 20 mg/ml solution, aliquoted and stored in a -80°C refrigerator. It was diluted to the corresponding concentration with 0.1% BSA saline before use. The Ab-10 antibody stock solution had a concentration of 16.3 mg/ml. It was diluted with 0.1% BSA saline and aliquoted and stored in a -80°C refrigerator.

3. Experimental Animals

BALB/cA-nude mice, 6-7 weeks old, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Production license number: SCXK(Shanghai)2013-0018; Animal qualification certificate number: 2013001814303. Housing environment: SPF grade.

4. Experimental Procedure

Nude mice were subcutaneously inoculated with human lung cancer NCI-H1993 cells. After tumors grew to 100-150 ^mm³ , the animals were randomly divided into groups (D0). The dosage and dosing schedule are shown in Table 19. Tumor volume was measured 2-3 times per week, and the mice were weighed and the data were recorded. Tumor volume (V) was calculated using the formula:

V = 1/2 × a × b ² where a and b represent length and width respectively.

T/C (%) = (TT ₀ ) / (CC ₀ ) × 100, wherein T and C are the tumor volumes at the end of the experiment; T ₀ and C ₀ are the tumor volumes at the beginning of the experiment.

5. Results

ADC-12 is an anti-c-Met antibody-toxin conjugate. ADC-12 (1, 3, and 10 mg/kg, IV, D0) dose-dependently inhibited the growth of subcutaneous xenografts of NCI-H1993 nude mice harboring a high-expression c-Met human lung cancer, with tumor inhibition rates of 45%, 63%, and 124%, respectively. Seven out of ten tumors in the 10 mg/kg group experienced partial regression (D21). Ab-10, the naked antibody used to prepare ADC-12, exhibited a 42% tumor inhibition rate against NCI-H1993 tumors (30 mg/kg, IV, twice weekly for a total of six doses). All tumor-bearing mice tolerated the drugs well, with no symptoms such as weight loss. ADC-12 demonstrated significantly greater efficacy against NCI-H1993 than Ab-10.

Table 19. Efficacy of ADC-12 and Ab-10 antibody stock solutions on subcutaneous transplanted tumors of human lung cancer NCI-H1993 in nude mice.

D0: time of first administration; P values are compared with the vehicle; **P < 0.01, compared with the Ab-10 antibody 30 mg/kg solution group; Student's t test was used for all tests. Number of mice at the start of the experiment: n = 10.

Discussion: ADC-12 (1, 3, and 10 mg/kg, IV, D0) dose-dependently inhibited the growth of subcutaneous xenografts in nude mice bearing NCI-H1993, a human lung cancer that overexpresses c-Met, and caused partial tumor regression. Ab-10 antibody stock solution (30 mg/kg, IV, twice weekly for a total of six doses) was also effective against NCI-H1993. ADC-12 demonstrated significantly greater efficacy than Ab-10 antibody stock solution against NCI-H1993. All drugs were well tolerated by tumor-bearing mice.

Claims (56)

