Silenced antibody-based Anti-met constructs for the treatment of tumors and metastasis

Abstract

The present disclosure relates to novel silent antibody-based therapeutic agents for the treatment of tumors and / or metastases. The therapeutic agents of the present disclosure are monovalent and specific for MET.

Description

Technical Field

[0001] This disclosure relates to novel silent antibody-based therapeutic agents for the treatment of tumors and / or metastases. The therapeutic agents of this disclosure are monovalent and specific to MET. Background Technology

[0002] Cancer is a hereditary disease caused by mutations in endogenous genes within somatic cells. Only a few genes—called oncogenes and tumor suppressor genes—are altered in cancer cells and drive tumorigenesis. Activated oncogenes act as promoters, while inactivated tumor suppressor genes act as brakes, preventing cancer cell growth. Following the discovery of oncogene mutations, a new concept emerged: "oncogene addiction," referring to cancer cells that, despite numerous genetic alterations, still rely on a single oncogene protein for continued proliferation or survival. Consequently, new therapeutic approaches have been developed to create "targeted therapies" for cancer treatment. Over the past two decades, pharmacological targeting of proteins encoded by oncogene mutations—through chemical drugs or antibodies—has represented the cutting edge in eliminating mutated cells and combating cancer. Targeted therapies promise to be more effective than conventional cytotoxic chemotherapy and often have fewer side effects. However, only patients whose tumors carry altered, specific target genes are likely to benefit from targeted therapies. Therefore, personalized medicine is needed in parallel to assess druggable genetic damage through comprehensive genomic mapping of a patient's tumor.

[0003] The MET oncogene encodes a unique receptor tyrosine kinase with pleiotropic functions. When genetic alterations occur (through point mutations, gene fusions, translocations, and / or amplifications), MET triggers cellular transformation by activating an aggressive growth program. Therefore, genetic damage to MET that leads to constitutive overactivation of the Met kinase can induce and maintain a transformative phenotype (“MET addiction”).

[0004] MET genetic damage occurs in most solid tumors, with an overall incidence of 1–4%, and can upregulate its kinase activity.1 Point mutations are concentrated in domains crucial for hepatocyte growth factor (HGF) ligand binding or receptor signaling (SEMA domain, juxtamembrane domain, and catalytic domain). Recently, next-generation sequencing revealed exon 14 splicing site mutations in 3% of non-small cell lung cancers,2 leading to exon skipping and deletion of the juxtamembrane region of the MET transcript, where the serine residue (Ser985) negatively regulates Met kinase activity,3 and the internalization and degradation of Met require the tyrosine residue (Tyr1003).4

[0005] In the “invasive growth” program induced by MET, the proliferative response is associated with migration, survival, extracellular matrix degradation, and cell polarity induction.5 Cells strive to perform these biological responses to adapt to adverse conditions and / or escape to find a more favorable environment. Under hostile conditions, Met is overexpressed via transcriptional upregulation due to various stimuli such as hypoxia, inflammatory cytokines, pro-angiogenic factors, mitogens, and even HGF itself. Finally, Met is overexpressed in the presence of radiation-induced DNA damage and helps resist radiotherapy by promoting the activation of DNA repair and the escape of programmed cell death in cancer cells.

[0006] Several Met-targeting molecules have been developed to eliminate highly active Met signaling in a selective, robust, and highly effective manner. These drugs include HGF antagonists (blocking antibodies or decoys), mAbs targeting the Met receptor, and chemical tyrosine kinase inhibitors (TKIs). Anti-Met mAbs potentially represent a major step forward in the fight against MET-driven cancers. Currently, four anti-Met mAbs are in early clinical trials: MetMab (Onartuzumab, Roche), LY2875358 (Emibetuzumab, Eli Lilly & Company), ARGX-111 (Argenx), SAIT301 (Samsung), and Sym015 (Symphogen A / S) – a mixture of two antibodies. They exert their effects by competitively blocking the binding of HGF to MET (onatuzumab, ARGX-111) and / or downregulating MET (imatuzumab, SAIT301, Sym015).

[0007] Mouse DN30 mAb (disclosed in WO 2007 / 090807) is an IgG2A that binds to the extracellular domain of the human Met receptor and induces only some Met-triggered biological effects.6 Due to its bivalent nature, allowing simultaneous binding to two different antigen molecules, it partially activates receptor phosphorylation, resulting in a stable receptor complex in a manner similar to that obtained with the natural ligand. This undesirable partial agonistic activity against Met has not been observed in the monovalent DN30 Fab fragment (MvDN30).7 Converting the bivalent DN30 parent antibody to the monovalent Fab fragment would unlock the therapeutic potential of DN30 anti-Met antibodies, resulting in a fully antagonistic molecule. However, the low molecular weight and short half-life of Fab severely limit its therapeutic deployment. The inventors have therefore developed a novel engineered molecule called DCD (Dual Constant Domain Fab), characterized by the replication of constant domains present in the DN30 Fab: DCD-1 (where replication occurs in tandem) and DCD-2 (where the constant domains of the light and heavy chains are interchanged) (disclosed in WO 2014 / 108829). Both novel recombinant molecules exhibit biochemical properties comparable to the original Fab in vitro and can act as complete Met antagonists. In vivo, after systemic administration, the novel recombinant molecules reduce the growth of Met-addictive tumors. DCD-1 and DCD-2 show improved pharmacokinetic properties compared to the original DN30 Fab, but neither achieved behavior comparable to the original mAb.

