Anti-c-MET antibodies and antibody-drug conjugates

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to mesenchymal-epithelial transition factor (c-Met). The present invention also relates to antibody-drug conjugates (ADCs) comprising these anti-c-Met antibodies or antigen-binding fragments, pharmaceutical compositions comprising the antibodies, antigen-binding fragments or ADCs, and their use in the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to mesenchymal epithelial conversion factor (c-Met). The present invention also relates to antibody-drug conjugates (ADCs) containing these anti-c-Met antibodies or antigen-binding fragments, pharmaceutical compositions containing the antibody, antigen-binding fragment, or ADC, and their use in the treatment of cancer. [Background technology]

[0002] Hepatocyte growth factor receptor, or mesenchymal epithelial transition factor (HGFR, c-Met), is a receptor tyrosine kinase encoded by the MET oncogene and expressed on the surface of various epithelial cells. The ligand for c-Met is hepatocyte growth factor (HGF), also known as a scattering factor (SF), a high molecular weight polypeptide known for its angiogenic and mitogenic properties.

[0003] The extracellular domain of mature human c-Met consists of three domains: 1) a semaphorin (SEMA) domain formed by the folding of the N-terminal 500 residues, containing the entire α-subunit and part of the β-subunit; 2) a PSI domain of approximately 50 residues containing four disulfide bonds (found in plexins, semaphorins, and integrins); and 3) four immunoglobulin-plexin transcription (IPT) domains linking the PSI domain to a transmembrane helix. Intracellularly, c-Met contains a characteristic near-membrane sequence and a tyrosine kinase catalytic domain adjacent to the carboxyl-terminal sequence. Within the folded structure of the c-Met protein, the SEMA domain forms a seven-bladed β-propeller.

[0004] c-Met is expressed in a variety of normal tissues (Human Protein Atlas, www.proteinatlas.org and our research findings). It is most prominently observed in gastrointestinal tissue (stomach, gallbladder, duodenum, small intestine, colon, rectum), female reproductive tissue (endometrium, cervix, vagina, placenta), and urogenital tissue (bladder, ureters, kidneys), and is also observed to some extent in the thyroid gland, skin, lungs, liver, breast, and esophagus. Furthermore, significant c-Met expression is present not only in the eyelids (lacrimal glands, meibomian glands, sebaceous glands, hair follicle sheath) but also in the eye (cornea and lens epithelium, limbus, and conjunctiva).

[0005] The binding of c-Met to HGF results in receptor dimerization, heteromerization, or multimerization, phosphorylation of multiple tyrosine residues in the intracellular domain, and activation of a wide range of distinct cellular signaling pathways, including those involved in proliferation, motility, migration, and invasion. While c-Met is important in regulating tissue homeostasis under normal physiological conditions, it is also clearly involved in the development and progression of malignancies due to mutations (in exon 14), gene amplification, or protein overexpression. Mechanisms associated with c-Met also appear to be involved in resistance to (chemo)therapy, such as therapies targeting other regulators of cell proliferation, including epidermal growth factor receptor (EGFR), transforming growth factor-β (TGFβ), and human epidermal growth factor receptor 3 (HER3).

[0006] The human c-Met signaling pathway is one of the most frequently dysregulated pathways in human cancers. It is involved in many types of solid tumors, and high c-Met expression is generally associated with a poor prognosis. For this reason, the c-Met and HGF / SF signaling pathways are targets for cancer therapy.

[0007] While a wide variety of c-Met inhibitors, ranging from small molecules to antibodies, are currently in clinical development, positive responses have only been observed in patients with c-Met signaling-dependent tumors, such as those with MET amplification or exon 14 mutations. In some cases, treatment has even worsened the patient's condition, resulting in disappointing clinical outcomes.

[0008] Approved small molecule c-Met inhibitors include tyrosine kinase inhibitors (TKIs), crizotinib (Xalkori®, Pfizer) and cabozantinib (Cometrix®, Ipsen Pharma; Cabometyx®, Ipsen Pharma). The former is used to treat anaplastic lymphoma kinase (ALK)-positive or ROS1 tyrosine kinase-positive non-small cell lung cancer (NSCLC), while the latter targets c-Met and VEGFR2 in particular and is used to treat medullary thyroid carcinoma and renal cell carcinoma (RCC).

[0009] In cancer treatment, any agonistic effect on c-Met should be avoided. Therefore, suitable therapeutic antibodies interact with c-Met in a way that avoids dimerization and activation (agonistic effect), instead inducing internalization and degradation. While agonist antibodies used in regenerative medicine can be systematically prepared (e.g., International Publication No. 2018 / 001909), these antibodies are often undesirable byproducts of searching for suitable therapeutic antibodies for treating cancers that have an antagonistic (i.e., inhibitory) effect on c-Met signaling.

[0010] Therefore, designing anti-c-Met antibodies for cancer treatment requires a delicate balance between desirable binding properties, inhibition of c-Met signaling, and acceptable pharmacokinetic and pharmacodynamic properties.

[0011] The search for antibodies that exhibit antagonistic effects but not agonistic effects is a complex task. Some antibodies may even reverse from antagonist to agonist as a result of humanization, for example, of mouse monoclonal antibodies.

[0012] Several c-Met-targeting antibodies for cancer treatment have been developed and clinical trials have begun. Such c-Met-specific antibodies include conventional anti-c-Met antibodies, as well as bispecific antibodies that target both c-Met and other signaling proteins such as EGFR.

[0013] Antagonistic antibodies against c-Met that have progressed to clinical development include, for example, onartuzumab (Genentech, International Publication No. 2006 / 015371), ARGX-111 (Argenx, International Publication No. 2012 / 059561), emibetuzumab (LY2875358; Eli Lilly, International Publication No. 2010 / 059654), SAIT-301 (Samsung, US Patent Application Publication No. 2014-0154251), terisotuzumab (antibody ABT-700; Abbott / Abbvie, Wang et al., BMC Cancer 2016, 16, 105-118; International Publication No. 2017 / 201204), and Sym015 (c-Met A mixture of two anti-c-Met monoclonal antibodies that bind to non-overlapping epitopes in ECD (Symphogen, International Publication No. 2016 / 042412).

[0014] Onartuzumab was the first anti-c-Met antibody developed, a humanized monovalent antagonist anti-c-Met antibody derived from the c-Met agonist antibody 5D5 (Spigel et al., J. Clin. Oncol. 2013, 31, 4105-4114; Xiang et al., Clin. Cancer Res. 2013, 19, 5068-5078). Despite promising experimental results, development of onartuzumab was terminated due to a lack of clinically meaningful efficacy in late-stage clinical trials (Spigel et al., J. Clin. Oncol. 2014, 32, abstract 8000; NCT01456325). The humanized IgG4, emibetuzumab (LY2875358), was included in a phase 2 clinical trial (NCT01900652) in patients with stage IV EGFR-mutated NSCLC. In this trial, no significant difference in median progression-free survival (PFS) was observed in the treatment population (9.3 months with emibetuzumab plus erlotinib compared to 9.5 months with erlotinib alone) (Scagliotti et al., J. Thorac. Oncol. 2020, 15, 80-90). The human IgG1, ARGX-111 (NCT02055066), the humanized IgG1, ABT-700 (terisotuzumab; NCT01472016), and the humanized IgG1 monoclonal antibody mixture, Sym015 (NCT02648724), are currently being evaluated in phase 1 clinical trials. Currently, there are no anti-c-Met antibodies approved for therapeutic use.

[0015] ADCs are an interesting alternative because they can kill cells that are not involved in the c-Met signaling pathway. The effectiveness of c-Met-targeting TKIs or monoclonal antibodies largely depends on the tumor / MET amplification caused by c-Met, while the effectiveness of ADCs containing antibodies against c-Met largely depends on the extracellular expression and internalization of c-Met, and the sensitivity to cytotoxic agents conjugated to the antibody in the ADC.

[0016] Therefore, anti-c-Met antibodies that are considered suitable for use in ADCs for cancer treatment must bind to c-Met with high affinity and have acceptable pharmacokinetic and pharmacodynamic properties, but must not have agonistic effects. In addition, they need to induce the internalization of c-Met.

[0017] As previously explained, the search for antibodies with antagonistic effects but without agonistic effects has already been a complex task. However, in the case of antibodies suitable for ADCs, this tendency is prominent because the epitopes of the extracellular domain (ECD) of c-Met that cause internalization rather than dimerization have not been fully identified.

[0018] Therefore, in ADCs, the efficacy depends not only on the binding properties of the antibody (affinity and specificity for the antagonistic effect against c-Met), but also on the extent to which the ADC is internalized and then processed by the cells. In the cells, the ADC releases biologically active drugs that exert a cytotoxic effect. Therefore, the sensitivity of tumor cells to cytotoxic drugs is also important.

[0019] ADCs based on antibodies specific for c-Met include telisotuzumab vedotin (ABBV-399; Abbvie, WO 2017 / 201204), TR1801-ADC (Tanabe Research, Gymnopoulos et al., Mol. Oncol. 2020, 14, 54-68), SHR-A1403 (Jiangsu HengRui Medicine Co., Yang et al., Acta Pharmacologica Sinica 2019, 40, 971-979) and hucMET-27-ADC (Immunogen Inc., WO 2018 / 129029).

[0020] Telisotuzumab vedotin is based on the c-Met antibody Telisotuzumab (ABT-700) conjugated to monomethyl auristatin E (MMAE) via a cleavable linker. TR1801-ADC is a site-specific conjugate of the humanized antibody hD12 and tesirine, a pyrrolobenzodiazepine (PBD) toxin-linker. SHR-A1403 consists of a humanized IgG2 monoclonal antibody against c-Met conjugated to a cytotoxic microtubule inhibitor. The immunogenic ADC is a conjugate of hucMET-27 and DGN549 or DM4, an indolinobenzodiazepine DNA alkylating agent.

[0021] Some ADCs in the preclinical stage have shown promising results, and some of them have advanced to clinical trials (NCT02099058, NCT03539536, NCT03859752, NCT03856541), but the life-prolonging effect has not yet been determined.

[0022] Despite the fact that development in this field has been ongoing for over 20 years, there are still no commercially available products for cancer treatment with antibodies specific to c-Met. The development of multiple clinical candidates has been discontinued, indicating that it is by no means easy to find an antibody that has sufficient specificity in the antagonistic effect rather than the agonistic effect against c-Met and has an acceptable therapeutic window (therapeutic time / concentration range).

[0023] However, considering the important role in cancer progression, c-Met remains an attractive target, and there is still a need for therapeutic antibodies and ADCs that have the desired selectivity, specificity, and efficacy, as well as an acceptable therapeutic window.

Summary of the Invention

[0026] In a second aspect, the invention relates to an ADC comprising an anti-c-Met antibody or antigen-binding fragment conjugated to a cytotoxic drug via a linker.