1. An antibody or its antigen-binding fragment that specifically binds to the c-Met receptor, comprising the following CDR region sequence: i) Antibody heavy chain variable region HCDR region sequence: SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; and antibody light chain variable region LCDR region sequence: SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or ii) Antibody heavy chain variable region HCDR region sequence: SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; and antibody light chain variable region LCDR region sequence: SEQ ID NO:12, SEQ ID NO:10 and SEQ ID NO:11. 2. The antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor is a murine antibody or a fragment thereof. 3. The antibody that specifically binds to the c-Met receptor or its antigen-binding fragment according to claim 2, wherein the variable region sequence of the mouse antibody heavy chain is: SEQ ID NO:4. 4. The antibody that specifically binds to the c-Met receptor or its antigen-binding fragment according to claim 2, wherein the variable region sequence of the murine antibody light chain is: SEQ ID NO:5. 5. The antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to any one of claims 2-4, wherein the heavy chain variable region of the murine antibody is: SEQ ID NO:4, and the light chain variable region is: SEQ ID NO:5. 6. The antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to claim 1, wherein it is a chimeric antibody or a humanized antibody or a fragment thereof. 7. The antibody that specifically binds to the c-Met receptor according to claim 6, or its antigen-binding fragment, wherein the heavy chain FR region sequence on the variable region of the humanized antibody heavy chain is derived from a human heavy chain sequence. 8. The antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to claim 7, wherein the human heavy chain is IGHV 3-33*01; wherein the FR region sequence of the heavy chain comprises the framework sequence or mutant sequence of the FR1, FR2, FR3 and FR4 regions of the human heavy chain IGHV 3-33*01. 9. The antibody that specifically binds to the c-Met receptor according to claim 8, or its antigen-binding fragment, wherein the mutated sequence of the human heavy chain IGHV 3-33*01 framework sequence comprises a reversion mutation of 0-10 amino acids. 10. The antibody that specifically binds to the c-Met receptor according to claim 7, or the antigen-binding fragment thereof, wherein the humanized antibody comprises a heavy chain variable region sequence selected from those shown in SEQ ID NO:13-15. 11. The antibody that specifically binds to the c-Met receptor according to claim 6, or its antigen-binding fragment, wherein the light chain FR region sequence on the variable region of the humanized antibody light chain is selected from human germline light chain sequences. 12. The antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to claim 11, wherein the human light chain is IGKV085 or IGKV 4-1*01, and wherein the FR region sequence of the light chain comprises the framework sequence or mutant sequence of the FR1, FR2, FR3 and FR4 regions of the human light chains IGKV085 and IGKV 4-1*01. 13. The antibody that specifically binds to the c-Met receptor according to claim 12, or its antigen-binding fragment, wherein the mutated sequence of the human light chain IGKV085 or IGKV 4-1*01 framework sequence comprises a reversion mutation of 0-10 amino acids. 14. The antibody that specifically binds to the c-Met receptor according to claim 11, or its antigen-binding fragment, wherein the humanized antibody comprises a light chain variable region sequence selected from those shown in SEQ ID NO:16-18. 15. The antibody that specifically binds to the c-Met receptor or its antigen-binding fragment according to any one of claims 6-14, wherein the humanized antibody comprises a heavy chain variable region sequence selected from SEQ ID NO:13-15 and a light chain variable region sequence selected from SEQ ID NO:16-18. 16. An antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to any one of claims 1 and 6-14, comprising a combination of a heavy chain variable region sequence and a light chain variable region sequence selected from any one of a) to c): a) The heavy chain variable region sequence of SEQ ID NO:13 and the light chain variable region sequence of SEQ ID NO:16; b) The heavy chain variable region sequence of SEQ ID NO:14 and the light chain variable region sequence of SEQ ID NO:17; or c) The heavy chain variable region sequence of SEQ ID NO:15 and the light chain variable region sequence of SEQ ID NO:18. 17. An antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to any one of claims 6-16, comprising a constant region of the antibody heavy chain, said constant region comprising a constant region derived from human IgG1 or a variant thereof, human IgG2 or a variant thereof, human IgG3 or a variant thereof, or human IgG4 or a variant thereof. 