WO2019212253A1 - Antibody specifically binding to c-met, and use thereof - Google Patents

Abstract

The present invention relates to an anti-c-Met antibody that is cross-reactive to human and mouse c-Met, and a use thereof and, more specifically, to: an anti-c-Met antibody or an antigen-binding fragment thereof; a bispecific antibody or an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof; a pharmaceutical composition for preventing or treating cancer, comprising same; a nucleic acid coding for the antibody or an antigen-binding fragment thereof; a vector and a host cell comprising the nucleic acid; a method for preparing an anti-c-Met antibody or an antigen-binding fragment thereof by using same; and a co-administered cancer treatment composition containing the anti-c-Met antibody or an antigen-binding fragment thereof and another cancer treatment agent. An antibody binding to c-Met and an antigen-binding fragment thereof, according to the present invention, can bind to human and mouse c-Met with high affinity, and thus more accurate preclinical results can be identified in an efficacy evaluation by using mouse tumor models. An antibody binding to c-Met and an antigen-binding fragment thereof, according to the present invention, can be effectively used in the prevention or treatment of target cancer.

Description

Antibodies that specifically bind to C-MET and uses thereof

The present invention relates to anti-c-Met antibodies cross-linking to human and mouse c-Met and uses thereof, and more particularly to anti-c-Met antibodies or antigen binding fragments thereof, such antibodies or antigen binding fragments thereof. Bispecific antibodies or antibody-drug conjugates comprising, pharmaceutical compositions for the prevention or treatment of cancer comprising the same, nucleic acids encoding the antibodies or antigen-binding fragments thereof, vectors and host cells comprising the nucleic acids, anti-c using the same A method for producing a -Met antibody or antigen-binding fragment thereof, and a combination dosage composition for treating cancer, comprising the anti-c-Met antibody or antigen-binding fragment thereof and other cancer therapeutic agents.

Various growths such as hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) Factors interact with receptor tyrosine kinases (RTKs) on the cell surface to induce important cytophysiological regulation, such as cell growth, differentiation, angiogenesis, tissue regeneration, as well as developmental stages. When these growth factors and receptors are deregulated in physiological aspects such as mutation, overexpression, and self-activation, they initiate and promote cancer development by causing abnormal cell growth or differentiation (Lemmon MA & Schlessinger J, Cell. 141: 1117-1134, 2010).

Met proto-oncogene (c-Met) protein is known to be a proto-oncogene that expresses hepatocyte growth factor (HGF) / scatter factor (SF) receptors (Dean M et al., Nature. 318: 385). -388, 1985, Gherardi et al., Nat. Rev. Cancer. 12: 89-103, 2012), interact with HGF, the only known ligand, to induce mesenchymalepithelial transition (MET), Promote the growth, penetration and metastasis of cancer cells. In the case of c-Met, it is considered to be an effective anticancer target because it is involved in the mechanism of development, metastasis, invasion, and neovascularization regardless of HGF, which is a ligand during the development of various tumors. The research on c-Met inhibitors such as is active (Comoglio PM et al., Nat. Rev. Drug. Discov. 7: 504-516, 2008).

The development of antagonistic antibodies against the anticancer target c-Met is a representative chemotherapy strategy by c-Met inhibition. In the case of anti-c-Met antibodies, it has been reported to inhibit the interaction of the ligand HGF and c-Met or to deactivate c-Met by inactivation. For example, the 'OA-5D5' one-armed antagonistic antibody developed as an anti-c-Met antibody is modified to have no side effects that induce c-Met dimerization as an agonist. It was developed as an antibody (Martens T et al., Clin. Cancer Res. 15: 6144-6152, 2006), and 'DN30' induces the inhibition of tumorigenesis by inactivating c-Met itself and losing its function. (Petrelli A et al., PNAS. 103: 5090-5095, 2006). However, single-armed antagonist antibody showed only minimal tumor suppression effect by treatment with antibody alone, but showed significant therapeutic effect when treated with chemotherapy, while c-Met inactivated antibody had low competition with ligand and agonist. It was confirmed that there is a drawback that shows the effect as) partially. Therefore, there is a continuous need for the development of therapeutic antibodies that inhibit the function of human c-Met.

In the development of anti-cancer target antibodies, in vivo preclinical efficacy evaluation using mouse tumor model as well as in vitro efficacy evaluation is required. In particular, the preclinical test results such as the ability to reduce tumor size and increase the number of days of survival, which can be identified when evaluating efficacy using a mouse tumor model, will mainly determine the therapeutic efficacy of the antibody. The mouse tumor model used at this time is made by injecting human-derived cancer cells that overexpress anticancer targets. Actually, the tumor microenvironment in mice is caused by the interference of not only the injected human tumor cells but also the mixed mouse-derived cells. In confirming the therapeutic effect of antibodies, there is a high probability that the correlation between preclinical and clinical results is low (Talmadge JE et al., Am. J. Pathol. 170: 793-804, 2007). Thus, not only antibodies that inhibit only human-derived anticancer targets, but also combination treatments with mouse-derived anticancer targets or antibodies that inhibit ligands, or antibodies specific for human / mouse heterologous anticancer targets, are more accurate in preclinical treatment in tumor models. You can show the result. For example, in the case of anti-Dll4 (delta like ligand 4) antibodies that inhibit angiogenesis in tumors, the antibody against mouse Dll4 as well as the antibody to human Dll4 is significantly combined with the mouse tumor model. Tumor size reduction has been reported (Hoey T et al., Cell Stem Cell. 5: 168-177, 2009). In addition, in the case of antibodies targeting vascular endothelial growth factor receptor 2 (VEGFR-2) or vascular endothelial growth factor (VEGF), antibodies that cross-reactive with human / mouse heterologous anticancer targets are mouse tumor models. The high tumor suppression effect suggested the need for cross-reactive antibody development (Huang J et al., Cytotechnology. 62: 61-71, 2010; Liang WC et al., J. Biol. Chem. 281: 951-). 961, 2006).

As such, anti-c-Met antibodies that inhibit only c-Met function have no mouse c-Met receptor inhibitory action on autocrine / paracrine action of human or mouse hepatocyte growth factor. There is a difficulty in evaluating the effects of preclinical efficacy evaluation in mouse tumor models. Human c-Met (P08581, UniProtKB / Swiss-Prot) consists of 1,390 amino acids and mouse c-Met (P16056, UniProtKB / Swiss-Prot) consists of 1,379 amino acids, which have more than 89% high sequence similarity with each other (Chan AML et al., Oncogene. 2: 593-599, 1988). Hepatocyte growth factor, a ligand, also has a very high sequence similarity of over 90% between humans and mice (Tashiro K et al., PNAS. 87: 3200-3204, 1990). Since it is a Sema domain, there is a high possibility of developing and applying a cross-reactive antibody. Therefore, the development of antibodies that cross-react to human / mouse c-Met, which inhibits cancer-specific ligand-receptor action in the tumor tumor microenvironment against human / mouse c-Met, confirms effective preclinical studies in mouse tumor models. need.