[0008] WO2020 / 074459 discloses monovalent agents specific to Met. In these agents, one arm of the antibody is molecularly engineered to improve in vivo stability due to the activity of the Fc domain binding to the Fc receptor expressed in the organ. One of these monovalent agents, hOA-DN30, is further described in J Exp Clin Cancer Res (2022) Mar29;41(1):112. Different single-arm anti-c-Met antibodies for the treatment of glioblastoma are disclosed in Clin Cancer Res (2006) 12, 6144. In this disclosure, such agents are further improved by introducing a silencing mutation in the Fc region. In addition to the effects usually attributed to such mutations, such mutations have important additional safety aspects. Eliminating Fcγ receptor binding reduces the residual risk of cMET dimerization resulting from cross-linking mediated by immune cell Fcγ receptors on the surface of tumor cells. Summary of the Invention

[0009] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, and wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain and a human constant CH3 domain.

[0010] In some embodiments, the humanized VL domain and the human CL domain are fused in the N-to-C direction. In some embodiments, in the N-to-C direction, the humanized VH domain is fused with the human CH1 domain, which is fused with the human hinge region, which is fused with the human CH2 domain, and which is fused with the human CH3 domain. In some embodiments, in the N-to-C direction, the human hinge region is fused with the CH2 domain, which is fused with the human CH3 domain, wherein the human hinge region is truncated at the N-end.

[0011] In some embodiments, the humanized VL domain has the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the humanized VH domain has the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the human CL domain is a human κ-type light chain domain. In some embodiments, the human hinge region and the human constant domains CH1, CH2, and CH3 are derived from human IgG1. In some embodiments, the two Fc peptides are linked in the hinge region by intermolecular disulfide bonds.

[0012] In some embodiments, the first Fc polypeptide and the second Fc polypeptide meet at an interface, one of the first and second Fc polypeptides contains a club at the interface, and the other of the first and second Fc polypeptides contains a mortar at the interface, wherein the club is localized into the mortar. In some embodiments, one of the first or second Fc polypeptides contains a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries an amino acid mutation at position 389, wherein the original amino acid at position 389 has been mutated to introduce an amino acid with a larger side chain volume than the original amino acid; and wherein the other Fc polypeptide contains a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries three amino acid mutations at positions 389, 391, and 438, wherein the original amino acid has been mutated to introduce an amino acid with a smaller side chain volume than the original amino acid, wherein the amino acid numbering is according to Kabat's EU numbering scheme. In some embodiments, the original amino acids at positions 389, 391, and 438 are threonine, leucine, and tyrosine, respectively; and in one or the second Fc polypeptide, threonine at position 389 has been mutated to tryptophan; and in another Fc polypeptide, threonine at position 389 has been mutated to serine, leucine at position 391 has been mutated to alanine, and tyrosine at position 438 has been mutated to valine.

[0013] In some embodiments, the human CL domain has the amino acid sequence shown in SEQ ID NO: 15, and the human CH1 domain has the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the first human Fc polypeptide has the amino acid sequence shown in SEQ ID NO: 17, and the second human Fc polypeptide has the amino acid sequence shown in SEQ ID NO: 18.

[0014] In some embodiments, the anti-Met antibody fragment of this disclosure induces the shedding of the extracellular domain of Met when it binds to Met.

[0015] In some embodiments, the Fc regions of the first and second Fc peptides contain mutations L234A, L235A, and P329A (according to the EU index), or mutations L234A, L235E, G237A, A330S, and P331S (according to the EU index). In some embodiments, the first and second Fc peptides contain mutations L234A, L235A, and P329A (according to the EU index).

[0016] In some embodiments, the first polypeptide contains the amino acid sequence SEQ ID NO: 19, the second polypeptide contains the amino acid sequence SEQ ID NO: 20, and the third polypeptide contains the amino acid sequence SEQ ID NO: 18.

[0017] In some embodiments, this disclosure relates to isolated nucleic acids encoding any one of the aforementioned anti-Met antibody fragments. In some embodiments, this disclosure relates to compositions comprising two or more recombinant nucleic acids that collectively encode the aforementioned anti-Met antibody fragments.

[0018] In some embodiments, this disclosure relates to the use of any of the aforementioned anti-Met antibody fragments in the treatment of tumors and / or metastases. In some embodiments, this disclosure relates to the use of any of the aforementioned anti-Met antibody fragments in the treatment of tumors and / or metastases in patients carrying a genetic alteration of the MET gene. In some embodiments, this disclosure relates to the use of any of the aforementioned anti-Met antibody fragments in the treatment of tumors and / or metastases in patients carrying the wild-type MET gene.