18. The antibody that specifically binds to the c-Met receptor according to claim 17, or its antigen-binding fragment, comprising a full-length heavy chain sequence selected from SEQ ID NO:23-25. 19. An antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to any one of claims 6-16, comprising a constant region of the antibody light chain, said constant region comprising a constant region selected from human κ or λ chains or variants thereof. 20. The antibody that specifically binds to the c-Met receptor according to claim 19, or the antigen-binding fragment thereof, comprising a full-length light chain sequence selected from SEQ ID NO:26-28. 21. The antibody that specifically binds to the c-Met receptor according to any one of claims 6-16, or the antigen-binding fragment thereof, wherein the humanized antibody comprises a combination selected from the following full-length light chain sequences and full-length heavy chain sequences: Ab-9: the heavy chain sequence of SEQ ID NO:23 and the light chain sequence of SEQ ID NO:26; Ab-10: the heavy chain sequence of SEQ ID NO:24 and the light chain sequence of SEQ ID NO:27; or Ab-11: the heavy chain sequence of SEQ ID NO:25 and the light chain sequence of SEQ ID NO:28. 22. A DNA molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor according to any one of claims 1-21. 23. An expression vector containing the DNA molecule according to claim 22. 24. A host cell transformed with the expression vector according to claim 23. 25. The host cell transformed by the expression vector according to claim 24, wherein the host cell is a mammalian cell. 26. The host cell transformed by the expression vector according to claim 25, wherein the host cell is a CHO cell. 27. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor as described in any one of claims 1-21, and one or more pharmaceutically acceptable excipients, diluents, or carriers. 28. An antibody-cytotoxic drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvent compound thereof: Ab-[(L ₂ )tL ₁ -D)]y (I) in: D represents the drug module; _L1 and _L2 are connector units; t is 0 or 1; y is 1-8; Ab is an antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor as described in any one of claims 1-17. 29. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein y is 2-5. 30. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein _-L2- is a compound of the following formula ( _-L2- ): in X ₁ is selected from hydrogen atom, halogen, hydroxyl, cyano, alkyl, alkoxy and cycloalkyl; X ₂ is selected from -alkyl-, -cycloalkyl-, and -heterocyclic-; m is 0-5; S is a sulfur atom. 31. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 30, or a pharmaceutically acceptable salt or solvent compound thereof, wherein m is 1-3. 32. The antibody-cytotoxic drug conjugate of formula (I) according to claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein the drug module D is a cytotoxic agent selected from toxins, chemotherapeutic agents, antibiotics, radioisotopes and ribolysins. 33. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein D is a compound of formula (D): Or in the form of its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: ^R1 - ^R7 are selected from hydrogen atoms, halogens, hydroxyl groups, cyano groups, alkyl groups, alkoxy groups, and cycloalkyl groups; ^R8 - ^R11 are selected from hydrogen atoms, halogens, alkenyl groups, alkyl groups, alkoxy groups, and cycloalkyl groups; ^R12 - ^R13 are selected from hydrogen atoms, alkyl groups, or halogens; R ¹⁴ is selected from aryl or heteroaryl, wherein the aryl or heteroaryl may optionally be further substituted with a substituent selected from hydrogen atom, halogen, hydroxyl, alkyl, alkoxy and cycloalkyl; R ¹⁵ can be selected from halogen, alkenyl, alkyl, cycloalkyl and COO; R ¹⁷ ; R ¹⁶ is selected from hydrogen atom, halogen, hydroxyl, cyano, alkyl, alkoxy and cycloalkyl; R ¹⁷ is selected from hydrogen atoms, alkyl groups, and alkoxy groups. 34. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 33, or a pharmaceutically acceptable salt or solvent compound thereof, wherein at least one of ^R8 - ^R11 is selected from halogen, alkenyl, alkyl and cycloalkyl, and the remainder are hydrogen atoms; Alternatively, any two of ^R8 - ^R11 can form a cycloalkyl group, and the remaining two groups can be selected from hydrogen atoms, alkyl groups, and cycloalkyl groups. 35. The antibody-cytotoxic drug conjugate of formula (I) according to claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein _L1 comprises a linker selected from Val-Cit, MC, PAB and MC-PAB. 36. The antibody-cytotoxic drug conjugate of formula (I) according to claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein D is maytansine alkaloid. 37. The antibody-cytotoxic drug conjugate of formula (I) according to claim 36, or a pharmaceutically acceptable salt or solvent compound thereof, wherein D is selected from DM1, DM3 and DM4. 