Under these technical backgrounds, the present inventors have developed an improved antibody having cross-linking ability to human and mouse c-Met, improved affinity with c-Met using affinity maturation process, and completed the present invention. .

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms a prior art known to those of ordinary skill in the art. You may not.

Summary of the Invention

It is an object of the present invention to provide an anti-c-Met antibody or antigen binding fragment thereof that specifically binds to c-Met.

Another object of the present invention is to provide a bispecific antibody or antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof.

It is another object of the present invention to provide a pharmaceutical composition and a method for treating or preventing cancer comprising the anti-c-Met antibody or antigen-binding fragment thereof and / or the bispecific antibody or antibody-drug conjugate. .

Still another object of the present invention is a nucleic acid encoding the anti-c-Met antibody or antigen-binding fragment thereof, a vector and host cell comprising the nucleic acid, a method for producing an anti-c-Met antibody or antigen-binding fragment thereof using the same. To provide.

It is another object of the present invention to provide a combination dosage composition for treating cancer and a method of treatment comprising the anti-c-Met antibody or antigen-binding fragment thereof and other cancer therapeutic agents.

Another object of the present invention is to provide the use of the antibody or antigen-binding fragment thereof or the bispecific antibody or the antibody-drug conjugate for the treatment of cancer.

Another object of the present invention is to provide the use of the antibody or antigen-binding fragment thereof or the bispecific antibody or the antibody-drug conjugate for the manufacture of a medicament for the treatment of cancer.

In order to achieve the above object, the present invention provides a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 27; A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-31; A heavy chain variable region comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 32 and 33; And a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 34 and 35; Light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 36 and 37; An anti-c-Met antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 38 and 39 is provided.

The present invention also provides a bispecific antibody or antibody-drug conjugate comprising said anti-c-Met antibody or antigen binding fragment thereof.

The present invention also provides pharmaceutical compositions and methods for the treatment or prevention of cancer comprising the anti-c-Met antibody or antigen-binding fragment thereof and / or the bispecific antibody or antibody-drug conjugate.

The present invention also provides a nucleic acid encoding the anti-c-Met antibody or antigen-binding fragment thereof, a vector and host cell comprising the nucleic acid, a method for producing an anti-c-Met antibody or antigen-binding fragment thereof using the same. do.

The present invention also provides a combination dosage composition for treating cancer and a method of treatment comprising the anti-c-Met antibody or antigen-binding fragment thereof and other cancer therapeutic agents.

The present invention also provides a method for treating cancer, characterized in that the antibody or antigen-binding fragment thereof or the bispecific antibody or the antibody-drug conjugate is administered.

The present invention also provides the use of the antibody or antigen-binding fragment thereof or the bispecific antibody or the antibody-drug conjugate for the treatment of cancer and the antibody or antigen-binding fragment thereof or the bispecific antibody for the manufacture of a medicament for the treatment of cancer. Or the use of such antibody-drug conjugates.

1 shows the heavy chain variable region sequence and CDR / Framework classification of the parent antibody (1F12).

2 shows the light chain variable region sequences and CDR / Framework classification of the parent antibody (1F12).

3 is a mutant library design of anti-c-Met antibodies.

Figure 4 shows the primer sequence for constructing a mutant library of anti-c-Met antibody.

5 shows the CDR sequences of anti-c-Met antibody affinity variants.

Figure 6 shows the results of the agonist activity analysis of 16 anti-c-Met antibody affinity variants.

Figure 7 shows affinity results of the anti-c-Met antibody.

Figure 8 shows the results of gastric cancer cell line growth inhibition pattern analysis.

Figure 9 shows the results of the combined efficacy analysis with anti-c-Met antibody alone and immunotherapy in colorectal cancer cell line.

Figure 10 shows the results of analyzing the combined efficacy of anti-c-Met antibody and radiation therapy in colorectal cancer cell line.

Figure 11 shows the results of analysis of immune cell distribution in colorectal cancer subcutaneous animal model.

12 shows the change of PD-L1 expression in tumor cells after administration of anti-c-Met antibody in colorectal cancer subcutaneous animal model.

Detailed Description of the Invention and Preferred Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Application of antibody engineering technology is required to improve antibody efficacy. Among them, there is an affinity maturation method for maturing the affinity of the antibody for the antigen. Affinity maturation refers to a technique for increasing the binding affinity of the antibody to the antigen by introducing a random mutation into the antibody gene, it can be very useful for the development of effective therapeutic and diagnostic antibody drugs. For in vitro affinity maturation, three approaches are commonly used. Error prone PCR, randomization of target residues using degraded oligonucleotides, and chain shuffling. The portion that can be selected as a target residue is the complementarity determining region (CDR), which in particular is a logical target for randomization since CDR-H3 and CDR-L3 tend to dominate antibody-antigen interactions. The binding affinity of an antibody is improved by changing the amino acid in the target antibody gene CDR region. This method has been reported to increase the binding affinity by 22 times by changing the amino acid in CDR-H3 of AKA (humanized antibody binding to tumor-associated glycoprotein 72) (Hong et al., J. Biol. Chem. 2006, 281, 6985-6992), antibodies developed against the Hepatitis B virus antigen also reported a 6-fold increase in binding affinity (Hong el al., J. Microbiol. 2007, 45, 528-533).

In one aspect, the present invention relates to an anti-c-Met antibody or antigen-binding fragment thereof that specifically binds to c-Met. Preferably, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 27; A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-31; A heavy chain variable region comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 32 and 33; And a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 34 and 35; Light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 36 and 37; An anti-c-Met antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 38 and 39.

In the present invention, the anti-c-Met antibody or antigen-binding fragment thereof is a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 27; A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-31; Heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 32 and 33, each having at least 80% sequence homology, preferably at least 90% sequence homology, more preferably 99% sequence homology An antibody or antigen-binding fragment thereof comprising the heavy chain variable region comprising a sequence and having the same properties as c-Met according to the present invention is also included in the scope of the anti-c-Met antibody or antigen-binding fragment thereof according to the present invention. do.

In addition, light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 34 and 35; Light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 36 and 37; Each having at least 80% sequence homology, preferably at least 90% sequence homology, and more preferably 99% sequence homology with a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 38 and 39 An antibody or antigen-binding fragment thereof comprising the light chain variable region comprising a sequence and having the same properties as c-Met according to the present invention is also included in the scope of the anti-c-Met antibody or antigen-binding fragment thereof according to the present invention. do.

In the present invention, the anti-c-Met antibody or antigen-binding fragment thereof is a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 40 and 42 to 48 and in the group consisting of SEQ ID NO: 41 and 49 to 54 It may be characterized by including the selected light chain variable region.

Meanwhile, at least 80% sequence homology, preferably at least 90% sequence homology, and more preferably 99% sequence homology with a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 and 42 to 48 An antibody or antigen-binding fragment thereof comprising the sequence having the same properties as c-Met according to the present invention is also included in the scope of the anti-c-Met antibody or antigen-binding fragment thereof according to the present invention.