[0019] In some embodiments, this disclosure relates to a method for preparing the aforementioned anti-Met antibody fragment, the method comprising the steps of: (i) synthesizing cDNA sequences of a first, second, and third polypeptide constituting the anti-Met antibody fragment; (ii) inserting the three cDNA sequences into one or more plasmids suitable for expression in mammalian cell lines; (iii) transiently or stably co-transfecting mammalian cell lines with the plasmids; (iv) collecting the culture supernatant; and (v) purifying the anti-Met antibody fragment by affinity chromatography. Attached Figure Description

[0020] Figure 1 The SCX chromatograms for the test formats are shown. All three formats show similar distributions of charged species. VERT-004 appears to be slightly more acidic.

[0021] Figure 2 The RP chromatogram is shown in the test form. VERT-004 shows a slightly different chromatogram compared to VERT-001 and VERT-002, indicated by the presence of multiple peaks.

[0022] Figure 3 The mass analysis of each individual peak present in VERT-001, VERT-002 and VERT-004 in the RP chromatogram is shown.

[0023] Figure 4 The binding of VERT001, VERT002, and VERT004 to the ECD of c-Met, as measured in an ELISA assay, is shown.

[0024] Figure 5The antiproliferative activity of VERT-001, VERT-002, and VERT- against Hs746T cells (left panel) and EBC-1 cells (right panel) was demonstrated.

[0025] Figure 6 The monomer, HMW, and LMW contents of VERT-001, VERT-002, and VERT-004 are shown, determined by SEC analysis. (RM = freshly dispensed reference material for VERT001).

[0026] Figure 7 The formation of charge variants as analyzed by strong cation exchange chromatography is shown. (Freshly dispensed reference material RM = VERT001).

[0027] Figure 8 The binding of VERT001, VERT002, and VERT004 to the ECD of c-Met after incubation at 5 °C and 37 °C for 4 weeks, compared to a freshly thawed reference material, is shown.

[0028] Figure 9 The analysis of VERT-002 under pH 3 stress is shown. The left side shows the SEC chromatogram, and the right side shows the SCX chromatogram.

[0029] Figure 10 Analysis of the antiproliferative activity of the stress samples VERT001, VERT002 and VERT004 is shown.

[0030] Figure 11 Female hairless SCID mice were subcutaneously implanted with Hs746T tumor cells on day 0 and treated for 28 days with an isotype control, VERT-001, VERT-002, or VERT-004. Tumor volume (mm3) data are shown as mean + / - standard error of mean (SEM); each group included 9 mice.

[0031] Figure 12 Female hairless SCID mice were subcutaneously implanted with Hs746T tumor cells on day 0 and treated with either an allotype control or VERT-002 antibody. Tumor volume (mm3) data are shown as mean + / - standard error of mean (SEM); each group included 9 mice. A) Dose response to tumor growth inhibition; B) In vivo tumor growth on day 35.

[0032] Figure 13Female naked Balb / c mice were subcutaneously implanted with EBC-1 tumor cells and chronically treated for 28 days with either an isotype control or VERT-002. Tumor volume (mm3) is shown as mean and SEM; 10 mice were treated per group. A) Dose-response to tumor growth inhibition by chronic administration at days 1, 4, 8, 11, 15, 18, 22, and 25 after randomization; B) In vivo tumor growth on day 28; C) Plasma levels of soluble extracellular domain of MET (sMET ECD) in response to various treatment conditions, as shown. Detailed Implementation

[0033] definition This disclosure relates to antibodies that specifically bind to Met and antibody-based constructs, as well as the uses of such constructs, particularly for therapeutic purposes, such as treating tumors and metastases.

[0034] The terms "Met," "cMET," "cMet," and "MET" refer to proteins also known as hepatocyte growth factor receptor, HGFR, or c-Met. Human Met has the following amino acid sequence (UniProt P08581): As used herein, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds that interacts with an antigen. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelified antibodies, and chimeric antibodies. Antibodies can be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgD, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0035] As used herein, the term "antibody fragment" refers to a portion of one or more antibodies that retains the ability to specifically interact with an antigen (e.g., through binding, steric hindrance, or stable spatial distribution). Examples of binding fragments include, but are not limited to: Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CHI domains; F(ab')2 fragments, which are bivalent fragments consisting of two Fab fragments linked by disulfide bonds in a hinge region; Fd fragments, which consist of VH and CHI domains; Fv fragments, which consist of VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; and segregated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombination methods, making it possible to fabricate them into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be covered in the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the methods for screening fragments for practical purposes are the same as for intact antibodies. Antibody fragments can also be incorporated into single-domain antibodies, macrobodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and dual scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23: 1126-1136). Antibody fragments can be grafted into peptide-based scaffolds, such as fibronectin type III (Fn3) (see, U.S. Patent No. 6,703,199, which describes a fibronectin peptide monoclonal antibody). Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv segments (VH-CH1-VH-CH1), which together with complementary light chain peptides form a pair of antigen-binding sites (Zapata et al., (1995) ProteinEng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0036] The structure and location of immunoglobulin variable domains, such as CDRs, can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, for example, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, (1991) ed., Kabat et al.; Lazikani et al. (1997) J. Mol. Biol. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia et al. (1987) J. Mol Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948; Annals of the New York Academy of Sciences, 764, 47-49 (1995); Nucleic Acids Research, 25, 206-211 (1997).