[0027] In a preferred embodiment, the ADC is given by equation (III)

[0028] [ka]

[0029] (In the formula, The HC variable region of Ab is shown by the amino acid sequence in SEQ ID NO: 16, and the LC variable region of Ab is shown by the amino acid sequence in SEQ ID NO: 20; Ab is an IgG1 antibody; The cytotoxic drug binds site-specifically to the recombinant cysteine ​​at position 41 of HC (in Kabat numbering) via the linker. It is represented as follows.

[0030] Other aspects of this invention include pharmaceutical compositions comprising antibodies, antigen-binding fragments, or ADCs, and the use of them as pharmaceuticals for treating cancer, particularly in monotherapy or in combination therapy. [Brief explanation of the drawing]

[0031] [Figure 1] Quantitative internalization of Alexa Fluor 488 (AF488)-labeled mAb3b in c-Met-positive MKN45 cells. (A) Internalization determined by monitoring the increase in the fluorescence signal taken up into the cytoplasm for 24 hours using an IncuCyte® S3 instrument. (B) Percentage of internalization measured by flow cytometry. [Figure 2] Cell binding of ADC3b and the corresponding unbound antibody mAb3b to c-Met-positive MKN45 cells. [Figure 3] cMet expression is associated with in vitro cytotoxicity of ADC3b in various levels of human tumor cell lines. [Figure 3-1] Continuation of Figure 3. [Figure 4] Bystander cytotoxicity in c-Met-negative Jurkat NucLight Red cells co-cultured 1:1 with c-Met-positive MKN45 cells and treated with 1 μg / mL ADC3b or unbound control ADC. [Figure 5] In vivo efficacy of chimeric ADCs (ADC1, ADC2, ADC3) and the solvent alone in a c-Met-positive MKN-45 tumor model of female ces1c KO mice. [Figure 6] In vivo efficacy of humanized ADC and solvent alone in a c-Met-positive MKN-45 tumor model in female ces1c KO mice. [Figure 7] In vivo efficacy of humanized ADCs (ADC3a, ADC3b, ADC3c) and solvents alone in a c-Met-positive MKN-45 tumor model of female ces1c KO mice. [Figure 8]Efficacy of ADC3b in head and neck cancer PDX models HNXF1905(A) and lung cancer PDX model LXFL1176(B) where MET is not amplified. Arrows indicate the randomization and administration time of mice. [Modes for carrying out the invention]

[0032] Antibodies and their antigen-binding fragments The present invention provides an antibody or antibody-binding fragment that exhibits no agonistic effect on c-Met, but rather an antagonistic or neutral effect. A neutral effect means that the antibody binds to c-Met, but this binding does not stimulate c-Met signaling.

[0033] This invention relates to an antibody or antigen-binding fragment thereof, identified by its specific complementarity-determining region (CDR), that specifically binds to c-Met, exhibits excellent affinity and good efficacy against c-Met of both human and cynomolgus monkeys (cyno), and provides an acceptable therapeutic time range / therapeutic concentration range (therapeutic window).

[0034] The antibody or antigen-binding fragment of the present invention includes an HC variable region comprising heavy chains (HC)CDR1 to 3, which are complementarity-determining regions (CDRs). The amino acid sequence of HC CDR1 includes the sequence shown in SEQ ID NO: 26, the amino acid sequence of HC CDR2 includes the sequence shown in SEQ ID NO: 27, and the amino acid sequence of HC CDR3 includes the sequence shown in SEQ ID NO: 28.

[0035] The antibody or antigen-binding fragment of the present invention further comprises an LC variable region containing light chains (LC) CDR1 to LC CDR3, which are complementarity-determining regions, wherein the amino acid sequence of LC CDR1 includes the sequence shown in SEQ ID NO: 29, the amino acid sequence of LC CDR2 includes the sequence shown in SEQ ID NO: 30, and the amino acid sequence of LC CDR3 includes the sequence shown in SEQ ID NO: 31.

[0036] Throughout this specification, the term “antibody” refers to a monoclonal antibody (mAb) comprising two heavy chains and two light chains. The antibody may be an isotype such as IgA, IgE, IgG, or IgM antibody. Preferably, the antibody is an IgG antibody, more preferably an IgG1 or IgG2 antibody. The antibody may be a chimeric, humanized, or human antibody. Preferably, the antibody of the present invention is humanized. Even more preferably, the antibody is a humanized or human IgG antibody, more preferably a humanized or human IgG1 antibody, most preferably a humanized IgG1 antibody. The antibody may have a κ or λ light chain, preferably a κ light chain, i.e., a humanized or human IgG1-κ antibody.

[0037] The antibody or its antigen-binding fragment may, where applicable, include (1) an engineered constant region, i.e., a constant region into which one or more mutations may be introduced to increase the half-life, provide a linker-drug binding site, and / or increase or decrease effector function; or (2) an engineered variable region, i.e., a variable region into which one or more mutations may be introduced to provide a linker-drug binding site. The antibody or its antigen-binding fragment may be manufactured by recombination, synthesis, or by other known suitable methods.

[0038] Throughout this specification, the term “antigen-binding fragment” includes Fab, Fab', F(ab')2, Fv, scFv, or reduced IgG (rIgG) fragments, single-chain (sc) antibodies, single-domain (sd) antibodies, diabodies, or minibodies.

[0039] The "humanization" of non-human (e.g., rodent) antibodies is an antibody with the smallest sequence derived from the non-human antibody (e.g., a non-human-human chimeric antibody). Various methods for humanizing non-human antibodies are known in the art. For example, the antigen-binding complementarity-determining regions (CDRs) in the variable regions (VRs) of the heavy chain (HC) and light chain (LC) are derived from non-human antibodies, usually from mice, rats, or rabbits. These non-human CDRs can be combined with human framework regions (FRs, i.e., FR1, FR2, FR3, and FR4) of the variable regions of the HC and LC so that the functional properties of the antibody, such as binding affinity and specificity, are preserved at least partially. Further improvements to the antibody's performance may be made, such as by replacing amino acids in the human FRs with amino acids from the corresponding non-human species to improve binding affinity while maintaining low immunogenicity. The humanized variable regions thus are usually combined with the human constant region. A representative method for humanizing non-human antibodies is the method developed by Winter and his collaborators (Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-327; Verhoeyen et al., Science 1988, 239, 1534-1536). Alternatively, non-human antibodies can be humanized by modifying their amino acid sequence to increase their similarity to antibodies naturally produced in humans. For example, the amino acids in the FR of a non-human species are replaced with corresponding human amino acids to reduce immunogenicity while maintaining antibody binding affinity. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-327 and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596.See also the following review articles and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1998, 1, 105-115; Harris, Biochem. Soc. Transactions 1995, 23, 1035-1038 and Hurle and Gross, Curr. Op. Biotech. 1994, 5, 428-433.

[0040] CDRs can be determined by the Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, NIH publication no. 91-3242, pp. 662, 680, 689 (1991)) method, the Chothia (Chothia et al., Nature 1989, 342, 877-883) method, or the IMGT (Lefranc, The Immunologist 1999, 7, 132-136) method. These methods yield CDRs of slightly different lengths, as shown by the underlined CDRs in sequence numbers 1-20 determined by the IMGT method, and by the CDR sequences determined by the Kabat method shown in sequence numbers 26-31.

[0041] In one embodiment, this invention relates to a humanized antibody or its antigen-binding fragment comprising HC CDR 1-3 and LC CDR 1-3. The amino acid sequence of HC CDR1 includes the sequence shown in SEQ ID NO: 26; The amino acid sequence of HC CDR2 includes the sequence shown in SEQ ID NO: 27; and, The amino acid sequence of HC CDR3 includes the sequence shown in SEQ ID NO: 28. The amino acid sequence of LC CDR1 includes the sequence shown in SEQ ID NO: 29; The amino acid sequence of LC CDR2 includes the sequence shown in SEQ ID NO: 30; and, The amino acid sequence of LC CDR3 includes the sequence shown in SEQ ID NO: 31.

[0042] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises the amino acid sequence of the HC variable region shown in SEQ ID NO: 16 and the amino acid sequence of the LC variable region shown in SEQ ID NO: 20.

[0043] In one embodiment, the antibody of the present invention is an intact IgG antibody, more preferably an IgG1 antibody.

[0044] In other embodiments, the antibody fragment of the present invention is a Fab, Fab', or F(ab')2 fragment, more preferably a Fab fragment.

[0045] The antibody of the present invention is particularly suitable for therapeutic applications due to its high specificity, excellent affinity for c-Met in both humans and cynomolgus monkeys, and the fact that it does not exhibit agonistic effects in either in vitro or in vivo.

[0046] Antibody-drug conjugates This invention further provides an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment of the present invention is bound to a cytotoxic drug, such as a low-molecular-weight cytotoxic drug, via a linker. The portion to which the linker and the cytotoxic drug are bound is the linker-drug (portion).

[0047] The linker structure is designed to readily chemically bind to low-molecular-weight cytotoxic drugs, and to allow for the easy binding of another substance, such as the antibody or antigen-binding fragment of the present invention, to the resulting linker-drug to form an antibody-drug conjugate. The choice of linker affects the stability of the conjugate in the bloodstream and the release mode of the low-molecular-weight drug. Suitable linkers include, for example, Ducry et al., Bioconjugate Chem. 2010, 21, 5-13, King and Wagner, Bioconjugate Chem. 2014, 25, 825-839, Gordon et al., Bioconjugate Chem. 2015, 26, 2198-2215, Tsuchikama and An (DOI: 10.1007 / s13238-016-0323-0), Polakis (DOI: 10.1124 / pr.114.009373), and Bargh et al. (DOI: This is described in International Publications 10.1039 / c8cs00676h, 2011 / 133039, 2015 / 177360, and 2018 / 069375. The linker may be cleavable or non-cleavable. A cleavable linker includes a portion that is cleaved, for example, by a lysosomal protease or when exposed to an acidic pH or a high reduction potential. Suitable cleavable linkers are known in the art and include, for example, di, tri, or tetrapeptides, i.e., peptides consisting of 2, 3, or 4 amino acid residues. Furthermore, a cleavable linker may include a self-destructing portion, such as an ω-amino-aminocarbonyl cyclization spacer (see Saari et al., J. Med. Chem., 1990, 33, 97-101), or an -NH-CH2-O- portion. Cleavage of the linker makes the drug in the ADC available around the delivered area. Uncleavable linkers can also effectively release drugs (or their derivatives) from the ADC if, for example, the polypeptide in the complex is degraded within the lysosome. Examples of uncleavable linkers include succinimidyl-4-(N-maleimidomethyl(cyclohexane)-1-carboxylate and maleimidocaproic acid and their analogues.

[0048] In this invention, either a cleavable linker or an incleavable linker may be used. Preferably, the linker has a chemical group that can react with the thiol group of the cysteine ​​residue, usually a maleimide or a haloacetyl group. More preferably, the linker is a cleavable linker.