A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 and 49 to 54 and having at least 80% sequence homology, preferably at least 90% sequence homology, more preferably 99% An antibody or antigen-binding fragment thereof comprising the light chain variable region comprising a sequence and having the same properties as c-Met according to the present invention is also included in the scope of the anti-c-Met antibody or antigen-binding fragment thereof according to the present invention. do.

In addition, the anti-c-Met antibody or antigen-binding fragment thereof according to the present invention, the anti-c-Met antibody or antigen-binding fragment thereof according to the present invention, an antibody or a portion thereof in which a part of the amino acid sequence is substituted through conservative substitution Antigen binding fragments are also included.

As used herein, “conservative substitutions” refers to modifications of a polypeptide comprising replacing one or more amino acids with amino acids having similar biochemical properties that do not cause loss of the biological or biochemical function of the polypeptide. A “conservative amino acid substitution” is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. A class of amino acid residues with similar side chains is defined in the art and is well known. These classes include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains Amino acids (eg threonine, valine, isoleucine) and amino acids having aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). It is anticipated that the antibodies of the invention may have conservative amino acid substitutions and still retain activity.

As used herein, the term “c-Met specific antibody” refers to an antibody that binds to c-Met resulting in inhibition of the biological activity of c-Met, and is used interchangeably with “anti-c-Met antibody”.

In the present invention, the "anti-c-Met antibody" is a concept including both a polyclonal antibody and a monoclonal antibody (monoclonal antibody, monoclonal antibody), preferably a monoclonal antibody, intact whole antibody ( whole antibody). The whole antibody is a structure having two full-length light chains and two full-length heavy chains, and includes a constant region, and each light chain is connected by heavy and disulfide bonds.

The total antibody of the anti-c-Met antibody according to the present invention includes the IgA, IgD, IgE, IgM and IgG forms, wherein IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.

The anti-c-Met antibody according to the invention is preferably, but not limited to, a fully human antibody selected from a human antibody library.

“Antigen binding fragment” of an anti-c-Met antibody according to the present invention means a fragment having the function of binding to the antigen of the anti-c-Met antibody, ie c-Met, Fab, Fab ', As a concept including F (ab ') ₂ , scFv (scFv) ₂ , scFv-Fc, Fv, and the like, the term “antibody fragment” is used interchangeably herein.

The Fab has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain, and a first constant region of the heavy chain (CH1 domain). Fab 'differs from Fab in that it has a hinge region comprising at least one cysteine residue at the C terminus of the heavy chain CH1 domain. F (ab ') ₂ antibody Fab' is a cysteine residue of the hinge region is generated yirumyeonseo a disulfide bond.

The variable fragment (Fv) refers to a minimum antibody fragment having only a heavy chain variable region and a light chain variable region. Double-chain Fv (dsFv) is a disulfide bond, which is linked to a heavy chain variable region and a light chain variable region, and short-chain Fv (scFv) is generally covalently linked to a heavy chain variable region and a light chain variable region through a peptide linker. . Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab, cleavage with pepsin yields F (ab ') ₂ fragments), Genetic recombination techniques (e.g., DNA encoding the heavy chain or variable region thereof and DNA encoding the light chain or variable region thereof as a template, and amplified by PCR (Polymerase Chain Reaction) method using primer pairs , Amplification by combining a DNA linking a peptide linker and a primer pair such that both ends are linked to a heavy chain or a variable region and a light chain or a variable region, respectively.

As used herein, the term “heavy chain” refers to a variable length domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 It means all fragments. The term “light chain” herein also refers to both the full-length light chain and fragment thereof including the variable region domain VL and the constant region domain CL, including the amino acid sequence having sufficient variable region sequence to confer specificity to the antigen.

As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequences of the hypervariable regions of immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (CDRH1, CDRH2 and CDRH3) and light chains (CDRL1, CDRL2 and CDRL3) each contain three CDRs. CDRs provide key contact residues for the antibody to bind antigen or epitope.

In the present invention, the anti-c-Met antibody or antigen-binding fragment thereof may be characterized by having a specific binding capacity to human c-Met. Preferably, it may be characterized by having cross-linking ability with respect to human c-Met and mouse c-Met, but is not limited thereto.

In another aspect, the present invention relates to an antibody-drug conjugate (ADC) in which a drug is conjugated to the anti-c-Met antibody or an antigen-binding fragment thereof.

Antibody-drug conjugates (ADCs) require the anticancer drug to be stably bound to the antibody until the anticancer drug is delivered to the target cancer cell. Drug delivered to the target must be released from the antibody to induce killing of the target cell. This requires that the drug binds to the antibody stably and at the same time has sufficient cytotoxicity to induce the death of the target cell when released from the target cell.

In the present invention, the cytotoxic substances including drugs such as the anti-c-Met antibody or antigen-binding fragment thereof and an anticancer agent are bound to each other (eg, by covalent bonds, peptide bonds, etc.) to conjugate or fusion proteins ( In the form of cytotoxic substances and / or markers). The cytotoxic substance may be any substance that is toxic to cancer cells, particularly solid cancer cells, and may be one or more selected from the group consisting of radioisotopes, cytotoxic compounds, cytotoxic proteins, anticancer agents, and the like. It is not limited to this. The cytotoxin protein is selected from the group consisting of lysine (ricin), saporin (saporin), gelonin (gelonin), momordin (momordin), deboganin (debouganin), diphtheria toxin, pseudomonas toxin, etc. It may be one or more, but is not limited thereto. The radioisotope may be at least one selected from the group consisting of 131I, 188Rh, 90Y, and the like, but is not limited thereto. The cytotoxin compound is duocarmycin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), N2'-diacetyl-N2 '-( 3-mercapto-1-oxopropyl) maytansine (N2'-deacetyl-N2 '-(3-mercapto-1-oxopropyl) maytansine; DM1), PBD (Pyrrolobenzodiazepine) dimer, and the like. However, it is not limited thereto.

In the present invention, the antibody-drug conjugate may be according to techniques well known in the art.

In the present invention, the antibody-drug conjugate may be characterized in that the antibody or antigen-binding fragment thereof is bound to the drug through a linker.

In the present invention, the linker may be a cleavable linker or a non-cleavable linker.

The linker is a linking site between the anti-c-Met antibody and the drug, for example the linker is in a form that is cleavable under intracellular conditions, i.e. the drug is released from the antibody through cleavage of the linker in the intracellular environment. do.

The linker may be cleaved by a cleavage agent present in an intracellular environment such as a lysosomal or endosome, and may be a peptide linker that may be cleaved by an intracellular peptidase or protease enzyme such as a lysosomal or endosomal protease. . Peptide linkers generally have at least two amino acids in length. The cleavage agent may include cathepsin B and cathepsin D, plasmin, and may hydrolyze the peptide to release the drug into target cells. The peptide linker may be cleaved by thiol dependent protease cathepsin-B, which is highly expressed in cancer tissue, for example Phe-Leu or Gly-Phe-Leu-Gly linkers can be used. In addition, the peptide linker may be cleaved by, for example, an intracellular protease, and may be a Val-Cit linker or a Phe-Lys linker.