[0037] As used herein, “human antibody” or “human antibody fragment” refers to antibodies and antibody fragments having variable regions, wherein both the frame and CDR regions are sequences derived from human sources. Human antibodies can also be isolated from synthetic libraries or from transgenic mice (e.g., Xenomouse, OmniMouse, Harbour mice, ATX-Gx mice, Trianni mice), provided that the antibodies produced by the respective systems have variable regions, wherein the frame and CDR regions are derived from human-derived sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from such sequences. Human-derived includes, for example, human germline sequences or mutant versions of human germline sequences, or antibodies containing a common frame sequence derived from human frame sequence analysis, as described, for example, in Knappik et al., (2000) J Mol Biol 296:57-86.

[0038] A “humanized antibody” or “humanized antibody fragment” is defined herein as an antibody molecule having a constant antibody region derived from a human sequence, and a variable antibody region, or a portion thereof or only the CDR, derived from another species. For example, a humanized antibody may be CDR-grafted, wherein the CDR of the variable domain is derived from a non-human source, while one or more frames of the variable domain are human, and the constant domain, if any, is human.

[0039] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule having a constant antibody region derived from or corresponding to a sequence found in one species and a variable antibody region derived from another species. Preferably, the constant antibody region is derived from or corresponds to a sequence found in humans, and the variable antibody region (e.g., VH, VL, CDR, or FR regions) is derived from a sequence found in non-human animals, such as mice, rats, rabbits, or hamsters.

[0040] As used herein, the term "antigen-binding arm" refers to a component of the antibody fragment of the present invention that has the ability to specifically bind to a target molecule. The antigen-binding arm is a complex of variable domain sequences (VL and VH) (including CDR and frame regions) of the immunoglobulin light and heavy chains with constant domain sequences (CL and CH) of the immunoglobulin light and heavy chains.

[0041] As used herein, “hinge region,” “hinge sequence,” and their variants have meanings known in the art, as described in, for example, Janeway et al., Immuno Biology: the immune system in health and disease (Elsevier Science Ltd., NY) (4th ed., 1999).

[0042] As used herein, the phrase "truncated hinge region" refers to a polypeptide containing part, but not all, of the hinge sequence. The truncated hinge region is capable of linking to the "first" Fc polypeptide. If the wild-type hinge sequence is absent, the remaining sequence in the "second" Fc polypeptide will contain components capable of linking to the "first" Fc polypeptide. For example, these components may be modifying residues capable of forming disulfide bonds or added cysteine ​​residues.

[0043] A "pestle" refers to at least one amino acid side chain that protrudes from the interface of the first Fc polypeptide and can therefore be positioned in a complementary mortise in the adjacent interface (i.e., the interface of the second Fc polypeptide) to stabilize the heteropolymer, thus favoring the formation of heteropolymers over, for example, the formation of homopolymers. The pestle can be present in the original interface or can be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the first polypeptide is altered to encode the pestle. For this purpose, at least one "original" amino acid residue in the interface encoding the first polypeptide is replaced with a nucleic acid encoding at least one "imported" amino acid residue having a side chain volume larger than the original amino acid residue. It should be understood that there can be more than one original and corresponding imported residue. The upper limit of the number of original residues replaced is the total number of residues in the interface of the first polypeptide.

[0044] A "mortise" refers to at least one amino acid side chain that recesses from the interface of the second Fc polypeptide and thus accommodates a corresponding pestle on the interface of the adjacent first Fc polypeptide. The mortise may be present in the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the second polypeptide is altered to encode the mortise. For this purpose, at least one "original" amino acid residue in the interface encoding the second polypeptide is replaced with a nucleic acid encoding at least one "imported" amino acid residue having a smaller side chain volume than the original amino acid residue. It should be understood that there may be more than one original and corresponding imported residue. The upper limit of the number of original residues replaced is the total number of residues in the interface of the second polypeptide.

[0045] The "locatable" position of the pestle within the mortar means that the spatial positions of the pestle and mortar at the interfaces of the first and second Fc peptides, respectively, and the dimensions of the pestle and mortar, allow the pestle to be positioned within the mortar without significantly interfering with the normal association of the first and second peptides at the interface. Since the pestle typically does not extend perpendicularly from the interface axis and has a preferred conformation, the alignment of the pestle with the corresponding mortar relies on modeling the pestle / mortar pair based on a three-dimensional structure, such as that obtained through X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using techniques generally accepted in the art.

[0046] The terms "isolated antibody" or "isolated antibody fragment" refer to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments with different antigen specificities. Furthermore, isolated antibodies or antibody fragments may be substantially free of other cellular material and / or chemicals. Therefore, in some respects, the antibody provided is an isolated antibody that has been separated from antibodies with different specificities. Isolated antibodies can be monoclonal antibodies. Isolated antibodies can be recombinant monoclonal antibodies. However, isolated antibodies that specifically bind to epitopes, isotypes, or variants of a target may have cross-reactivity with other relevant antigens (e.g., those from other species, such as species homologs).