[0049] Cytotoxic drugs conjugated to the antibody or antigen-binding fragment of the present invention are suitable for the treatment of cancer. Suitable cytotoxic drugs include, but are not limited to, duocalmycin, calicheamicin, pyrrolobenzodiazepine (PBD) dimers, maytansinoids (e.g., DM1 or DM4), and auristatin (e.g., MMAE or MMAF) derivatives. Preferably, the cytotoxic drug is a duocalmycin derivative.

[0050] Duocalmycins, first isolated from Streptomyces cultures, are a type of antitumor antibiotic encompassing duocalmycin A, duocalmycin SA, and CC-1065. These highly potent drugs are believed to derive their biological activity from their ability to initiate a series of events that terminate in apoptosis, specifically by selectively alkylating the N3 position of adenine in the secondary groove.

[0051] International Publication No. 2011 / 133039 discloses a series of linker-drugs, including the duocalmycin derivative CC-1065. The duocalmycin derivatives usable in this invention are disclosed on pages 182-197. Many of the chemical synthesis methods of these linker-drugs are described in Examples 1-12 of International Publication No. 2011 / 133039.

[0052] In a preferred embodiment, the present invention relates to an ADC in which a linker-drug is bound via a cysteine ​​residue of the antibody or antigen-binding fragment of the present invention.

[0053] In one embodiment, this invention is based on formula (I) [ka] (In the formula, Ab is the antibody or antigen-binding fragment of the present invention; n is an integer between 0 and 3; m represents the average DAR of 1 to 6; R 1 teeth, [ka] Selected from the group consisting of; y is an integer between 1 and 16; R 2 teeth, [ka] (Selected from the group consisting of...) This relates to ADCs represented by [the specified format].

[0054] In the structural formulas shown in this specification, n represents an integer from 0 to 3, and m represents the mean drug-antibody ratio (DAR) from 1 to 6. It is well known in the art that DAR and drug load distribution can be determined, for example, by hydrophobic interaction chromatography (HIC) or reversed-phase high-performance liquid chromatography (RP-HPLC). HIC is particularly suitable for determining the mean DAR.

[0055] In a particular embodiment, the present invention relates to an ADC represented by formula (I), where n is 0 or 1, m represents the average DAR of 1 to 6, preferably 1 to 4, more preferably 1 to 2, and most preferably 1.5 to 2, and R 1 teeth, [ka] Selected from the group consisting of; y is an integer between 1 and 16, preferably between 1 and 4; R 2 teeth, [ka] That is the case.

[0056] In certain embodiments, this invention relates to an ADC represented by formula (I), where n is 0 or 1, m represents the average DAR of 1.5 to 2, and R 1 teeth [ka] and; y is between 1 and 4; R 2 teeth [ka] That is the case.

[0057] In a preferred embodiment, the ADC is given by equation (II) [ka] The compound is represented by the formula, where Ab is the antibody or antigen-binding fragment of the present invention, and 1 to 4 represent the average DAR of the compound.

[0058] In a particularly preferred embodiment, the ADC is given by equation (III) [ka] The compound is represented by , where Ab is the antibody or antigen-binding fragment of the present invention, and 1.5-2 indicates the average DAR of the compound.

[0059] The ADC of the present invention may be wild-type or site-specific, and can be manufactured by known methods in the art, such as those exemplified below.

[0060] Wild-type ADCs may be produced, for example, by conjugating a linker-drug with an antibody or its antigen-binding fragment via the lysine ε-amino group of the antibody, preferably using a linker-drug containing an amine-reactive group such as an activated ester, and the ADC is obtained by contacting the activated ester with the antibody or its antigen-binding fragment. Alternatively, wild-type ADCs can be produced by conjugating a linker-drug using a free thiol in the side chain of cysteine ​​generated by the reduction of interchain disulfide bonds, using methods and conditions known in the art (see, for example, Doronina et al., Bioconjugate Chem. 2006, 17, 114-124). This production method involves partial reduction of interchain disulfide exposed to a solvent and modification of the resulting thiol with a Michael receptor-containing linker-drug (e.g., maleimide-containing linker-drug, α-haloacetamide, or ester). In this cysteine ​​conjugation strategy, up to two linker-drugs can be obtained for each reduced disulfide. Most human IgG molecules have four disulfide bonds exposed to the solvent, and therefore, 0 to 8 linker-drugs are possible per antibody. The exact number of linker-drugs per antibody depends on the degree of disulfide reduction and the molar equivalents of the linker-drug used in the subsequent binding reaction. Complete reduction of all four disulfide bonds yields a homogeneous construct with 8 linker-drugs per antibody, while partial reduction typically results in a heterogeneous mixture with 0, 2, 4, 6, or 8 linker-drugs per antibody.

[0061] Site-specific ADCs are preferably produced by linking the linker-drug to the antibody or its antigen-binding fragment via the side chain of a recombinant cysteine ​​residue at an appropriate position on the antibody or its antigen-binding fragment. The recombinant cysteine ​​is usually capped with cysteine ​​or another thiol such as glutathione to form a disulfide. These capped residues must be decapsulated before linking with the linker-drug. Linking of the recombinant residue to the linker-drug is achieved by (1) reducing both the native interchain disulfide and the mutant disulfide, then reoxidizing the native interchain cysteine ​​using a mild oxidizing agent such as CuSO4 or dehydroascorbic acid, and then linking the uncapped recombinant cysteine ​​to the linker-drug in a standard manner, or (2) using a mild reducing agent that reduces the mutant disulfide more than the interchain disulfide bond, and then linking the uncapped recombinant cysteine ​​to the linker-drug in a standard manner. Under optimal conditions, two linker-drugs bind to one antibody or its antigen-binding fragment (i.e., the drug-to-antibody ratio DAR is 2) (when one cysteine ​​is incorporated into the HC or LC of the mAb or fragment). Suitable methods for site-specific linker-drug binding are described, for example, in International Publication 2015 / 177360 (describes methods of reduction and reoxidation), International Publication 2017 / 137628 (describes methods using mild reducing agents), and International Publication 2018 / 215427 (describes methods for binding reduced interchain cysteine ​​to uncapped recombinant cysteine).

[0062] In this invention, the term "recombinant cysteine" means an antibody in which an amino acid other than cysteine ​​in the heavy or light chain is substituted with cysteine. As is known to those skilled in the art, substitutions can be made at the amino acid level or at the DNA level, for example, by site-directed mutation.

[0063] In one embodiment, the present invention relates to an ADC in which a linker-drug is site-specifically bound to the antibody or antigen-binding fragment of the present invention via a recombinant cysteine ​​residue introduced into the variable or constant region of the heavy chain or light chain.

[0064] In a preferred embodiment, the present invention relates to an ADC in which a linker-drug is site-specifically bound to an antibody or antigen-binding fragment of the present invention via recombinant cysteine ​​at one or more positions selected from the HC variable region 40, 41, and 89 (Kabat numbering) and the LC variable region 40 and 41 (Kabat numbering) of the antibody or antigen-binding fragment. Preferably, the recombinant cysteine ​​is located at position 41 of the HC or at position 40 or 41 of the LC, more preferably at position 41 of the HC.

[0065] In certain embodiments, the HC variable region of Ab is represented by the amino acid sequence of SEQ ID NO: 16, and the LC variable region of Ab is represented by the amino acid sequence of SEQ ID NO: 20. Preferably, the ADC is an ADC represented by formula (III). More preferably, the cytotoxic drug site-specifically binds to the recombinant cysteine ​​at position 41 (Kabat numbering) of the HC of Ab via a linker. Even more preferably, Ab is an IgG1 antibody. Most preferably, Ab is an IgG1 antibody having a κ light chain.

[0066] The toxicity profile of the ADC, which includes the HC variable region shown in SEQ ID NO: 16 and the LC variable region shown in SEQ ID NO: 20, and in which the vc-seco-DUBA drug is site-specifically bound via recombinant cysteine ​​at the HC41 position, was remarkably favorable in cynomolgus monkeys, considering that many diverse normal tissues express c-Met. Despite the widespread expression of c-Met, the tolerability of this ADC is surprisingly high. The maximum dose (HNSTD) of the ADC that does not cause serious toxicity is estimated to be 15 mg / kg once every three weeks.

[0067] Pharmaceutical composition The present invention further relates to a pharmaceutical composition comprising the anti-c-Met antibody or its antigen-binding fragment or anti-c-Met ADC described herein and one or more pharmaceutically acceptable additives. Common pharmaceutical formulations of therapeutic proteins such as mAbs, fragments, and (monoclonal) ADCs take the form of lyophilized cakes (lyophilized powders) that require dissolution (i.e., reconstitution) (in water) before intravenous infusion, or frozen (water) solutions that require thawing before use.

[0068] Generally, pharmaceutical compositions are provided in the form of lyophilized cakes. Suitable pharmaceutically acceptable additives for incorporation into the (pre-lyophilized) pharmaceutical compositions of this invention include buffer solutions (e.g., citrate, histidine, or succinate-containing salts in water), lyophilization protectants (e.g., sucrose, trehalose), osmotic regulators (e.g., sodium chloride), surfactants (e.g., polysorbates), and bulking agents (e.g., mannitol, glycine). Additives used in lyophilized protein formulations are selected based on their ability to prevent protein denaturation during lyophilization and storage. As an example, a single-dose sterile lyophilized powder formulation of Kadcyla® (Roche), reconstituted with sterile or sterile water for injection (BWFI or SWFI), contains 20 mg / mL trastuzumab emtansine, 0.02% w / v polysorbate 20, 10 mM sodium succinate, and 6% w / v sucrose, with a pH of 5.0.

[0069] Pharmaceutical use In another aspect, the present invention provides the previously described pharmaceutical compositions for use as anti-c-Met antibodies or their antigen-binding fragments, ADCs, or, in particular, pharmaceuticals for the treatment of cancer.

[0070] In this invention, cancer is preferably a tumor that expresses c-Met. Such tumors may be c-Met-positive solid tumors or hematopoietic tumors caused by MET. Examples of solid tumors or hematopoietic tumors that can be treated with this invention, as described above, include breast cancer; brain tumors (e.g., glioblastoma multiforme (GBM) or medulloblastoma); head and neck cancers; thyroid cancers; salivary gland cancers (e.g., parotid gland cancer); adrenal cancers (e.g., neuroblastoma, paraganglioma, or pheochromocytoma); bone cancers (e.g., osteosarcoma); soft tissue sarcomas (STS); eye cancers (e.g., uveal melanoma); esophageal cancers; gastric cancers (GC); small intestine cancers; colorectal cancers (CRC); urothelial cell carcinomas (e.g., bladder cancer, penile cancer, ureteral cancer, or kidney cancer); ovarian cancers; uterine cancers (e.g., endometrial cancers). This may include, but is not limited to, malignant tumors of the lymphatic system (e.g., mature T and NK tumors) or myeloid tumors (multiple myeloma).