38. The antibody-cytotoxic drug conjugate of general formula (I) according to claim 36 or 37, or a pharmaceutically acceptable salt or solvent compound thereof, wherein _L1 is selected from N-succinimino 4-(2-pyridylthio)valerate (SPP), N-succinimino 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate (SMCC), and N-succinimino (4-iodo-acetyl)aminobenzoate (SIAB). 39. The antibody-cytotoxic drug conjugate of formula (I) according to claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein D is a camptothecin alkaloid. 40. The antibody-cytotoxic drug conjugate of formula (I) according to claim 39, or a pharmaceutically acceptable salt or solvent compound thereof, wherein D is selected from CPT, 10-hydroxy-CPT, CPT-11 (irinotecan), SN-38, and topotecan. 41. The antibody-cytotoxic drug conjugate of formula (I) according to claim 39 or 40, or a pharmaceutically acceptable salt or solvent compound thereof, wherein the linker _L1 comprises a structure optionally selected from Val-Cit, MC, PAB, MC-PAB, and MC-vc-PAB. 42. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein the conjugate of formula (II) or a pharmaceutically acceptable salt or solvent compound thereof is: in: R ² - R ¹⁶ as defined in claim 33; Ab, t, y, _L1 , _L2 as defined in claim 28. 43. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein the conjugate drug of formula (III) or a pharmaceutically acceptable salt or solvent compound thereof is: in: R ² - R ¹⁶ as defined in claim 33; Ab,y is as defined in claim 28; n is 3-6. 44. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 43, or a pharmaceutically acceptable salt or solvent compound thereof, wherein n is 5. 45. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein the conjugate is of formula (IV) or a pharmaceutically acceptable salt or solvent compound thereof: in: R ² - R ¹⁶ as defined in claim 33; Ab,y is as defined in claim 28; n is as defined in claim 43; ^X1 , ^X2 , m are as defined in claim 30. 46. The antibody-cytotoxic drug conjugate of formula (I) as described in claim 28, or a pharmaceutically acceptable salt or solvent compound thereof, wherein the conjugate is of formula (V) or a pharmaceutically acceptable salt or solvent compound thereof: in: Ab, D, y are as defined in claim 28; n is as defined in claim 43; ^X1 , ^X2 , m are as defined in claim 30. 47. The antibody-cytotoxic drug conjugate of general formula (I) according to any one of claims 28-46, wherein the antibody-cytotoxic drug conjugate or its pharmaceutically acceptable salt or solvent compound is selected from: Ab-9, Ab-10, Ab-11 as defined in claim 21, wherein the range of y is 1-8. 48. The antibody-cytotoxic drug conjugate of formula (I) according to claim 47, or a pharmaceutically acceptable salt or solvent compound thereof, wherein y is 2-5. 49. A method for preparing a conjugated drug of formula (V) according to claim 46 or a pharmaceutically acceptable salt or solvent compound thereof, the method comprising: A compound of general formula (Ab-L2) reacts with a compound of general formula (L1-D) in an organic solvent to give a compound of general formula (V); wherein the organic solvent is; in: Ab is an antibody or antigen-binding fragment thereof that specifically binds to the c-Met receptor as described in any one of claims 1-21; X ₁ is selected from hydrogen atom, halogen, hydroxyl, cyano, alkyl, alkoxy and cycloalkyl; X ₂ is selected from alkyl, cycloalkyl, and heterocyclic groups; X is 0-5; m is 0-5. 50. A method for preparing a conjugated drug of general formula (V) according to claim 49 or a pharmaceutically acceptable salt or solvent compound thereof, wherein X is 1-3 and/or wherein m is 1-3. 51. A method for preparing a conjugated drug of formula (V) according to claim 49 or 50, or a pharmaceutically acceptable salt or solvent compound thereof, wherein the organic solvent is acetonitrile or ethanol. 52. A pharmaceutical composition comprising an antibody-cytotoxic drug conjugate of formula (I) according to any one of claims 28-48 or a pharmaceutically acceptable salt or solvent compound thereof and a pharmaceutically acceptable excipient, diluent or carrier. 53. Use in the preparation of a medicament for treating c-Met-mediated diseases or conditions, of an antibody that specifically binds to the c-Met receptor according to any one of claims 1-21 or an antigen-binding fragment thereof, or a pharmaceutical composition according to claim 27, or an antibody-cytotoxic drug conjugate of general formula (I) according to any one of claims 28-48 or a pharmaceutically acceptable salt or solvent compound thereof, or a pharmaceutical composition according to claim 52. 54. The use according to claim 53, wherein the disease or condition is cancer. 55. The use according to claim 53, wherein the disease or condition is cancer expressing c-Met. 56. The use according to claim 53, wherein the disease or condition is gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, kidney cancer, melanoma, or non-small cell lung cancer.