[0047] As used herein, the term "recombinant antibody" or "recombinant antibody fragment" encompasses all antibodies or antibody fragments prepared, expressed, constructed, or isolated from non-naturally occurring devices. Examples include antibodies isolated from host cells transformed to express antibodies, antibodies selected and isolated from recombinant, combined human antibody libraries, and antibodies prepared, expressed, constructed, or isolated by any other device involving splicing all or part of the human immunoglobulin gene, sequence, or other DNA sequences, or antibodies isolated from transgenic or transchromosomally transfected animals (e.g., mice) carrying the human immunoglobulin gene or hybridomas thus prepared. Preferably, such recombinant antibodies have variable regions, wherein the framework region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies can be mutagenized in vitro (or, when using animals transgenic with human Ig sequences, in vivo somatic cell mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, while derived from and associated with human germline VH and VL sequences, can exist non-naturally in vivo in a human antibody germline repertoire. Recombinant antibodies can be monoclonal antibodies.

[0048] As used herein, the term “monoclonal” has the meaning commonly given in the art as a single clone derived from an antibody-producing cell and an antibody or antibody fragment (or its corresponding functional fragment) that recognizes a single epitope on the bound antigen.

[0049] As used herein, if an antibody can distinguish an antigen from one or more reference antigens, then such an antibody "specifically binds," "specifically binds," "is specific to," or "specifically recognizes" the antigen (such as human Met), because binding specificity is not an absolute but a relative characteristic. For example, standard ELISA or standard flow cytometry assays can be performed. Scoring can be done using standard colorimetric methods (e.g., using a secondary antibody with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide) or by binding a secondary antibody labeled with PE or other dyes or labels. Reactions in some wells are scored by optical density (OD), for example, at 450 nm, or by mean or median fluorescence intensity (MFI) in flow cytometry. Typical background (i.e., a negative reaction) can be 0.1 OD; typical positive reactions can be 1 OD. Background and positive reaction MFI are highly dependent on instrument settings. Positive / negative differences can exceed 10-fold. Typically, binding specificity is not determined using a single reference antigen, but rather using a group of approximately three to five unrelated antigens (e.g., milk powder, BSA, transferrin, etc.). For flow cytometry, a variety of antigen-negative cells can be used. However, antibodies that specifically bind to antigens can have cross-reactivity with their respective orthologous antigens from other species (e.g., species homologs). In some embodiments, such cross-reactivity with orthologous antigens is even preferred.

[0050] As used in this article, an antibody is considered "cross-reactive" or "cross-reactive" if it binds to an orthologous antigen from another species. For example, an antibody is cross-reactive if it binds to human Met and cynomolgus monkey Met.

[0051] As used herein, the term "affinity" refers to the strength of the interaction between a peptide and its target at a single site. Within each site, the peptide's binding region interacts with its target at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0052] The term "epitope" includes any protein region that is specifically recognized by an antibody or its antibody fragment, or otherwise interacts with a molecule. Typically, an epitope is a chemically active surface group of a molecule (such as an amino acid or carbohydrate or sugar side chain) and may typically have specific three-dimensional structural features as well as specific charge characteristics. Those skilled in the art will understand that in practice, anything that can be specifically bound by an antibody can be an epitope.

[0053] The term "domain" or "protein domain" refers to the region of a protein polypeptide chain that forms a functional unit and / or independently forms a three-dimensional structure.

[0054] The “compositions” disclosed herein can be used for therapeutic or preventative applications. Therefore, this disclosure includes pharmaceutical compositions comprising antibodies or antibody fragments as disclosed herein, and pharmaceutically acceptable carriers or excipients thereof. In a related aspect, this disclosure provides methods for treating inflammatory diseases, autoimmune diseases, hematologic malignancies, and other underlying diseases. Such methods include the step of administering an effective amount of a pharmaceutical composition comprising an antibody or antibody fragment as described herein to a subject in need of it.

[0055] The treatment methods disclosed herein include administering a therapeutically effective amount of an antibody or antibody fragment as disclosed herein to a subject requiring such treatment. As used herein, "therapeutically effective amount" or "effective amount" means the amount necessary for an anti-Met antibody to elicit the desired biological response. According to this subject matter disclosure, a therapeutically effective amount is the necessary amount of an anti-Met antibody for the treatment and / or prevention of disease.

[0056] "Administered" or "applied" includes, but is not limited to, delivery of the drug via injectable form (such as intravenous, intramuscular, intradermal, or subcutaneous routes) or mucosal route (e.g., as a nasal spray or aerosol for inhalation) or as an ingestible solution, capsule, or tablet. Preferably, administration is via injectable form.

[0057] As used herein, "treatment," "treatment," or "management" refers to a clinical intervention aimed at altering the natural course of disease in a subject being treated, and may be performed in prevention or during clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, antibodies or antibody fragments according to this disclosure are used to delay disease development or slow disease progression.