[0071] In one embodiment, the present invention relates to an anti-c-Met antibody or its antigen-binding fragment, ADC, or a previously described pharmaceutical composition for use in the treatment of c-Met-positive human solid tumors or hematopoietic malignancies caused by MET.

[0072] In a preferred embodiment, the present invention relates to an anti-c-Met antibody or its antigen-binding fragment, an ADC, or a pharmaceutical composition described herein, particularly an ADC comprising a duocalmycin derivative linker-drug for use in the treatment of c-Met-positive human solid tumors selected from the group consisting of breast cancer; brain tumors; head and neck cancers; thyroid cancer; salivary gland cancer; soft tissue sarcoma (STS); eye cancer; esophageal cancer; gastric cancer (GC); small intestine cancer; colorectal cancer (CRC); urothelial cell carcinoma (UCC); ovarian cancer; uterine cancer; endometrial cancer; cervical cancer; lung cancer (particularly non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)); melanoma; liver cancer; pancreatic cancer; non-melanoma skin cancer; prostate cancer; germ cell cancer; and cancer of unknown primary origin (CUP).

[0073] In a more preferred embodiment, the present invention relates to anti-c-Met antibodies or their antigen-binding fragments, ADCs, or the pharmaceutical compositions described herein, particularly for breast cancer; glioblastoma multiforme (GBM); medulloblastoma; head and neck cancer; papillary thyroid cancer; salivary gland cancer; soft tissue sarcoma (STS); uveal melanoma; esophageal cancer; gastric cancer (GC); small intestine cancer; colorectal cancer (CRC); urothelial cell carcinoma (UCC); bladder cancer; urinary tract cancer; penile cancer; papillary renal cell carcinoma (PRCC); and other cancers. This relates to an ADC containing a duocalmycin derivative linker-drug for use in the treatment of c-Met-positive human solid tumors selected from the group consisting of cystic renal cell carcinoma (CCRCC); non-clear cell renal cell carcinoma; nephroblastoma; ovarian cancer; uterine cancer; endometrial cancer; cervical cancer; non-small cell lung cancer (NSCLC); small cell lung cancer (SCLC); melanoma; hepatocellular carcinoma (HCC); pancreatic cancer; non-melanoma skin cancer; prostate cancer; germ cell cancer; and cancer of unknown primary origin (CUP).

[0074] In a more preferred embodiment, the present invention relates to an anti-c-Met antibody or its antigen-binding fragment, an ADC, or a pharmaceutical composition described herein, in particular an ADC comprising a duocalmycin derivative linker-drug for use in the treatment of hematopoietic malignancies caused by MET, wherein the hematopoietic malignancies caused by MET are lymphoid or myeloid malignancies, more preferably mature T and NK tumors or multiple myeloma.

[0075] The anti-c-Met antibodies or their antigen-binding fragments, ADCs, or pharmaceutical compositions described herein may be used for the manufacture of the pharmaceuticals described herein. Preferably, the anti-c-Met antibodies or their antigen-binding fragments, ADCs, or pharmaceutical compositions described herein are for therapeutic methods in which the anti-c-Met antibodies or their antigen-binding fragments, ADCs, or pharmaceutical compositions are administered in a therapeutically effective amount to a subject, particularly to a subject in need of treatment. Therefore, selectively, or in combination with other embodiments, in some embodiments, this invention relates to the use of the anti-c-Met antibodies or their antigen-binding fragments, ADCs, or pharmaceutical compositions described herein for the manufacture of pharmaceuticals for the treatment of cancer. For examples of cancers treated by this invention, which are not limited to this invention, please refer to the foregoing description.

[0076] Selectively, or in combination with other embodiments, in some embodiments, the present invention relates to a method for treating cancer, the method comprising administering to a subject in need of treatment a therapeutically effective amount of the previously described anti-c-Met antibody or its antigen-binding fragment, ADC, or pharmaceutical composition. Examples of cancers treated by the present invention, but not limited to these, should be seen in the preceding description.

[0077] The anti-c-Met antibodies or their antigen-binding fragments, ADCs, or pharmaceutical compositions described herein are intended for administration to a subject. These anti-c-Met antibodies or their antigen-binding fragments, ADCs, or pharmaceutical compositions can be used in the therapeutic methods described herein by administering an effective amount of the composition to the subject requiring it. In this specification, the term “subject” refers to all animals classified as mammals, including but not limited to primates and humans. The subject is preferably human. The expression “therapeutic effective dose” means an amount sufficient to produce a desired response or to improve a symptom or sign. The therapeutic effective dose for a particular subject may vary depending on factors such as the symptoms, the subject’s overall condition, the method, route and dose of administration, and the severity of side effects.

[0078] The present invention also relates to the use of the anti-c-Met antibodies or antigen-binding fragments thereof, anti-c-Met ADCs or pharmaceutical compositions described above, administered sequentially or simultaneously, in combination with one or more other therapeutic agents.

[0079] Suitable chemotherapeutic agents include alkylating agents such as nitrogen mustards, hydroxyurea, nitrosoureas, tetrazines (e.g., temozolomide) and aziridines (e.g., mitomycin); drugs that interfere with DNA damage response such as PARP inhibitors, ATR and ATM inhibitors, CHK1 and CHK2 inhibitors, DNA-PK inhibitors, WEE1 inhibitors; antimetabolites such as folic acid antagonists (e.g., pemetrexed), fluoropyrimidines (e.g., gemcitabine), deoxynucleoside analogs, thiopurines; antimicrotubule agents such as vinca alkaloids and taxanes; topoisomerase I and II inhibitors; cytotoxic antibiotics such as anthracyclines and bleomycins; DNA methylation inhibitors such as decitabine and azacitidine; histone deacetylase inhibitors; all-trans retinoic acid; and arsenic trioxide. Suitable radiotherapeutic agents include radioisotopes such as metaiodobenzylguanidine (MIBG)( 131 I), sodium phosphate( 32 P), radium chloride( 223 Ra), strontium chloride( 89 Sr) and samarium ethylene diamine tetramethylene phosphonic acid (EDTMP) samarium( 153 Sm). Suitable agents used as hormonal therapy drugs include hormone synthesis inhibitors such as aromatase inhibitors and GnRH analogs; hormone receptor antagonists such as selective estrogen receptor modulators (e.g., tamoxifen and fulvestrant), and antiandrogen drugs such as bicalutamide, enzalutamide, flutamide; CYP17A1 inhibitors such as abiraterone; and somatostatin analogs.

[0080] Targeted therapies are treatments that inhibit specific proteins involved in tumorigenesis and proliferation, and may include small molecule drugs; proteins such as therapeutic antibodies; peptides and peptide derivatives; or hybrids of proteins and small molecules such as ADCs. Examples of targeted small molecule drugs include mTor inhibitors such as everolimus, temsirolimus, and rapamycin; kinase inhibitors such as imatinib, dasatinib, and nilotinib; VEGF inhibitors such as sorafenib and regorafenib; EGFR / HER2 inhibitors such as gefitinib, lapatinib, and erlotinib; and CDK4 / 6 inhibitors such as palbociclib, ribociclib, and abemaciclib. Examples of therapies that target peptides or peptide derivatives include proteasome inhibitors such as bortezomib and carfilzomib.

[0081] Appropriate anti-inflammatory agents include D-penicillamine, azathioprine and 6-mercaptopurine, cyclosporine, anti-TNF biological agents (e.g., infliximab, etanercept, adalimumab, golimumab, certolizumab or certolizumab pegol), leflunomide, abatacept, tocilizumab, anakinra, ustekinumab, rituximab, daratumumab, ofatumumab, obinutuzumab, secukinumab, apremilast, acitretin, and JAK inhibitors (e.g., tofacitinib, baricitinib or upadacitinib).

[0082] Immunotherapy agents include drugs that induce, enhance, or suppress the immune response, such as cytokines (IL2 and IFNα); immunomodulatory imides (e.g., thalidomide, lenalidomide, pomalidomide, or imiquimod); therapeutic cancer vaccines (e.g., tarimodine laherparepbec); and cell-based immunotherapies (e.g., dendritic cell vaccines, adoptive T cells, or chimeric antigen receptor-modified T cells); and therapeutic antibodies that, when bound to a ligand that binds to the cell membrane, can induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cell-mediated cytotoxicity (CDC) via their Fc region.

[0083] In some embodiments, the present invention relates to the use of a combination of the previously described anti-c-Met antibody or its antigen-binding fragment, anti-c-Met ADC, or pharmaceutical composition administered sequentially or simultaneously, and therapeutic antibodies, chemotherapeutic agents, and / or ADCs against cancer-related targets other than the c-Met antigen for the treatment of human solid tumors or hematopoietic malignancies, as previously described.

[0084] The therapeutically effective dose of the anti-c-Met antibody or its antigen-binding fragment or ADC of this invention is approximately 0.01 to approximately 15 mg / kg body weight, more specifically approximately 0.1 to approximately 10 mg / kg body weight, and more specifically approximately 0.3 to approximately 10 mg / kg body weight. This latter range roughly corresponds to a constant dose of 20 to 800 mg of the antibody or ADC. The compounds of this invention can be administered weekly, bi-weekly, every three weeks, monthly, or every six weeks. The appropriate treatment method depends on the severity of the disease, the patient's age, the compound administered, and other factors considered by the treating physician.

[0085] In this invention, treatment preferably involves preventing, reversing, curing, relieving, and / or delaying cancer. This may mean that the severity of at least one symptom of cancer is reduced, and / or that at least one parameter related to cancer is improved.

[0086] General definition In this specification and claims, the verb "comprise" and its conjugations are used in a non-restrictive sense, meaning that the item following the word is included, and not excluding items not mentioned. In addition, reference to an element with the indefinite article "a, an" does not exclude the possibility of multiple elements being present unless the context explicitly requires that there be only one. Thus, the indefinite article "a, an" usually means "at least one."

[0087] When the word "about" or "approximately" is used with a number (e.g., about 10), it preferably means that the number may be within ±1% of that number.

[0088] All patents and documents cited in this specification are incorporated herein by reference in their entirety.

[0089] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. [Examples]

[0090] Hydrophobic interaction chromatography (HIC) for characterizing ADCs For HIC analysis, 5–10 μL of sample (1 mg / mL) was injected into a TSKgel Butyl-NPR column (4.6 mm diameter × 3.5 cm length, Tosoh Bioscience, catalog number 14947). Elution was performed using a linear gradient from 100% buffer A (25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 6.95) to 100% buffer B (25 mM sodium phosphate, pH 6.95, 20% isopropanol) at a rate of 0.4 mL / min for 20 minutes. A Waters Acquity H-Class UPLC system equipped with a PDA detector and Empower software was used. Absorbance was measured at 214 nm, and the retention time of the ADC was determined.