In the present invention, the cleavable linker is pH sensitive, and may be sensitive to hydrolysis at a specific pH value. In general, it is shown that pH sensitive linkers can be hydrolyzed under acidic conditions. For example, acid labile linkers that can be hydrolyzed in lysosomes such as hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, Ketal and the like.

The linker may be cleaved under reducing conditions, for example disulfide linkers. N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl-3- (2-pyridyldithio) butyrate (SPDB), and N-succinimidyl-oxycarbonyl SMPT Various alpha disulfide bonds can be formed using -alpha-methyl-alpha- (2-pyridyl-dithio) toluene).

In the present invention, the drug and / or drug-linker may be conjugated randomly through lysine of the antibody or through cysteine which is exposed when the disulfide bond chain is reduced. In some cases, the linker-drug may be bound via a genetically engineered tag, such as cysteine present in a peptide or protein. The genetically engineered tag, eg, peptide or protein, may comprise an amino acid motif that can be recognized by, for example, an isoprenoid transferase. The peptide or protein has a deletion at the carboxy terminus of the peptide or protein, or has an addition via covalent attachment of a spacer unit to the carboxy (C) terminus of the peptide or protein. The peptide or protein may be directly covalently linked to an amino acid motif or covalently linked to a spacer unit to be linked to an amino acid motif. The amino acid spacer unit is composed of 1 to 20 amino acids, of which a glycine unit is preferable.

The linker may comprise a beta-glucuronide linker which is present in a large number in lysosomes or is hydrolyzed by beta-glucuronidase which is overexpressed in some tumor cells. Unlike the peptide linker, the hydrophilicity is high, and when combined with drugs having high hydrophobic properties, the solubility of the antibody-drug complex can be increased.

In this regard, the present invention relates to a beta-glucuronide linker disclosed in Korean Patent Publication No. 2015-0137015, for example, a beta-glucuronide linker comprising a self-immolative group. Can be used.

In addition, the linker may be, for example, a non-cleavable linker, and the drug is released through only one step of antibody hydrolysis to produce, for example, an amino acid-linker-drug complex. This type of linker may be a thioether group or maleimidocaproyl, and may maintain stability in blood.

In the present invention, the drug may be characterized as a chemotherapeutic agent, toxin, micro RNA (miRNA), siRNA, shRNA or radioisotope. The drug may be bound to the antibody with an agent that exhibits a pharmacological effect.

The chemotherapeutic agent may be a cytotoxic agent or an immunosuppressant. Specifically, it may include a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can function as a DNA intercalator. It may also include immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents or combinations thereof.

Such drugs include, for example, maytansinoids, orstatin, aminopterin, actinomycin, bleomycin, thalidomide, camptocecin, N8-acetyl spermidine, 1- (2 chloroethyl) -1,2- Dimethyl sulfonyl hydrazide, esperamycin, etoposide, 6-mercaptopurine, dolastatin, tricortesene, calicheamicin, taxol, taxanes, paclitaxel, docetaxel, methotrexate, Vincristine, vinblastine, doxorubicin, melphalan, chlorambucil, duocarmycin, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea Cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin; mitomycin; mitomycin A, Mitomycin C), Dhaka Dacarbazine, procarbazine, topotecan, topotecan, nitrogen mustard, cytoxan, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan ), Camptothecin, idarubicin, daunorubicin, dactinomycin, plicamycin, asparaginase, vinorelbine, Chlorambucil, melphalan, carmustine, lomustine, busuLfan, treosulfan, decarbazine, teniposide , Topotecan, 9-aminocamptothecin, 9-aminocamptothecin, crisnatol, trimmetrexate, mycophenolic acid, tiazofurin, ribavirin ( ribavirin), EICAR (5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), hydroxyurea Hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytosine arabinoside (cytosine) arabinoside, fludarabine, tamoxifen, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide , Bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, deferon-hypocrellin A Interferon-α, Interferon-γ, tumor necrosis factor, gemcitabine, velcade, Revlimid, lovastatin, 1 -Methyl-4-phenylpyridinium ion (1-methyl-4-phenylpyridiniumion), staurosporine , Actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, bleomycin B2, peplomycin, epirubicin, pyrubicin pirarubicin, zorubicin, mitosantron, mitoxantrone, verapamil and thapsigargin, nucleic acid degrading enzymes and toxins derived from bacteria or plants, but not limited thereto. It doesn't happen.

In the present invention, the drug is an amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate which can react to form covalent bonds with electrophilic groups on linkers and linker reagents. And one or more nucleophilic groups selected from the group consisting of arylhydrazide groups.

In another aspect, the present invention relates to a bispecific antibody comprising the anti-c-Met antibody or antigen-binding fragment thereof.

In the present invention, the bispecific antibody is one of two arms of the antibody, one arm comprises an anti-c-Met antibody or antigen-binding fragment thereof according to the invention, and the other cancer ( arm) comprises a form comprising an antibody specific for an antigen other than c-Met, preferably an cancer-associated antigen or an immune gateway protein antigen, or an antibody or antigen-binding fragment thereof that specifically binds to an immune cell-associated antigen. it means.

The antigen to which the antibody other than the anti-c-Met antibody included in the double antibody binds is preferably a cancer-associated antigen or an immune gateway protein antigen, HGF, EGFR, EGFRvIII, Her2, Her3, IGF-1R, VEGF, VEGFR. -1, VEGFR-2, VEGFR-3, Ang2, Dll4, NRP1, FGFR, FGFR2, FGFR3, c-Kit, MUC1, MUC16, CD20, CD22, CD27, CD30, CD33, CD40, CD52, CD70, CD79, DDL3 , Folate R1, Nectin 4, Trop2, gpNMB, Axl, BCMA, PD-1, PD-L1, PD-L2, CTLA4, BTLA, 4-1BB, ICOS, GITR, OX40, VISTA, TIM-3, LAG-3 , KIR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, EphA2, EphA4, EphB2, E-selectin, EpCam, CEA, PSMA, PSA, c-MET, and the like, and TCR / CD3, CD16 (FcγRIIIa) CD44, CD56, CD69, CD64 (FcγRI), CD89, CD11b / CD18 (CR3), and the like, may be selected as antigens related to immune cells. However, the present invention is not limited thereto.

In another aspect, the present invention relates to a pharmaceutical composition for preventing and / or treating cancer comprising the anti-c-Met antibody or antigen-binding fragment thereof.

In another aspect, the present invention relates to a pharmaceutical composition for preventing and / or treating cancer comprising the bispecific antibody or antibody-drug conjugate.

In the present invention, the cancer may be related to the expression or overexpression of c-Met.

In the present invention, "cancer" and "tumor" are used in the same sense and refer to or mean the physiological state of a mammal, which is typically characterized by unregulated cell growth / proliferation.

In the present invention, the anti-c-Met antibody inhibits the growth of cancer cells derived from various carcinomas due to high anti-c-Met binding and thus suppression of c-Met function, and the Inhibition of phosphorylation inhibits c-Met signaling and inhibits neovascularization. Therefore, the antibodies of the present invention are very effective for the prevention and treatment of cancer.

The cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited and includes both solid and hematological cancers. Examples of such cancers include breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, ovarian cancer, rectal cancer, colon cancer, vaginal cancer, small intestine cancer, endocrine Cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone marrow cancer, but is not limited thereto. The cancer may be primary or metastatic cancer. More preferably, the cancer that can be prevented or treated by the pharmaceutical composition may be characterized as a c-Met expressing cancer.

In the present invention, the pharmaceutical composition may be used in combination treatment with radiation.

In another aspect, the present invention requires a therapeutically effective amount of the anti-c-Met antibody or antigen-binding fragment thereof and / or the bispecific antibody or antibody-drug conjugate, for the prevention and / or treatment of c-Met related diseases. It relates to a method of preventing and / or treating c-Met-related diseases, comprising administering to a patient. The prevention and / or treatment method may further comprise identifying a patient in need of prevention and / or treatment of the disease prior to the administering step.

In the present invention, the method of treatment comprises the steps of administering a pharmaceutical composition comprising an anti-c-Met antibody or antigen binding fragment thereof; And irradiating the radiation; may be characterized in that it comprises a.

The radiation may be characterized in that the irradiation (irradiation) is 2Gy ~ 10Gy, but is not limited thereto.

In the method of the present invention, the number of times of radiation treatment and the amount of time between the radiation treatment and the administration of the pharmaceutical composition may vary according to the method of the present invention, but preferably the administration of the pharmaceutical composition or the administration of the pharmaceutical composition simultaneously with the irradiation. It may be characterized by irradiating radiation after 10 to 20 days, but is not limited thereto.

In another aspect, the present invention relates to the use of said antibody or antigen-binding fragment thereof, said bispecific antibody or antibody-drug conjugate for the treatment of cancer.

In another aspect, the invention relates to the use of said antibody or antigen-binding fragment thereof said bispecific antibody or antibody-drug conjugate for the manufacture of a medicament for the treatment of cancer.

In the pharmaceutical composition, treatment method and use according to the present invention, the anti-c-Met antibody or antigen-binding fragment thereof is provided as a single active ingredient, administered in combination with a cytotoxic substance such as an anticancer agent, or a cell such as an anticancer agent. It may be provided in the form of a conjugate (antibody-drug conjugate (ADC)) conjugated with a toxic substance.

The anti-c-Met antibody or antigen-binding fragment thereof according to the present invention, and a pharmaceutical composition comprising the same, can be used for use in combination with a conventional therapeutic agent. That is, the anti-c-Met antibody or antigen-binding fragment thereof, and the pharmaceutical composition comprising the same according to the present invention may be used at the same time or sequentially administered with a conventional therapeutic agent such as an anticancer agent.

In the present invention, the pharmaceutical composition may be characterized by comprising a therapeutically effective amount of an anti-c-Met antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" is a substance that can be added to the active ingredient to help formulate or stabilize the formulation and does not cause significant deleterious toxic effects on the patient. Pharmaceutically acceptable carriers are conventionally used in the formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

The pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the invention may be administered parenterally, for example by intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion and the like.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, and usually The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to prevent or treat cancer.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or by incorporating into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, suppositories, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

In another aspect, the invention relates to a nucleic acid encoding an anti-c-Met antibody according to the invention. As used herein, nucleic acids may be present in cells, cell lysates, or in partially purified or substantially pure form. Nucleic acids are prepared by other cellular components or other contaminants, for example, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. When purified from nucleic acids or proteins of other cells, they are "isolated" or "substantially pure." Nucleic acids of the invention may be, for example, DNA or RNA, and may or may not include intron sequences.

In the present invention, the nucleic acid encoding the anti-c-Met antibody may be characterized in that it comprises a sequence selected from the group consisting of SEQ ID NO: 55 to 69. Specifically, the polynucleotide sequence encoding the heavy chain of the antibody according to the present invention is SEQ ID NO: 55 or 57 to 63 and / or the polynucleotide sequence encoding the light chain of the antibody according to the present invention is SEQ ID NO: 56 or 64 to 69.

In another aspect, the present invention relates to a recombinant expression vector comprising the nucleic acid. For the expression of anti-c-Met antibodies or antigen-binding fragments thereof according to the invention, DNA encoding partial or full-length light and heavy chains can be prepared using standard molecular biology techniques (e.g., PCR amplification or hybrid expression of the desired antibody). CDNA cloning using a cutting board), and DNA can be “bind to work” into transcription and translation control sequences and inserted into the expression vector.

As used herein, the term “binding to work” may mean that the gene encoding the antibody is ligated into the vector such that the transcriptional and translational control sequences in the vector serve the intended function of regulating the transcription and translation of the antibody gene. . The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The light chain gene of the antibody and the heavy chain gene of the antibody are inserted into separate vectors, or both genes are inserted into the same expression vector. Antibodies are inserted into expression vectors by standard methods (eg ligation of complementary restriction enzyme sites on antibody gene fragments and vectors, or blunt terminal ligation if no restriction enzyme sites are present). In some cases, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide binds to the amino terminus of the antibody chain gene in frame. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide derived from an immunoglobulin non-protein). In addition, the recombinant expression vector has a regulatory sequence that controls the expression of the antibody chain gene in the host cell. A “regulatory sequence” can include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Those skilled in the art can recognize that the design of the expression vector can vary by differently selecting regulatory sequences depending on factors such as the selection of host cells to be transformed, the expression level of the protein, and the like.

In another aspect, the present invention relates to a host cell comprising the nucleic acid or the vector. The host cell according to the present invention is preferably selected from the group consisting of animal cells, plant cells, yeast, E. coli and insect cells, but is not limited thereto.

Specifically, the host cell according to the present invention is E. coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphyllo Prokaryotic cells, such as the Staphylococcus sp. Also, fungi such as Aspergillus sp., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. And Neuro Eukaryotic cells such as yeast, such as Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as cells from insects.

It can also be derived from plants or mammals. Preferably, monkey kidney cells (COS7) cells, NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells , MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells and the like are available, but are not limited to these. Especially preferably CHO cells can be used.

The nucleic acid or the vector is transfected or transfected into a host cell. Many different types of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for “transfection” or “transfection”, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection may be used. Various expression host / vector combinations can be used to express anti-glycancan 3 antibodies according to the present invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. . Expression vectors that can be used in bacterial hosts include broader hosts such as bacterial plasmids derived from Escherichia coli, such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, and derivatives thereof. Plasmids with ranges, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as λgt10 and λgt11, NM989, and other DNA phages such as M13 and filamentary single-stranded DNA phages. Useful expression vectors for yeast cells are 2 ° C. plasmids and derivatives thereof. A useful vector for insect cells is pVL941.

In another aspect, the present invention provides a method for producing an anti-c-Met antibody or antigen-binding fragment thereof, comprising the step of culturing the host cell to express an anti-c-Met antibody or antigen-binding fragment thereof according to the present invention. It is about.

When a recombinant expression vector capable of expressing the anti-c-Met antibody or antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody is for a period of time sufficient to allow the antibody to be expressed in the host cell, or more preferably in the host cell. Can be prepared by culturing the host cell for a period sufficient to allow the antibody to be secreted into the culture medium in which is cultured.