[0058] "Prevention" or "avoidance" means reducing the risk of contracting or developing a disease (i.e., preventing the development of at least one clinical symptom of the disease in a subject who may be exposed to the causative agent or who is susceptible to the disease before its onset). "Prevention" also refers to methods aimed at preventing the onset of a disease or its symptoms, or delaying the onset of a disease or its symptoms.

[0059] As used in this context, “subject” or “species” refers to any mammal, including rodents such as mice or rats and primates such as cynomolgus monkeys (long-tailed macaques). Macaca fascicularis ), marmoset (Jerusalem's pinniped ( Callithrix jacchus ), rhesus monkey (common macaque ( Macaca mulatta )) or humans (Homo sapiens ( Homo sapiens Preferably, the subjects are primates, and most preferably, humans.

[0060] The term "effective function" refers to those biological activities attributable to the Fc region of an antibody, which varies with antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and direct cell activation or direct cell inhibition.

[0061] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a form of cytotoxicity in which antibodies bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. The primary cells mediating ADCC (NK cells) express only FcγRIII, while monocytes / macrophages express FcγRI, FcγRII, and FcγRIII.

[0062] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is triggered by the binding of the first component of the complement system (C1q) to the antibody (appropriate subclass) of this disclosure bound to its homologous antigen.

[0063] Antibody-dependent phagocytosis (ADCP) refers to the elimination of antibody-coated target cells by phagocytes (such as macrophages or dendritic cells) through internalization.

[0064] Throughout this specification, unless the context otherwise requires, the words “comprise,” “have,” and “include,” and their respective variations such as “comprises,” “comprising,” “has,” “having,” “include,” and “including” will be understood to imply coverage of the stated element or integer, or a group of elements or integers, but do not exclude any other element or integer, or a group of elements or integers.

[0065] As used herein, the terms "engineered" or "modified" include manipulation of nucleic acids or peptides by synthetic means (e.g., by recombinant technology, in vitro peptide synthesis, by enzymatic or chemical conjugation of peptides, or some combination of these techniques). Preferably, the antibody or antibody fragment according to this disclosure is engineered or modified to improve one or more properties, such as antigen binding, stability, half-life, effector function, immunogenicity, safety, etc.

[0066] As used herein, “variant” refers to a polypeptide that differs from a reference polypeptide through one or more modifications (e.g., amino acid substitution, insertion, or deletion). Variant polypeptides typically retain most of the properties of the reference polypeptide, such as binding to the target antigen, but introduce new, additional features or properties, such as a higher affinity for the target antigen compared to the reference polypeptide, or a humanized version of the reference polypeptide.

[0067] As used herein, the term "amino acid mutation" is intended to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification may be performed, provided the final construct possesses the desired properties, such as reduced binding to the Fc receptor. Amino acid sequence deletions and insertions include the deletion and insertion of amino acid residues at the N and / or C ends. A specific amino acid mutation is an amino acid substitution. Amino acid substitution includes replacement with a non-naturally occurring amino acid or a naturally occurring amino acid derivative of one of the twenty standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods other than genetic engineering (e.g., chemical modification) to alter the side chain groups of amino acid residues may also be used. As used herein, various nomenclatures may be used to refer to the same amino acid mutation. For example, the substitution of glycine at position 237 of the antibody Fc region with alanine may be represented as 237A, G237A, or Gly237Ala.

[0068] As used in this article, "EC50" refers to the concentration of an antibody or antibody fragment that induces half of the response between baseline and maximum value in the assay. Therefore, it represents the antibody or ligand concentration at which 50% of the maximum effect is observed.

[0069] The term "Ka" as used in this article refers to the binding rate of a specific antibody-antigen interaction.

[0070] As used herein, the term "Kd" refers to the dissociation rate of a specific antibody-antigen interaction. The Kd value of an antibody can be determined using methods known in the art.

[0071] As used herein, the term "KD" refers to the dissociation constant of a specific antibody-antigen interaction, which is obtained as the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration. A preferred method for determining antibody Kd is to use surface plasmon resonance, preferably employing a biosensor system such as the Biacore system, or using biolayer interference with the Octet BLI instrument.

[0072] The terms “inhibition” or “inhibit,” “reduction” or “reduce,” or “neutralization” or “neutralize” refer to a reduction or cessation of any phenotypic characteristic (such as binding or biological activity or function), or a reduction or cessation of the occurrence, extent, or likelihood of that characteristic. “Inhibition,” “reduction,” or “neutralization” need not be complete, as long as it is detectable by appropriate assay. In some embodiments, “reduction” or “inhibition” or “neutralization” refers to the ability to cause a reduction of 20% or greater. In another embodiment, “reduction” or “inhibition” or “neutralization” refers to the ability to cause a reduction of 50% or greater. In yet another embodiment, “reduction” or “inhibition” or “neutralization” refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or greater.

[0073] As used herein, the term "antagonistic" antibody refers to an antibody or antibody fragment that interacts with an antigen to partially or completely inhibit or neutralize the biological activity or function of the target antigen or any other phenotypic characterization.