[0091] Size exclusion chromatography (SEC) for characterizing ADCs For SEC analysis, 5 μL of sample (1 mg / mL) was injected into a TSKgel G3000SWXL column (5 μm, 7.8 mm diameter × 30 cm length, Tosoh Bioscience, catalog number 08541) linked to a TSKgel SWXL Guard column (7 μm, 6.0 mm diameter × 4.0 cm length, Tosoh Bioscience, catalog number 08543). Elution was performed using 50 mM sodium phosphate, 300 mM NaCl, pH 7.5, at a rate of 0.6 mL / min for 30 minutes. The column temperature was maintained at 25°C. A Waters Acquity H-Class UPLC system with a PDA detector and Empower software was used. Absorbance was measured at 214 nm to quantify the HMW.

[0092] Immunization protocols and selections Mice were repeatedly immunized with recombinant human HGFR / c-Met ECD-Fc fusion protein. B cells were harvested from the spleen and used to prepare 57 hybridomas. These hybridomas were prepared by polyethylene glycol (PEG)-mediated cell fusion using B cells and mouse myeloma cells (accession number CVCL_J288) with a selection step in HAT medium (hypoxanthine-aminopterin-thymidine medium). The supernatant of immortalized antibody-secreting hybridoma cultures was analyzed for IgG production, antibody isotype, and specific binding to HGFR / c-Met using a Luminex bead assay immobilized with c-Met-Fc.

[0093] Screening of functional mouse antibodies Antibody function was determined by analyzing the stimulation of HepG2 cells by hepatocyte growth factor (HGF) in the presence or absence of the antibody. Immunoblotting identified antibodies that antagonized HGF-induced c-Met and protein kinase B (PKB) phosphorylation and were positive by HepG2 flow cytometry. Based on neutral or antagonistic properties, only 11 of the initial 57 hybridomas were selected and sequenced. One agonist hybridoma was selected and sequenced as a positive control. Some amino acid sequences were mutated to more germline-like sequences to avoid rare, potentially unstable, and low-expression antibody chain sequences. Prior to codon optimization, one or more amino acid residues in the adjacency region of the VL domain were replaced with amino acid residues from known germline sequences in the international ImMunoGeneTics (IMGT) database (Scaviner et al., Exp. Clin. Immunogenetics 1999, 16.4, 234-240). This process of reverse mutation into a germline sequence, also known as germlineization, was performed on several VL domain sequences that were part of Framework 1 or J segment, or mouse IGKV-FR1 or IGKJ, respectively. Eleven heavy chain variable domains (VH) and thirteen light chain variable domains (VL) were obtained.

[0094] Based on the amino acid sequence, the corresponding DNA coding sequence was codon-optimized for expression in human cells, synthesized, and fused to DNA sequences encoding the constant portion of the human IgG1 subclass (HC SEQ ID NO: 22, LC SEQ ID NO: 23). A batch of 14 chimeric antibodies was prepared by expressing transgenes using antibody sequences encoding plasmids expressed in Expi293F cells. The antibodies were purified from the cell supernatant by protein A affinity purification.

[0095] The affinity of chimeric antibodies to full-length human and cynomolgus monkey (cyno) c-Met expressed on the cell surface was tested. For this purpose, ExpiCHO-S cells were transiently transfected with plasmid vectors encoding full-length human and cynomolgus monkey c-Met receptors before use in antibody binding experiments and cultured according to the manufacturer's instructions. As a standard plasmid backbone, commercially available mammalian expression vector pcDNA3.4 (ThermoFisher Scientific) containing full-length human or cynomolgus monkey c-Met antigen coding sequences with a human CMV promoter (accession numbers P08581 (SEQ ID NO: 24) and A0A2K5UM05 (SEQ ID NO: 25), respectively) was used.

[0096] Humanization Humanized antibodies were prepared by CDR transplantation. CDRs from two antagonistic clones and three neutral clones were identified using CDR definitions from IMGT (Lefranc and Marie-Paule. Immunologist 1999, 7.4, 132-136) and the Kabat numbering system. Using mouse VH domains, candidate human variable domains were identified by searching online public databases of human IgG sequences using the BLAST search algorithm. For each variable domain, five candidates were selected based on criteria such as framework homology, presence of major framework residues, standard loop structure, and immunogenicity. The same procedure was repeated for the VL domains of the antibodies. All humanized VH variants were combined with all humanized VL variants to obtain 25 humanized variants for each antibody, totaling 125 variants.

[0097] Humanized variants containing the heavy chain 41C (HC-41C) mutation were synthesized using the following method, and their affinity for human or cynomolgus monkey c-Met was measured using ExpiCHO-S cells expressing human or cynomolgus monkey c-Met.

[0098] Expression of antibody and c-Met antigen transgenes a) Preparation of cDNA constructs and expression vectors The mouse amino acid sequence of the heavy chain variable domain (VH) was ligated to the HAVT20 reader sequence (SEQ ID NO: 21) at the N-terminus and to the human IgG1 heavy chain constant region (SEQ ID NO: 22) at the C-terminus. The resulting chimeric amino acid sequence was back-translated into a cDNA sequence and codon-optimized for expression in human cells.

[0099] Similarly, chimeric cDNA sequences of light chain (LC) constructs were obtained by concatenating the appropriate secretory signal sequence (including the HAVT20 reader sequence), the variable region (VL) of the mouse amino acid sequence, and the constant region of the human IgGκ light chain (SEQ ID NO: 23), and then back-translating the resulting amino acid sequence into a codon-optimized cDNA sequence for expression in human cells.

[0100] cDNA sequences encoding LC and HC of a humanized variant with the HC-41C mutation were obtained by the same method. The HC and LC sequences were ligated at the N-terminus to the HAVT20 reader sequence (SEQ ID NO: 21) and at the C-terminus to the human IgG1 heavy chain constant region (SEQ ID NO: 22) or the human IgGκ light chain constant region (SEQ ID NO: 23).

[0101] b) Vector construction and cloning strategies Commercial mammalian expression vector pcDNA3.4 (ThermoFisher Scientific) containing a CMV:BGHpA expression cassette was used for the expression of antibody chains and c-Met antigen. HC, LC, or antigen cDNA was ligated to the pcDNA3.4 vector at restriction sites AscI and NheI. The final vectors containing either the HC, LC, or c-Met expression cassette (CMV:HC:BGHpA and CMV:LC-BGHpA, respectively) were used to transform E. coli NEB 5-α cells. Large-scale preparation of the final expression vectors for transfection was performed using the Maxiprep kit or the Megaprep kit (Qiagen).

[0102] c) Transient expression of antibodies in mammalian cells Commercially available Expi293F cells (ThermoFisher Scientific) were transfected with an expression vector using ExpiFectamine transfection agent, following the manufacturer's instructions as follows: 75 × 10⁻¹⁴ 7 Cells were seeded in 300 mL of FortiCHO medium, and 300 μg of expression vector was added to the cells in combination with 800 μL of ExpiFectamine transfection agent. One day after transfection, 1.5 mL of enhancer 1 and 15 mL of enhancer 2 were added to the culture. Six days after transfection, the cell culture supernatant was collected by centrifugation at 4,000 g for 15 minutes, and the clarified collection was filtered through a PES bottle filter / MF75 filter (Nalgene).

[0103] d) Transient expression of c-Met in mammalian cells Commercially available ExpiCHO-S cells (ThermoFisher Scientific) were transfected with an expression vector using the ExpiFectamineCHO transfection agent, following the manufacturer's instructions as follows: 1.2 × 10⁶ cells 9 Cells were seeded in 200 mL of ExpiCHO expression medium, and 200 μg of expression vector was added to the cells in combination with 640 μL of ExpiFectamineCHO transfection agent. One day after transfection, the cell cultures were used for dose-dependent cell binding analysis.

[0104] Cell binding experiment Chimeric antibodies Cell binding of 14 wild-type chimeric anti-c-Met antibodies and 14 HC-41C chimeric anti-c-Met antibodies was experimentally investigated using human c-Met-positive tumor cell lines MKN45, NCI-H596, and PC3, rhesus monkey c-Met-positive tumor cell line 4MBr-5, and human c-Met-negative tumor cell line MDA-MB-175-VII. Cells approximately 90% confluent were used during the assay, detached with trypsin-Barsen® (EDTA) (Lonza) for 5-10 minutes, washed, and diluted with ice-cold FACS buffer (PBS 1×, 0.1% v / w BSA, 0.02% v / v sodium azide (NaN3)) to a concentration of 1×10⁻⁶. 6 The solution was adjusted to the number of cells / mL. To prevent alteration and internalization of surface antigens, the cells were stained in 96-well round-bottom microtiter plates using ice-cold reagents / solutions at 4°C. 100,000 cells per well (100 μL / well) were added to the 96-well plates and centrifuged at 300 × g for 3 minutes. For pre-incubation with HGF (paracrine cell lines only), cells were resuspended in 50 ng / mL recombinant human HGF in FACS buffer (50 μL / well), incubated at 4°C for 30 minutes, and then washed twice with 150 μL of FACS buffer. The supernatant was discarded, and the cells were stained with 50 μL of each antibody for 30 minutes. Serial dilutions were performed with ice-cold FACS buffer. The cells were washed twice by centrifugation at 300 × g for 3 minutes and resuspended in 50 μL of 6000-fold diluted secondary F(ab')2 goat anti-human IgG (Fc fragment specific) antibody APC conjugate (Jackson ImmunoResearch). After incubation on ice for 30 minutes, the cells were washed twice again and resuspended in 150 μL of ice-cold FACS buffer for FACS analysis. Median fluorescence intensity (MFI) was obtained to analyze the data.

[0105] All chimeric antibodies bound well to human c-Met in the tested cell lines. Only five chimeric antibodies bound well to rhesus monkey c-Met in the 4MBr-5 cell line. As expected, there was no difference in binding characteristics between wild-type chimeric antibodies and their HC-41C counterparts. None of the antibodies bound to c-Met-negative cell lines, indicating that all antibodies specifically recognize the c-Met receptor. Based on their high affinity for both human and rhesus monkey c-Met, three wild-type chimeric antibodies and their corresponding HC-41C chimeric antibodies were selected for further development (see Table 1). In paracrine cell lines, HGF was EC1 of the six selected chimeric antibodies. 50 The values ​​were varied, indicating that HGF competed for the binding of these antibodies. This effect was not observed with the two non-selective chimeric antibodies and their HC-41C counterparts.

[0106] [Table 1]

[0107] [Table 2]

[0108] Hybridomas Hyb1 and Hyb2 were initially tested (in vitro) and classified as antagonists (Table 2), while the corresponding chimeric antibodies wt / 41C-chi-mAb1 and wt / 41C-chi-mAb2 were evaluated as partial agonists in vitro (in proliferation stimulation experiments in NCI-H596 cells). From this perspective, all three wt / 41C chimeric mAbs were humanized and further experimented with, but wt / 41C-chi-mAb3 is the most preferred chimeric anti-c-Met antibody for further development as an ADC to treat cancer indications.