In some cases, the expressed antibody may be purified from the host cell to be homogeneous. Separation or purification of the antibody can be carried out by separation, purification methods, such as chromatography, which are used in conventional proteins. The chromatography can include, for example, affinity chromatography comprising a Protein A column, Protein G column, ion exchange chromatography or hydrophobic chromatography. In addition to the above chromatography, the antibody can be separated and purified by further combining filtration, ultrafiltration, salting out, dialysis and the like.

The anti-c-Met antibody or antigen-binding fragment thereof and the pharmaceutical composition comprising the same according to the present invention can be used for use in combination with a conventional therapeutic agent.

Thus, in another aspect, the present invention relates to a combination dosage composition for treating cancer and a method of treatment comprising the anti-c-Met antibody or antigen-binding fragment thereof and other cancer therapeutic agents.

The other cancer therapeutic agent means all therapeutic agents that can be used for cancer treatment, in addition to the anti-c-Met antibody or antigen-binding fragment thereof according to the present invention. In the present invention, the cancer treatment agent may be characterized in that the immune gateway inhibitor, but is not limited thereto.

The body's immune system has an immunoassay system to suppress the hyperimmune response caused by T-cell overproliferation. The immune checkpoint system is called an immune checkpoint, and the proteins involved in the immune checkpoint are called immune checkpoint proteins. In essence, immune gates function to inhibit hyper-immune responses caused by T-cell overactivation and / or hyperproliferation, but cancer cells exploit these immune barriers to prevent T-cells from attacking themselves. It is free from the attack, and ultimately the cancer progresses.

It is already known in the art to treat such diseases as cancer using inhibitors of such immune gates, and antibody pharmaceuticals targeting immunogate protein are commercially available and various immunogate inhibitors are under development.

The first type of immunoblock inhibitor, the therapeutic agent ipilimumab, a cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) -specific monoclonal antibody, has been shown to be effective in metastatic malignant melanoma. Subsequently, monoclonal antibodies specific for programmed cell death-1 (PD-1) and programmed death ligand-1 (PD-L1), ligands for PD-1, are being developed. Representative examples include nivolumab, pembrolizumab, avelumab, atezolizumab and durvalumab. PD-1 or PD-L1 inhibitors are found in malignant melanoma as well as in tumors whose effects vary.

In the present invention, the immune gateway inhibitor means an immune checkpoint inhibitor or a checkpoint inhibitor, and may be characterized in that the anti-CTLA-4 antibody, anti-PD-1 antibody or anti-PD-L1 antibody, but is not limited thereto. In particular, Ipilimumab, Nivolumab, Pembrolizumab, Atezlizumab, Avelumab or Durvalumab may be used. However, the present invention is not limited thereto.

In the present invention, the cancer is breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, ovarian cancer, rectal cancer, colon cancer, vaginal cancer, small intestine Cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer or bone marrow cancer, but is not limited thereto.

“Combination” means that the anti-c-Met antibody or antigen-binding fragment thereof and each of the other cancer therapeutic agents may be administered simultaneously, sequentially, or in reverse order, and administered in a combination of an appropriate effective amount within the scope of those skilled in the art. Can be.

In one embodiment of the present invention, when the anti-PD-L1 antibody and the anti-c-Met antibody according to the present invention in combination, it was confirmed that further suppress the growth of the tumor.

The combination dosage composition includes an anti-c-Met antibody, and the configuration thereof is the same as the composition included in the composition for preventing or treating cancer described above, and thus the description of each composition is equally applicable to the composition for combination administration. .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Mutation Library Construction for Affinity Maturation

The c-Met target antibody 1F12 was selected using phage display technology, and directed evolution was used to improve the affinity of the antibody. The variable region of the antibody is divided into a complementarity-determining region (CDR) and a framework region, and CDRs contribute greatly to the antigen-antibody binding. The heavy chain variable region and the light chain variable region of the parent antibody are as shown in FIGS. 1 and 2. The CDR and framework regions of antibodies were classified based on KABAT numbering (Table 1).

Ten mutant libraries (1F12-H1mut, 1F12-H2-1mut, 1F12-H2-2mut, 1F12-H3-) using NNK degenerate codons in a total of six CDRs in the variable region of the parent antibody (1F12) heavy and light chains 1mut, 1F12-H3-2mut, 1F12-L1-1mut, 1F12-L1-2mut, 1F12-L2mut, 1F12-L3-1mut, 1F12-L3-2mut) were prepared (FIG. 3). The DNA sequence of the 1F12 mutant single chain variable fragment (sfiI-VH-linker-VL-sfiI) in which the CDRs used for constructing mutant libraries were randomized was obtained by using overlapping extension PCR using primers shown in Table 2 below. .

Cysteine is present at VH99 and VH100d of heavy chain CDR-H3 (Fig. 1), and these two positions form an interchain disulfide bond, which stabilizes the structure of CDR-H3. Therefore, these two sites do not use NNK codons. TGT codons were used to retain the residues. pComb3X scFv expression vector (OmpA leader sequence-sfiI-VH-linker-VL-sfiI-His-HA-Amber codon-pIII) was linearized by removing the insert sequence using sfiI (NEB) restriction enzyme. Mutant libraries were constructed by inserting sfiI-VH-linker-VL-sfiI into which the position of each CDR was 20 amino acids by NNK degenerate codon into the linearized vector by using T4 ligase (NEB) into the linearized vector. Completed.

Example 2: Screening for Affinity Modified Antibodies

The constructed mutant libraries used TG1 E. coli as host cells, and had a transformant of about 3.10 x 1010. The mutant libraries were recovered in phage form, and the phage display technology was used to enrich the antibody pool with higher binding capacity to c-Met, and affinity variants were selected by screening using ELISA. For the first time, 13 affinity clones were selected, and the clones were 1F12_H35H, 1F12_H53D, 1F12_H57K, 1F12_H58D, 1F12_H60N, 1F12_H100eH, 1F12_H100hR 1F12_L26D, 1F12_L27BD, 1F12F1F1F12F. 1F12-H35H means that the amino acid at position 35 of the 1F12 heavy chain is replaced with Histidine (H), and 1F12_L26D means a mutation in which the amino acid at position 26 of the 1F12 light chain is substituted with Aspartic acid. These 13 affinity-mutant antibodies were constructed to produce production vectors for the production of full-length human IgG1 in mammalian cells, and were performed according to the manufacturer's manual using the Expi293 expression system (Gibco). As a result, 1F12_H100hR (a clone in which the amino acid at the heavy chain 100h position was replaced with Arginine in the parent antibody) did not all express the antibody in repeated preparation. Six heavy chain CDR residue-substituted antibodies and six light chain CDR residue-substituted antibodies of 1F12 were obtained, and four antibodies in combination thereof were further prepared (1F12_H2L3, 1F12_H2L6, 1F12_H3L5, 1F12_H6L5). 1F12_H2L3 is an antibody prepared by combining a heavy chain expression vector in which the heavy chain variable region position 53 is substituted with Aspartic acid and a light chain expression vector in which the light chain variable region position 50 is substituted with Glutamic acid (7 heavy chain mutation expression vectors and 6 The light chain mutant expression vectors of the species were prepared as the second and third expression vectors, respectively). In conclusion, in FIG. 5, except for 1F12_H100hR, antibodies were expressed and purified in mammalian cells and used in subsequent experiments.