[0074] “Wild-type” proteins are versions or variants of naturally occurring proteins. The amino acid sequence of a wild-type protein (e.g., the Fc region of a human IgG1 antibody) is the same as that of a naturally occurring protein. Due to allotype differences, wild-type proteins can have more than one amino acid sequence. For example, there are several allotypes of the naturally occurring human IgG1 heavy chain constant region (see, for example, Jeffries et al., (2009) mAbs 1:1).

[0075] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin typically contains two constant domains: a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the amino acid residues in the Fc region are numbered according to the EU numbering system, also known as the EU index, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, National Institutes of Health Public Health Service, Bethesda, MD, 1991. Various Fc modifications are commonly used. See reviews, for example, Antibodies (2020) 9: 64.

[0076] As used herein, the terms “silent” or “silent” in the context of a silent antibody or an antibody containing a silencing mutation refer to a mutation in the Fc domain of such an antibody that partially or completely reduces binding to one or more cell surface Fcγ receptors, thereby reducing or inhibiting (in some embodiments substantially eliminating) one or more Fc-mediated antibody effector functions, such as ADCC, ADCP, and CDC complement responses (see, e.g., Kang and Jung, Experimental and Molecular Medicine (2019) 51:138). Silent effector functions can be obtained through mutations in the Fc region of an antibody, as described in the prior art (e.g., Strohl, Biotechnology 20: 685-91 describes LALA and N297A; Baudino et al., J. Immunol. 181: 6664-69 describes D265A). Other exemplary Fc silencing mutations include amino acid substitutions at one or more of the following positions: E233, L234, L235, G236, N297, P331, and P329 (see, for example, U.S. Patent Nos. 6,737,056, 7,332,581; WO 2004 / 056312, WO2021 / 234402; and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). Silent mutations also include (according to EU index number) LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A) and PG-LALA (L234A / L235A / P329G) mutations, as well as AEASS mutations (L234A / L235E / G237A / A330S / P331S).

[0077] Embodiments of the present invention In the following description, numerous specific details are given to provide a thorough understanding of the implementation scheme. The implementation scheme can be practiced without one or more of these specific details, or with the availability of other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the implementation scheme.

[0078] This disclosure relates to novel therapeutic agents for treating tumors and / or metastases.

[0079] It is estimated that over 200,000 patients become “MET addicted” each year, and Met inhibition could potentially lead to remission of the disease. Given that mutations accumulate with age and the elderly population is projected to increase over the next 20 years, the burden of cancer is expected to rise, significantly impacting global patient management healthcare resources. Genetic alterations leading to “MET addiction” have been identified in gastric, esophageal, colorectal, renal, and lung cancers, melanoma, and brain tumors. Furthermore, a number of MET genetic lesions have been identified as an acquired mechanism of resistance to many other targeted therapies in colorectal and non-small cell lung cancer (NSCLC).

[0080] The role of metastasis is also related to the ability of Met to help cells adapt to harsh environments. Met-driven metastatic capacity depends not only on genetic and epigenetic alterations, but also on the paracrine secretion of HGF from the tumor stroma, which is composed of a variety of different cell types, including fibroblasts, resident epithelial cells, pericytes, myofibroblasts, vascular and lymphatic endothelial cells, and infiltrating cells of the immune system.

[0081] Today, Met has been identified as a cancer-specific target for: (i) personalized treatment of tumors with MET mutations / amplifications (“MET addiction”); (ii) prevention / reversal of Met-driven primary and secondary resistance to other targeted cancer therapies; and (iii) prevention / reversal of Met-driven invasive / metastatic phenotypes.

[0082] Monovalent antibodies, for example, are described in WO2005 / 063816 and Proc Natl Acad Sci USA (2013)110, E2987. A “single-arm” anti-Met antibody, named “hOA-DN30,” is described in WO2020 / 074459. hOA-DN30 is a highly stable humanized monovalent antibody that blocks Met activation via shedding mechanisms, including (i) removal of Met from the cell surface via the 'shedding' of the extracellular domain; (ii) isolation of the HGF ligand; (iii) inhibition of homo- or hetero-dimerization of the Met receptor on the membrane; and (iv) stimulation of receptor degradation. This disclosure provides significant additional improvements to hOA-DN30. The introduction of silencing mutations not only endows the antibody with effects associated with the corresponding mutation but also provides an additional important safety aspect—the elimination of the Fcγ receptor reduces the residual risk of MET dimerization caused by immune cell Fcγ receptor-mediated hypercrosslinking on the tumor cell surface.

[0083] Anti-Met antibody fragment In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain and a human constant CH3 domain.

[0084] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The humanized VL domain and the human CL domain are fused in the direction from the N to the C ends.

[0085] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. In the direction from N to C, the humanized VH domain merges with the human CH1 domain, the human CH1 domain merges with the human hinge region, the human hinge region merges with the human CH2 domain, and the human CH2 domain merges with the human CH3 domain.

[0086] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The humanized VL domain and the human CL domain are fused at the N to C ends, and In the direction from N to C, the humanized VH domain merges with the human CH1 domain, the human CH1 domain merges with the human hinge region, the human hinge region merges with the human CH2 domain, and the human CH2 domain merges with the human CH3 domain.