[0109] Humanized antibodies Cell binding of a total of 75 humanized anti-c-Met antibodies (derived from three selected chimeric antibodies) was experimentally performed on human c-Met-positive tumor cell lines MKN45, NCI-H596, and PC-3, rhesus monkey c-Met-positive tumor cell line 4MBr-5, and human c-Met-negative tumor cell line MDA-MB-175-VII. Cells approximately 90% confluent were used during the assay, detached with trypsin-Barthen® (EDTA) (Lonza) for 5-10 minutes, washed, and diluted with ice-cold FACS buffer (PBS 1×, 0.1% v / w BSA, 0.02% v / v sodium azide (NaN3)) to a concentration of 1×10⁻⁶. 6 Cells / mL were adjusted. To prevent alteration and internalization of surface antigens, the cells were stained in a 96-well round-bottom microtiter plate using ice-cold reagents / solutions at 4°C. 100,000 cells per well (100 μL / well) were added to the 96-well plate and centrifuged at 300 × g for 3 minutes. For pre-incubation with HGF, cells were resuspended in 50 ng / mL recombinant human HGF in FACS buffer (50 μL / well), incubated at 4°C for 30 minutes, and then washed twice with 150 μL of FACS buffer. The supernatant was discarded, and the cells were stained with 50 μL of each antibody for 30 minutes. Serial dilutions were performed with ice-cold FACS buffer. Cells were washed twice by centrifugation at 300 × g for 3 minutes and resuspended in 50 μL of 6000-fold diluted secondary F(ab')2 goat anti-human IgG (Fc fragment specific) antibody APC conjugate (Jackson ImmunoResearch). After incubation on ice for 30 minutes, the cells were washed twice again and resuspended in 150 μL of ice-cold FACS buffer for FACS analysis. Median fluorescence intensity (MFI) was obtained to analyze the data.

[0110] None of the humanized antibodies bound to c-Met-negative cell lines. All humanized antibodies derived from wt / 41C-chi-mAb2 and wt / 41C-chi-mAb3 bound well to both human c-Met and rhesus monkey c-Met in the tested cell lines, whereas only 5 of the 25 humanized antibodies derived from wt / 41C-chi-mAb1 bound well. Despite the good binding properties of the humanized antibodies derived from wt / 41C-chi-mAb2, the hydrophobicity of 20 of the 25 humanized antibodies dramatically increased in HIC. This increase in hydrophobicity is undesirable because higher hydrophobicity in antibody-containing biological compounds leads to more rapid removal from the bloodstream (in animal models). Similar results were obtained with 14 of the 25 humanized antibodies derived from wt / 41C-chi-mAb1. Humanization of wt / 41C-chi-mAb3 did not appear to affect binding properties or hydrophobicity.

[0111] To further develop the cell binding EC of nine HC-41C humanized antibodies (Table 3) selected for further study. 50 The values ​​are shown in Table 4. From the results shown for the paracrine cell lines in Table 4, HGF is the EC of humanized antibodies. 50 By varying the values, this indicated that HGF competed for the binding of these antibodies.

[0112] Cell binding (EC) of humanized mAb1a (derived from chimeric antibody wt / 41C-chi-mAb1) was measured in human c-Met antigen-expressing MKN45 cells, NCI-H596 cells, and PC-3 cells, as well as rhesus monkey c-Met antigen-expressing 4MBr-5 cells. 50 The concentrations were 0.16 μg / mL (95% CI: 0.12~0.21 μg / mL), 0.04 μg / mL (95% CI: 0.006~0.225 μg / mL), 0.08 μg / mL (95% CI: 0.02~0.25 μg / mL), and 0.07 μg / mL (95% CI: 0.05~0.09 μg / mL), respectively. HGF binding altered cell binding in paracrine cell lines by 1.1 to 2.5 times.

[0113] Cell binding (EC) of humanized mAb2a, mAb2b, and mAb2c (derived from the chimeric antibody wt / 41C-chi-mAb2) was measured in human c-Met antigen-expressing MKN45 cells, NCI-H596 cells, and PC-3 cells, as well as rhesus monkey c-Met antigen-expressing 4MBr-5 cells. 50 The concentrations were 0.09–0.14 μg / mL, 0.02–0.04 μg / mL, 0.03–0.05 μg / mL, and 0.06–0.09 μg / mL, respectively. HGF binding altered cell binding in paracrine cell lines by 1.2–4.0 times.

[0114] Cell binding (EC) of humanized mAb3a, mAb3b, mAb3c, mAb3d, and mAb3e (derived from the chimeric antibody wt / 41C-chi-mAb3) was measured in human c-Met antigen-expressing MKN45 cells, NCI-H596 cells, and PC-3 cells, as well as rhesus monkey c-Met antigen-expressing 4MBr-5 cells. 50 The concentrations were 0.05–0.07 μg / mL, 0.02–0.03 μg / mL, 0.03–0.03 μg / mL, and 0.02–0.04 μg / mL, respectively. HGF binding altered cell binding in paracrine cell lines by 0.8–7.5 times.

[0115] [Table 3]

[0116] [Table 4-1] [Table 4-2]

[0117] Measured binding affinity (K) of unbound antibody mAb3b to human and cynomolgus monkey c-Met extracellular domain (ECD). D Table 5 shows the results of this low K D -obs(0.01nM) indicates a high affinity between the antibody and either human or cynomolgus monkey c-Met ECD.

[0118] Binding analysis was performed at 37°C using a surface plasmon resonance spectrometer (Biacore® T200 system, GE Life Sciences). Biotinylated human c-Met or cynomolgus monkey c-Met was captured on the surface of a CAPchip (Sensor Chip CAP, GE Life Sciences) suitable for capturing biotinylated molecules by injecting biotinylated capture reagent at 2 μL / min for 300 seconds into flow cells 1 and 2. Biotinylated c-Met antigen, diluted in running buffer (10 mM HEPES buffer (pH 7.4) at 25°C containing 150 mM NaCl, 3 mM EDTA, and 0.005% v / v polyoxyethylene (20) sorbitan monolaurate (Surfactant P20)), was injected at various contact times to obtain various capture levels of 5 μL / min. The dilution of the c-Met variant and contact time were estimated with a target capture level of approximately 20 RU. After a 1-minute baseline, the dissociation time was set to 900 seconds, and the mAb3b sample was injected at 30 μL / mL in five progressively increasing concentrations (0.037 nM, 0.11 nM, 0.33 nM, 1 nM, and 3 nM) for 60 seconds. The R of the interaction was... max The values ​​were 5-10 RU. Regeneration was performed with 6 M guanidine hydrochloride, 0.25 M NaOH solution (flow rate 30 μL / min for 120 seconds). Double blank subtraction was performed on the obtained sensorgrams by subtracting the signals from the blank reference flow channel and running buffer injection. Sensorgrams were created using Biacore® T200 evaluation software (v3.1). The sensorgrams were fitted to a 1:1 Langmuir coupled model, and the goodness of fit (χ²) and uniqueness of fit (U value) were evaluated by visual inspection and biological relevance.

[0119] [Table 5]

[0120] Internalization experiment Internalization of mAb3b was investigated in human c-Met-positive tumor cell lines MKN45, NCI-H441, and PC-3. Cells (100,000 cells per well in a 96-well round-bottom microtiter plate) were incubated at 4°C for 30 minutes with 50 μL of 10 μg / mL mAb3b or appropriate unbound control antibody. After washing with ice-cold complete growth medium (CGM) consisting of RPMI (Lonza) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS) (Gibco) and 80 U / mL penicillin / streptomycin (Lonza), the cells were incubated at 4°C for 30 minutes with 50 μL of Alexa Fluor 488 (AF488)-labeled Fab fragment goat anti-human IgG (1:600 ​​dilution) (Jackson Immunoresearch). After washing with ice-cold continuous glucose (CGM), the cells were resuspended in 150 μL of pre-warmed CGM. One cell was transferred to a polypropylene tube at each time point (0h, 0.5h, 1h, 3h, 24h) and placed in a 37°C water bath to initiate internalization. After incubation for the specified time, the cells were washed once with ice-cold FACS buffer consisting of 1× PBS (Lonza) supplemented with 0.1% v / w BSA (Sigma) and 0.02% sodium azide solution (Sigma) to stop internalization. After quenching at 4°C for 10 minutes using 50 μL of anti-Alexa Fluor 488 rabbit IgG antibody (1:30 dilution in ice-cold FACS buffer) (Molecular Probes, Life Technologies), the remaining surface expression was visualized. An additional 100 μL of ice-cold FACS buffer was added before measurement. Total fluorescence was determined at each time point for the same unquenched tube. Fluorescence intensity was determined by flow cytometry (BD FACSVerse, Franklin Lakes, NJ) and is shown as the median fluorescence intensity (MFI). Internalization was quantified by calculating the proportion of internalization using the following formula. Internalization rate (%)=1-(N1-Q1) / N1-(N1*Q0 / N0)*100% N1 = Unquenched MFI at each time point Q1 = Quenched MFI at each point in time Q0 = Quenched MFI at time 0 N0 = Unquenched MFI at time 0

[0121] A limitation of this method is the incomplete quenching of AF488-labeled mAbs bound to the cell surface. This is consistent with the supplier's description that the maximum quenching of the AF488 dye is at most 90% or less with bound AF488 dye.

[0122] As shown in Figure 1B for MKN45 cells, efficient internalization of mAb3b was demonstrated in each cell line, with maximum internalization occurring at 24 hours.

[0123] Real-time monitoring of mAb3b internalization was performed in MKN45 cells. Cells (18,750 cells / well) in complete growth medium were seeded into 96-well plates (50 μL / well). After incubation overnight at 37°C and 5% CO2, 3 μg / mL of unlabeled mAb3b containing human FabFluor-pH red fluorescent dye (Sartorius) was added to the cells (total volume 100 μL / well). Real-time live-cell analysis of internalization was performed by imaging the plates every 30 minutes for 48 hours using an IncuCyte® S3 instrument, and evaluating the phase and red fluorescence of two images per well using a 10x objective lens. The red fluorescence area and total cell area were masked and counted using the Cell-by-Cell adherent module of the IncuCyte® S3 software, and the (FabFluor red area / MKN45 area) was plotted against time (Figure 1A). The intensity of FabFluor increases during the pH-dependent pathway to lysosomes, showing the highest fluorescence at pH 4.7.