The CDR sequences of a total of 17 improved antibodies are shown in Table 3, and the heavy and light chain variable region sequences are shown in Table 4. In addition, the polynucleotide sequence encoding each improved antibody is shown in Table 5.

Example 3: Agonist activity analysis

Agonist activity analysis is essential for the development of c-Met antibodies. Typical antibodies have a bivalent structure with two paratopes that recognize the target in Y-form. As a result, one c-Met antibody binds to two target antigens, c-Met, which induces agonist activity that induces c-Met dimerization, activating a lower signal transduction pathway. Genentech developed the 5D5 antibody using Hybridoma technology, but the antibody binds to c-Met and shows a similar effect to HGF / SF, the ligand of c-Met, resulting in agonist activity that enhances the signal transduction pathway. To minimize this phenomenon, the 5D5 antibody was modified to OA-5D5, a one-arm form, and agonist activity was minimized.

Akt phosphorylation was measured to quantify the agonist activity of the parental antibody 1F12 and 16 affinity variants. Specifically, Serum starvation proceeded for 24 hours when the Caki-1 renal cell carcinoma cell line reached a confluency of about 70% per well in RPMI1640 complete medium (+ 10% FBS) in a 96 well cell culture plate. The parent antibody and 16 affinity variants were treated. As a control, 5D5, a c-Met agonist antibody, OA-5D5 with minimal agonist activity, and HGF / SF (R & D systems), a ligand for c-Met, were used. PBS was treated with the same volume as the vehicle treated samples, antibodies were treated at 10 μg / mL, HGF / SF was treated at 50 ng / L. The treatment time of antibody and ligand was 30 minutes, and the experiment was carried out in three iterations. 30 minutes after each sample treatment, the cells were washed once with 1X PBS and cell lysis was performed using lysis buffer. Subsequently, Akt phosphorylation was measured using a PathScan® Phospho-Akt Sandwich ELISA Kit (Cell signaling technology) according to the manufacturer's manual. The luminescent signal for each well was measured, and the PBS treatment group was 0%, HGF / SF. Treatment groups were converted to 100% to quantify the agonist activity of each antibody. 5D5 c-Met agonist antibody induced 86.01% of Akt phosphorylation and showed agonist activity similar to that of HGF. The modified OA-5D5 one-armed monovalent antibody showed a 30.46% reduced agonist activity to minimize agonist activity. In comparison, agonist activity of the 1F12 parent antibody was found to be 18.23%, and agonist activity of the 16 affinity variants is shown in Table 6 below (FIG. 6).

Example 4: Affinity Assay

ELISA-based affinity analysis was performed in three iterations. A 96-well ELISA plate (Costar) was coated with recombinant human c-Met (Sino biological) at 50 ng / well overnight at 4 ° C. The next day after blocking for 1 hour with 3% skim milk solution, it was washed three times using 1X PBST (Cell signaling technology). Each antibody was diluted 1/2 to 200 nM in PBS (Gibco) and treated in each well in a volume of 100 μL and allowed to stand for 1 hour at room temperature. After washing three times using 1X PBST (Cell signaling technology), anti-human Fab-HRP (Thermo scientific) secondary antibody diluted in 3% skim milk solution at 1: 10000 was added to each well at 100 μL for 1 hour. It was allowed to stand at room temperature. Each well was washed 5 times using 1X PBST (Cell signaling technology), and then 100 μL of TMB solution (Thermo scientific) was treated to each well. The reaction was terminated by 100 μL of signaling technology). The OD450 was measured using a UV / VIS spectrophotometer, and normalization of this value resulted in an increase in the affinity of the 1F12_H2L3, 1F12_H2L6, 1F12_H3L5, and 1F12_H6L5 clones compared to the parent antibody (1F12), with the highest affinity of the 1F12_H3L5 clone. It was confirmed (FIG. 7).

Example 5 Analysis of Cancer Cell Growth Inhibition

MKN45 is a c-Met amplified gastric cancer cell line, obtained from JCRB Cell Bank (Japan), and cultured in a medium in which 10% FBS was added to RPMI1640 to maintain the cells. Onartuzumab (OA-5D5; monovalent c-Met Antibody, Genentech) was used as a control antibody, and an antibody expression vector was prepared based on published antibody sequences. Transient expression using the Expi293 expression system (Gibco) was performed, and purification was performed by Affinity chromatography equipped with Mabselect sure (GE) on AKTA avant (GE), and purity of 98% or higher was confirmed by SE-HPLC analysis. ELISA and SPR analysis confirmed the similarity with the literature. For cell growth inhibition assay, 3000 cells / well were aliquoted into a complete culture medium containing RPMN1640 + 10% FBS in a 96 well assay plate (Corning, 3610), the cells were cultured overnight, and then the medium was removed. Antibodies were diluted in complete culture medium up to 100/5 from 100 nM and treated with 100 μL of medium. After 72 hours, Cell Titer Glo (Promega) was treated with 100 μL in each well, and cell viability was analyzed with Infinite M200 Pro (TECAN). As a result, it was confirmed that the efficacy of the 1F12_H2L3, 1F12_H2L6, 1F12_H3L5 and 1F12_H6L5 antibody is better than the antibody (1F12) and the control antibody onartuzumab (FIG. 8).

Example 6: Assessment of Tumor Growth Inhibition in Animal Models

In order to evaluate the tumor growth inhibitory ability in the animal model transplanted with tumor cells, MC38 cells, which are mouse colon cancer tumor cells, were transplanted into mice to prepare tumor animal models and evaluated tumor growth inhibitory ability according to 1F12_H3L5 administration.

MC38 was prepared to have 200000 cells per 100 μL, using Hank's Salt (HBSS) solution (Gibco) and Basal Matrigel (Corning®) in a 1: 1 mixture. Prepared cells were transplanted using a 1cc syringe (26G) at 100 μL in the region of the right back of 7-8 week old C57BL / 6 female mice.

After transplanting the MC38 cell lines into mice, the administration of 1F12_H3L5 (20 mg / kg, ip) and Atezolizumab (5 mg / kg, ip) was started twice a week from the 5th day. To evaluate the responsiveness of co-administration, 1F12_H3L5 (20 mg / kg, ip) and Atezolizumab (5 mg / kg, ip) were co-administered with the single-administered group at 5 days after tumor transplantation. (20 mg / kg, ip) was first administered followed by Atezolizumab (5 mg / kg, ip). Tumor size was measured by calculating the long axis and short axis in millimeters (mm) using a caliper to calculate the formula ((long axis) × (short axis) ² × 0.5).

The results showed that no significant tumor suppression was observed in the MC38 tumor animal model following the administration of 1F12_H3L5 (20 mg / kg, ip), and about 40-50% of the tumor was suppressed in the Atezolizumab (5 mg / kg, ip) group. Could. In the co-administration group (1F12_H3L5 + Atezolizumab) in the same model, it was confirmed that tumors did not occur compared to the control group (day 22 after tumor transplantation). Subjects continued to observe no tumor formation.

Animal test results conducted in MC38 tumor animal model suggest that 1F12_H3L5 can be enhanced by co-administration of 1F12_H3L5 with Atezolizumab (PD-L1 inhibitor), which is known as a representative immune gateway inhibitor (FIG. 9).

Example 7 Evaluation of Combination Therapy Reactivity with Radiation Therapy in Animal Models

In order to evaluate the tumor growth inhibitory ability in the animal model transplanted with tumor cells, MC38 cells, which are mouse colon cancer tumor cells, were transplanted into mice to prepare tumor animal models and evaluated tumor growth inhibitory ability according to 1F12_H3L5 administration.

MC38 was prepared to have 200000 cells per 100 μL, wherein Hank's Salt (HBSS) solution (Gibco) and Basal Matrigel (Corning®) were used in a 1: 1 mixture. Prepared cells were transplanted using a 1cc syringe (26G) at 100 μL in the region of the right back of 7-8 week old C57BL / 6 female mice. Tumor size was measured by calculating the long axis and short axis in millimeters (mm) using a caliper to calculate the formula ((long axis) × (short axis) ² × 0.5). For radiotherapy, mice were anesthetized at 20 days after tumor implantation and irradiated with 2Gy single doses of the subcutaneous tumor using a radiation irradiator.

As a result of the test, nearly 80% of tumor growth inhibition was observed in the radiation group at the end of the test, and nearly 90% of tumor growth inhibition was observed when 1F12_H3L5 or Atezolizumab was administered together with irradiation (Fig. 10). ).

Example 8 Analysis of Immune Cell Expression in MC38 Animal Model Tumors

In order to analyze the mechanism of the co-administration strategy with the immuno-checkpoint inhibitor, an MC38 animal model was prepared and analyzed for the expression of immune cells in tumor tissues.

MC38 was prepared to have 200000 cells per 100 μL, wherein Hank's Salt (HBSS) solution (Gibco) and Basal Matrigel (Corning®) were used in a 1: 1 mixture. Prepared cells were transplanted using a 1cc syringe (26G) at 100 μL in the region of the right back of 7-8 week old C57BL / 6 female mice. Tumor tissue was secured by autopsy at the time of tumor size of about 800-1000 mm ³ and analyzed by RNA sequencing using isolated tumor tissue. For the analysis, the gene expression signature of each major immune cell reported in the paper was analyzed.

As a result of confirming the major immune cell expression in MC38 tumor tissue, it is confirmed that tumors with a large number of immune cell infiltration (Fig. 11). Therefore, it is highly likely that the MC38 tumor model is likely to show immunoreactivity suppressor treatment and tumor immunotherapy-related responsiveness, and it is expected that future responsiveness will be predicted according to the tendency of tumor cell expression.

Example 9: Immune Checkpoint PD-L1 Analysis in Tumors

In order to confirm the expression changes of PD-L1, an immune checkpoint in tumor cells of 1F12_H3L5 and non-administered mice in the MC38 model, tumor tissues were separated into single cells. Analysis of PD-L1 in tumor cells was performed by staining CD45 and PD-L1, which are immune cell markers in single cells. In order to exclude immune cells invading tumor cells, expression of PD-L1 was confirmed in cells without expression of CD45.

As a result, it was confirmed that the cells expressing PD-L1 more than doubled in mouse subjects to which 1F12_H3L5 was administered (FIG. 12). This suggests that sensitivity to 1F12_H3L5 and PD-L1 inhibitors may be increased.

Antibodies or antigen-binding fragments thereof that bind to c-Met according to the present invention can bind to human and mouse c-Met with high affinity, thereby confirming more accurate preclinical results in efficacy evaluation using a mouse tumor model. Antibodies or antigen-binding fragments thereof that bind to c-Met according to the present invention can be usefully used for the prevention or treatment of a desired cancer.

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (20)

1. Heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 27; A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 28-31; A heavy chain variable region comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 32 and 33; And

   Light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 34 and 35; Light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 36 and 37; An anti-c-Met antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 38 and 39.
2. The method of claim 1, wherein the heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 40 and 42 to 48 and the light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41 and 49 to 54 An anti-c-Met antibody or antigen-binding fragment thereof.
3. The anti-c-Met antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a monoclonal antibody.
4. The method of claim 1, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv) ₂ , scFv-Fc, Fab, Fab 'and F (ab') ₂ of the anti-c-Met antibody. An anti-c-Met antibody or antigen-binding fragment thereof.
5. The anti-c-Met antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof cross-links to human c-Met and mouse c-Met.
6. A bispecific antibody or antibody-drug conjugate comprising the anti-c-Met antibody of claim 1 or an antigen binding fragment thereof.
7. A pharmaceutical composition for preventing or treating cancer, comprising the anti-c-Met antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient.
8. A pharmaceutical composition for preventing or treating cancer, comprising the bispecific antibody or antibody-drug conjugate of claim 6 as an active ingredient.
9. The method of claim 7 or 8, wherein the cancer is breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, ovarian cancer, rectal cancer, colon Pharmaceutical composition, characterized in that cancer, vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer or bone marrow cancer.
10. The pharmaceutical composition according to claim 7 or 8, which is used for combination treatment with radiation.
11. A nucleic acid encoding an anti-c-Met antibody or antigen binding fragment thereof according to claim 1.
12. A recombinant expression vector comprising the nucleic acid according to claim 11.
13. A host cell transformed with the recombinant expression vector according to claim 12.
14. The host cell according to claim 13, wherein the host cell is selected from the group consisting of animal cells, plant cells, yeast, E. coli and insect cells.
15. The method of claim 13, wherein monkey kidney cells (COS7) cells, NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) ) Cells, MDCK, myeloma cell line, HuT 78 cells and HEK293 cells, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis Or the genus Staphylococcus sp., Aspergillus sp., Pichia pastoris, Saccharomyces cerevisiae, Schizocaromas genus (Schizosaccharomyces sp.) And neurosporacrasa (Neurosporacrassa) host cell, characterized in that selected from the group consisting of.
16. A method for preparing an anti-c-Met antibody or antigen-binding fragment thereof, comprising culturing the host cell according to any one of claims 13 to 15.
17. A combination dosage composition for treating cancer, comprising the anti-c-Met antibody or antigen-binding fragment thereof of claim 1 and another cancer therapeutic agent.
18. 18. The combination dosage composition of claim 17, wherein the other cancer therapeutic agent is an immune gateway inhibitor.
19. 19. The combination dosage composition of claim 18, wherein the immune gateway inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody.
20. The method of claim 17, wherein the cancer is breast cancer, colon cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, ovarian cancer, rectal cancer, colon cancer, vaginal cancer, Concomitant administration composition characterized in that it is small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer or bone marrow cancer.

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