[0124] Site-specific binding and wild-type binding protocols General binding protocols for binding via partially reduced endogenous disulfide (wild-type (wt) binding) The antibody solution (5-10 mg / mL in 4.2 mM histidine, 50 mM trehalose, pH 6) was diluted with EDTA (25 mM aqueous solution) (4% v / v). The pH was adjusted to approximately 7.4 using TRIS (1 M aqueous solution, pH 8), and then TCEP (10 mM aqueous solution, 1-3 equivalents depending on the antibody and desired DAR) was added. The resulting mixture was incubated at room temperature for 1-3 hours. Dimethylacetamide (DMA) was added, followed by the linker-drug solution (10 mM DMA solution). The final DMA concentration was 5-10%. The resulting mixture was incubated at room temperature for 1-16 hours under light protection. Activated carbon was added to remove excess linker-drug, and the mixture was incubated at room temperature for 1 hour. The activated carbon was removed using a 0.2 μm PES filter, and the resulting ADC was formulated in 4.2 mM histidine, 50 mM trehalose, pH 6 using a Vivaspin centrifugal concentrator (molecular weight cutoff 30 kDa, PES). Finally, the ADC solution was sterile filtered using a 0.22 μm PES filter.

[0125] General site-specific binding protocols Site-specific conjugates were synthesized using the procedures of Protocol A or Protocol B. 1) Protocol A A solution of recombinant cysteine ​​antibody (5-10 mg / mL in 4.2 mM histidine, 50 mM trehalose, pH 6) was diluted with EDTA (25 mM aqueous solution) (4% v / v). The pH was adjusted to approximately 7.4 using TRIS (1 M aqueous solution, pH 8), and then TCEP (10 mM aqueous solution, 20 equivalents) was added. The resulting mixture was incubated at room temperature for 1-3 hours. Excess TCEP was removed using 4.2 mM histidine, 50 mM trehalose, pH 6 with a PD-10 desalting column or centrifugal concentrator (Vivaspin filter, molecular weight cutoff 30 kDa, PES).

[0126] The pH of the obtained antibody solution was raised to approximately 7.4 using TRIS (1M aqueous solution, pH 8), then dehydroascorbic acid (10 mM aqueous solution, 20 equivalents) was added, and the resulting mixture was incubated at room temperature for 1-2 hours. At room temperature or 37°C, DMA was added, followed by a linker-drug solution (10 mM DMA solution). The final DMA concentration was 5-10%. The resulting mixture was incubated in the dark at room temperature or 37°C for 1-16 hours. To remove excess linker-drug, activated carbon was added, and the mixture was incubated at room temperature for 1 hour. The activated carbon was removed using a 0.2 μm PES filter, and the resulting ADC was formulated in 4.2 mM histidine, 50 mM trehalose, pH 6 using a Vivaspin centrifugal concentrator (molecular weight cutoff 30 kDa, PES). Finally, the ADC solution was sterile filtered using a 0.22 μm PES filter.

[0127] 2) Protocol B A solution of recombinant cysteine ​​antibody (40 mg / mL in 500 μL of 15 mM histidine, 50 mM sucrose, and 0.01% polysorbate-20, pH 6) was diluted with 100 mM histidine (pH 5, 1300 μL). 2-(diphenylphosphino)benzenesulfonic acid (diPPBS) (426 μL of 10 mM aqueous solution, 32 equivalents) was added, and the resulting mixture was incubated at room temperature for 16–24 hours. Excess diPPBS was removed using a centrifugal concentrator (Vivaspin filter, molecular weight cutoff 30 kDa, PES) with 4.2 mM histidine, 50 mM trehalose, pH 6.

[0128] The pH of the obtained antibody solution was raised to approximately 7.4 using TRIS (1M aqueous solution, pH 8). DMA, followed by a linker-drug solution (10 mM DMA solution), was added at room temperature or 37°C. The final DMA concentration was 5–10%. The resulting mixture was incubated in the dark at room temperature or 37°C for 1–16 hours. Activated carbon was added to remove excess linker-drug, and the mixture was incubated at room temperature for 1 hour. The activated carbon was removed using a 0.2 μm PES filter, and the resulting ADC was formulated in 4.2 mM histidine, 50 mM trehalose, pH 6 using a Vivaspin centrifugal concentrator (molecular weight cutoff 30 kDa, PES). Finally, the ADC solution was sterile filtered using a 0.22 μm PES filter.

[0129] Site-specific binding protocol for mAb3b To a solution of mAb3b (10-12 mg / mL in 100 mM histidine, pH 5), diPPBS (10 mM aqueous solution, 16-32 equivalents) was added, and the resulting mixture was incubated overnight at room temperature. Excess diPPBS was removed using a Vivaspin centrifuge (cutoff molecular weight 30 kDa, PES) with 4.2 mM histidine, 50 mM trehalose, pH 6. DMA was added, followed by a linker-drug solution (10 mM DMA solution). The final DMA concentration was 10%. The resulting mixture was incubated overnight at room temperature in the dark. Activated carbon was added to remove excess linker-drug, and the mixture was incubated at room temperature for 1 hour. The activated carbon was removed using a 0.2 μm PES filter, and the resulting ADC was formulated using a Vivaspin centrifuge (cutoff molecular weight 30 kDa, PES) in 4.2 mM histidine, 50 mM trehalose, pH 6. Finally, the ADC solution was sterile filtered using a 0.22 μm PES filter.

[0130] In vitro cytotoxicity of ADCs As shown in Figure 2 for antibody mAb3b and antibody-drug conjugate ADC3b, the binding affinity to c-Met-expressing MKN45 cells was comparable for the unbound antibody and ADC (mean ± standard error of two double-drug experiments). Therefore, the antigen-binding properties of the ADC were not affected by the bound duocalmycin derivative linker-drug.

[0131] The in vitro cytotoxicity of chimeric and humanized anti-c-Met ADCs (Table 6) was determined in human tumor cell lines with varying levels of c-Met expression. Cells in complete growth medium were seeded (90 or 80 μL / well) into 96-well plates at the following cell densities and incubated at 37°C and 5% CO2: 2500 MKN-45 cells, 1000 PC-3 cells, 2500 NCI-H596 cells, and 5000 MDA-MB-175-VII cells per well. After overnight incubation, 10 μL of ADC, or 10 μL of ADC and 10 μL of HGF (500 ng / mL) were added. Serial dilutions of ADC were prepared using the medium. After 6 days, cell viability was assessed using the CellTiter-Glo® (CTG) luminescence assay kit (Promega Corporation, Madison, WI) according to the manufacturer's instructions. The survival rate was calculated by dividing the measured luminescence values ​​at each ADC concentration by the average value of untreated cells (growth medium only) and multiplying by 100.

[0132] The results are shown in Table 7. As expected, the unbound control ADC (rituximab-vc-seco-DUBA) affected the proliferation of c-Met-expressing tumor cells only at high concentrations. All anti-c-Met ADCs were inactive against the c-Met-negative human tumor cell line MDA-MB-175-VII (IC). 50 It was >10nM).

[0133] There were no significant differences in the efficacy of HC-41C humanized anti-c-Met ADC across various cell lines. HGF (50 ng / mL) did not affect cytotoxicity against PC-3 cells. The efficacy in HGF-stimulated NCI-H596 cells was at the same level as in unstimulated NCI-H596 cells. Proliferation was induced in NCI-H596 cells stimulated with 50 ng / mL of HGF compared to cells without HGF. HGF did not induce cytotoxicity.

[0134] [Table 6]

[0135] [Table 7]

[0136] In another experiment, the in vitro cytotoxicity of ADC3b was determined in human tumor cell lines with varying levels of c-Met expression. Cells in full growth medium were seeded (45 μL / well) into 384-well plates at the following cell densities and incubated at 37°C and 5% CO2: 650 MKN-45 cells, 600 EBC-1 cells, 250 PC-3 cells, 400 KP-4 cells, 2000 NCI-H441 cells, 2500 Hep-G2 cells, 4000 A2780 cells, 1500 HCC-1954 cells, and 600 Jurkat NucLight Red cells per well. After overnight incubation, 5 μL of ADC3b was added. Serial dilutions of ADC were prepared using the medium. Six days later, cell viability was evaluated using fluorescence-based PrestoBlue® Cell Viability Reagent (Invitrogen, ThermoFisher Scientific, USA) and fluorescence-based CyQUANT® Cell Proliferation Assay (Invitrogen, ThermoFisher Scientific, USA), following the manufacturer's instructions. Viability was calculated by dividing the measured luminescence values ​​at each ADC concentration by the average value of untreated cells (growth medium only) and multiplying by 100.

[0137] In all methods, ADC3b was shown to induce cytotoxicity in human tumor cell lines with high, moderate, and low c-Met expression (Figure 3 shows the results of the CyQUANT® cell proliferation assay). Cell viability is expressed in Figure 3 as the mean ± standard error of two triple experiments. No cytotoxicity was observed in the unbound control. No cytotoxicity was observed with ADC3b in c-Met-negative A2780 cells (approximately 35 cMet antigen-binding sites per cell) or Jurkat NucLight Red cells (no cMet antigen-binding sites).

[0138] ADC bystander cytotoxicity Bystander cytotoxicity was determined using c-Met-negative Jurkat NucLight Red cells co-cultured in a 1:1 ratio with c-Met-positive MKN45 cells. 5000 cells / well of each cell type (1:1 ratio) were placed in their respective culture media and added to 96-well plates pre-coated with poly-L-ornithine (0.1 mg / mL, Sigma). After incubation overnight at 37°C and 5% CO2, 10 μL of 1 μg / mL ADC3b or unbound control ADC (rituximab-vc-seco-DUBA) was added. Live cell analysis of the proliferation of co-cultured c-Met-negative Jurkat NucLight Red cells was performed for 6 days, imaging the plates every 6 hours using an IncuCyte® S3 instrument. Phase and fluorescence scanning was performed on 4 images per well using a 10x objective lens.

[0139] ADC3b can induce bystander-killing effects in adjacent cells that do not express c-Met (Figure 4).

[0140] Evaluation of in vivo chimeric antibody-drug conjugates (ADCs) in a subcutaneous xenograft model of gastric MKN-45 tumors in female ces1c KO mice. The in vivo efficacy of chimeric anti-c-Met ADCs was evaluated in a xenograft model of the MKN-45 (gastric adenocarcinoma) cell line in B6.Ces1ctm1.1 Loc.Foxn1nu mice. These mice lack exon 5 of the Ces1c gene, resulting in a loss of enzyme function. In this cell line, the MET gene was amplified; and high expression of c-Met on the cell surface was confirmed by immunohistochemical staining.

[0141] Using a syringe with a 30G needle, 5 × 10 in 200 μL of PBS:Matrigel in a 1:1 ratio. 6 Tumors were induced by subcutaneous injection of MKN-45 tumor cells into the left flank of all mice. Animal body weight was measured three times a week. Primary tumors were measured using calipers, and tumor volume was calculated using formula W. 2 The calculation was performed using the formula ×L / 2 (L = tumor length, W = tumor vertical width, L > W). The primary tumor was approximately 100 mm. 3After reaching a certain stage, the animals were randomized across the entire treatment group according to tumor volume and administered a single injection of 10 mg / kg of ADC1 (DAR 1.5), ADC2 (DAR 1.7), or ADC3 (DAR 1.1) on the same day or the following day. Mice were administered the drug on the day of or the day after transplantation.

[0142] As shown in Figure 5, the antitumor activity of all three chimeric ADCs tested was significant.

[0143] Evaluation of in vivo humanized antibody-drug conjugates (ADCs) in a subcutaneous xenograft model of gastric MKN-45 tumors in female ces1c KO mice. The in vivo efficacy of humanized anti-c-Met ADCs was evaluated in a xenograft model of the MKN-45 (gastric adenocarcinoma) cell line in B6.Ces1ctm1.1 Loc.Foxn1nu mice using the protocol described previously.

[0144] The primary tumor was approximately 100 mm in size. 3 After reaching a certain stage, the animals were randomized as a whole treatment group according to tumor volume, and on the day of randomization or the following day, they received a single injection of ADC1a derived from ADC1, ADC2a, ADC2b, and ADC2c derived from ADC2, and ADC3a, ADC3b, ADC3c, ADC3d, and ADC3e derived from ADC3.

[0145] As shown in Figure 6, the antitumor activity was highest in ADCs containing humanized antibodies derived from the chimeric mAb used in ADC3.

[0146] Evaluation of the antitumor efficacy of humanized antibody-drug conjugates (ADCs) in the patient-derived breast cancer xenograft model MAXF574. The in vivo efficacy of three humanized anti-c-Met ADCs (ADC3a, ADC3b, ADC3c) was investigated at B6-Ces1ctm1.1 Loc.CB17 Prkdc scidThe study evaluated the MET gene in a xenograft model of patient-derived invasive ductal carcinoma MAXF574 in a mouse strain. These mice lacked exon 5 of the Ces1c gene, resulting in a loss of enzyme function. The MET gene was not amplified in these tumors; however, moderate to high expression of c-Met was confirmed by immunohistochemical staining on the cell surface.

[0147] Tumor fragments were obtained from xenografts serially passaged in nude mice. After removal from donor mice, the tumors were fragmented (edge ​​length 3-4 mm) and placed in PBS containing 10% penicillin / streptomycin. Recipient animals were anesthetized by isoflurane inhalation, and the tumor fragments were transplanted subcutaneously on one side of the flank. Tumor growth was monitored twice a week. Tumor volume was determined by two-dimensional measurement using digital calipers. Tumor volume is given by the formula (l × w). 2 The calculation was performed using the formula ) × 0.5 (l = tumor length, w = tumor width (mm)). The volume of the transplanted tumor was targeted at 80-250 mm. 3 As the mice approached the target time, they were randomized across the treatment groups so that the median and mean tumor volumes were similar across the groups. On the same day or the following day, mice were injected with a single dose of 3 mg / kg of ADC3a, ADC3b, or ADC3c, including the solvent alone and unbound control ADCs. As shown in Figure 7, the unbound control ADCs showed some antitumor activity, which is bystander activity. The three anti-c-Met ADCs showed additional target-mediated antitumor activity. Of the three ADCs examined, ADC3b showed the highest antitumor activity.

[0148] Dose-response experiments of ADC3b in xenograft models of patient-derived cancers (LXFL1176 (lung) and HNXF1905 (head and neck)). Dose-response studies were conducted using two patient-derived xenograft models: 1) LXFL1176, a large cell carcinoma originating from lymph node metastasis, and 2) HNXF1905, a primary squamous cell carcinoma originating from the supraorbital region. Tumor exgrafts were prepared using the protocol described previously, using B6-Ces1ctm1.1 Loc.CB17 Prkdc scidThe MET gene was transplanted into mice. The MET gene was not amplified in any of the tumors; immunohistochemical staining revealed high expression of c-Met on the cell surface in the LXFL1176 model and moderate expression in the HNXF1905 model.

[0149] On the day of or the day after randomization, mice were injected with a single dose of 0.3, 1, 3, or 10 mg / kg of ADC3b. This included the solvent alone and unbound isotype control ADCs. The unbound control ADCs showed little to no antitumor activity, indicating little bystander activity in these models. Dose-dependent antitumor activity was demonstrated in all models. The efficacy of ADC3b in the head and neck cancer PDX model HNXF1905 is shown in Figure 8A, and the efficacy in the lung cancer PDX model LXFL1176 is shown in Figure 8B.

[0150] In vivo toxicity experiments in cynomolgus monkeys The in vivo toxicity of ADC3b was evaluated in male and female cynomolgus monkeys. Monkeys (5 males + 5 females per group) were administered ADC3b intravenously (5, 15, or 25 mg / kg once every 3 weeks).

[0151] The toxicity profile of ADC3b in cynomolgus monkeys in four cycles of critical toxicity and PK studies was remarkably mild, considering that many different normal tissues express c-Met. Binding of ADC3b to c-Met in cynomolgus monkeys and humans is comparable (as previously shown). It should be noted that c-Met is not activated in these normal tissues, as c-Met phosphorylation is not observed in these tissues. Despite the widespread expression of c-Met, the tolerability of ADC3b is surprising. The maximum dose (HNSTD) of ADC3b without serious toxicity is estimated to be 15 mg / kg once every three weeks. Given that many of the toxic effects of ADCs are observed in (normal) tissues expressing the target (Masson Hinrichs and Dixit, AAPS J. 2015, 17, 1055-1064), the excellent tolerability of ADC3b, an ADC targeting c-Met, is remarkable.

[0152] in vivo PKPD experiment Pharmacokinetic / pharmacodynamic experiments were conducted on the anti-c-Met ADC ADC3b in CES1c KO SCID mice with tumors. These mice lack exon 5 of the Ces1c gene, resulting in a loss of enzyme function. Further pharmacokinetic data were obtained from toxicity experiments of ADC3b in cynomolgus monkeys.

[0153] Mice were administered ADC3b (0.3, 1, 3, or 10 mg / kg, intravenous (iv) bolus injection), and plasma was collected 1, 48, 168, 336, and 504 hours after administration. Monkeys were administered ADC3b (5, 15, or 25 mg / kg, intravenous infusion), and plasma was collected 1, 6, 24, 48, 96, 192, 360, and 504 hours after administration. Total antibodies were quantified by LC-MS / MS-based assays, and bound antibodies were quantified by ligand-binding assays. Assays on bound antibodies capture ADCs with at least one linker-drug. The results shown in Tables 8, 9 (mice) and 10 (monkeys) indicate that ADCs are very stable, have a long half-life, and are removed slowly.

[0154] Table 8

[0155] Table 9

[0156] Table 10

[0157]

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[0158]

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[0159]

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[0160]

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change

[10] An antibody or antigen-binding fragment thereof as described in any one of [1] to [5], an antibody-drug conjugate as described in any one of [6] to [8], or a pharmaceutical composition as described in [9], for use as a pharmaceutical.

[11] The antibody or antigen-binding fragment, antibody-drug conjugate or pharmaceutical composition described in

[10] for use in the treatment of c-Met-positive human solid tumors or hematopoietic malignancies with MET.

[12] The c-Met-positive human solid tumor is selected from the group consisting of breast cancer; brain tumor; head and neck cancer; thyroid cancer; salivary gland cancer; soft tissue sarcoma; eye cancer; esophageal cancer; gastric cancer; small intestine cancer; colorectal cancer; urothelial cell carcinoma; ovarian cancer; uterine cancer; endometrial cancer; cervical cancer; lung cancer (especially non-small cell lung cancer and small cell lung cancer); melanoma; liver cancer; pancreatic cancer; non-melanoma skin cancer; prostate cancer; germ cell cancer; and cancer of unknown primary origin, as described in

[11] .

[13] The hematopoietic malignancy caused by the MET is a malignant tumor of the lymphoid system, preferably a mature T and NK tumor, as described in

[11] , an antibody or antigen-binding fragment, antibody-drug conjugate or pharmaceutical composition.

[14] A combination of an antibody or antigen-binding fragment thereof as described in any one of [1] to [5], an antibody-drug conjugate as described in any one of [6] to [8], or a pharmaceutical composition as described in [9], and a therapeutic antibody, chemotherapeutic agent, and / or antibody-drug conjugate against cancer-related targets other than the c-Met antigen for use in the treatment of c-Met-positive human solid tumors.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to mesenchymal epithelial conversion factor (c-Met), wherein the amino acid sequence of the HC variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the LC variable region is shown in SEQ ID NO:

20.

2. The antibody according to claim 1, which is an intact IgG1 antibody.

3. The antigen-binding fragment according to claim 1, which is a Fab fragment.

4. An antibody-drug conjugate comprising the antibody or antigen-binding fragment according to claim 1, conjugated to a cytotoxic drug via a linker, preferably a cleavable linker.

5. The antibody-drug conjugate according to claim 4, wherein the cytotoxic drug is a duocalmycin derivative.

6. Formula (III) 【Chemistry 1】 (In the formula, Ab is an IgG1 antibody; the cytotoxic drug site-specifically binds to recombinant cysteine ​​at position 41 of HC via a linker, according to Kabat numbering.) The antibody-drug conjugate according to claim 4, as represented by [the specified formula].

7. A pharmaceutical composition comprising an antibody or its antigen-binding fragment according to any one of claims 1 to 3, or an antibody-drug conjugate according to any one of claims 4 to 6, and one or more pharmaceutically acceptable additives, preferably in the form of a lyophilized powder.

8. The pharmaceutical composition according to claim 7, for use in the treatment of c-Met-positive human solid tumors or hematopoietic malignancies caused by MET.

9. The pharmaceutical composition according to claim 8, wherein the c-Met-positive human solid tumor is selected from the group consisting of breast cancer; brain tumor; head and neck cancer; thyroid cancer; salivary gland cancer; soft tissue sarcoma; eye cancer; esophageal cancer; stomach cancer; small intestine cancer; colorectal cancer; urothelial cell carcinoma; ovarian cancer; uterine cancer; endometrial cancer; cervical cancer; lung cancer (especially non-small cell lung cancer and small cell lung cancer); melanoma; liver cancer; pancreatic cancer; non-melanoma skin cancer; prostate cancer; germ cell cancer; and cancer of unknown primary origin.

10. The pharmaceutical composition according to claim 8, wherein the hematopoietic tumor caused by MET is a malignant tumor of the lymphatic system, preferably a mature T and NK tumor.

11. A combination pharmaceutical product comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or an antibody-drug conjugate according to any one of claims 4 to 6, and a therapeutic antibody, chemotherapeutic agent, and / or antibody-drug conjugate against cancer-related targets other than the c-Met antigen for use in the treatment of c-Met-positive human solid tumors.

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