[0087] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. In the direction from N to C, the human hinge region merges with the CH2 domain, which in turn merges with the human CH3 domain, wherein the human hinge region is truncated at the N end.

[0088] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The humanized VL domain and the human CL domain are fused in the direction from the N to the C ends.

[0089] In the direction from N to C, the humanized VH domain merges with the human CH1 domain, the human CH1 domain merges with the human hinge region, the human hinge region merges with the human CH2 domain, and the human CH2 domain merges with the human CH3 domain. In the direction from N to C, the human hinge region merges with the CH2 domain, which in turn merges with the human CH3 domain, wherein the human hinge region is truncated at the N end.

[0090] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The humanized VL domain has an amino acid sequence as shown in SEQ ID NO: 13.

[0091] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The humanized VH domain has the amino acid sequence shown in SEQ ID NO: 14.

[0092] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The humanized VL domain has the amino acid sequence shown in SEQ ID NO: 13, and The humanized VH domain has the amino acid sequence shown in SEQ ID NO: 14.

[0093] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The human CL domain is a human κ-type light chain domain.

[0094] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The hinge region and human constant structural domains CH1, CH2 and CH3 of this human are derived from human IgG1.

[0095] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The human CL domain is a human κ-type light chain domain, and The hinge region and human constant structural domains CH1, CH2 and CH3 of this human are derived from human IgG1.

[0096] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The two Fc polypeptides are connected by intermolecular disulfide bonds in the hinge region.

[0097] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The first Fc polypeptide and the second Fc polypeptide meet at the interface, one of the first and second Fc polypeptides contains a pestle at the interface, and the other of the first and second Fc polypeptides contains a mortar at the interface, wherein the pestle can be positioned in the mortar.

[0098] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The first or second Fc polypeptide contains a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries an amino acid mutation at position 389, wherein the original amino acid at position 389 has been mutated to introduce an amino acid with a larger side chain volume than the original amino acid; and the other Fc polypeptide contains a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries three amino acid mutations at positions 389, 391 and 438, wherein the original amino acids have been mutated to introduce amino acids with a smaller side chain volume than the original amino acid, wherein the amino acid numbering is according to Kabat's EU numbering scheme.

[0099] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The original amino acids at positions 389, 391, and 438 are threonine, leucine, and tyrosine, respectively; and in the first or second Fc polypeptide, threonine at position 389 has been mutated to tryptophan; and in the other Fc polypeptide, threonine at position 389 has been mutated to serine, leucine at position 391 has been mutated to alanine, and tyrosine at position 438 has been mutated to valine.

[0100] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The human CL domain has the amino acid sequence shown in SEQ ID NO: 15, and the human CH1 domain has the amino acid sequence shown in SEQ ID NO: 16.

[0101] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The first human Fc polypeptide has the amino acid sequence shown in SEQ ID NO: 17, and the second human Fc polypeptide has the amino acid sequence shown in SEQ ID NO: 18.

[0102] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. When the anti-Met antibody fragment binds to Met, it induces the shedding of the extracellular domain of Met.

[0103] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The Fc regions of the first and second Fc peptides contain mutations L234A, L235A, and P329A (according to the EU index), or mutations L234A, L235E, G237A, A330S, and P331S (according to the EU index).

[0104] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The Fc regions of the first and second Fc peptides contain mutations L234A, L235A, and P329A (according to the EU index).

[0105] In some embodiments, this disclosure relates to an anti-Met antibody fragment comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc peptides, wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The first polypeptide contains the amino acid sequence SEQ ID NO: 19, the second polypeptide contains the amino acid sequence SEQ ID NO: 20, and the third polypeptide contains the amino acid sequence SEQ ID NO: 18.

[0106] Nucleic acid In some embodiments, this disclosure relates to isolated nucleic acids encoding anti-Met antibody fragments comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc polypeptides, and wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. In some embodiments, the humanized VL domain is fused with the human CL domain in the N-to-C-terminal direction. In some embodiments, in the N-to-C-terminal direction, the humanized VH domain is fused with the human CH1 domain, which is fused with the human hinge region, which is fused with the human CH2 domain, and which is fused with the human CH3 domain. In some embodiments, in the N-to-C-terminal direction, the human hinge region is fused with the CH2 domain, which is fused with the human CH3 domain, wherein the human hinge region is truncated at the N-terminus. In some embodiments, in the N-to-C-terminal direction, the humanized VL domain is fused with a human CL domain, the humanized VH domain is fused with a human CH1 domain, the human CH1 domain is fused with a human hinge region, the human hinge region is fused with a human CH2 domain, and the human CH2 domain is fused with a human CH3 domain. Furthermore, in the N-to-C-terminal direction, the human hinge region is fused with a CH2 domain, and the CH2 domain is fused with a human CH3 domain, wherein the human hinge region is truncated at the N-terminus. In some embodiments, the humanized VL domain has the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the humanized VH domain has the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the humanized VL domain has the amino acid sequence shown in SEQ ID NO: 13, and the humanized VH domain has the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the human CL domain is a human κ-type light chain domain. In some embodiments, the human hinge region and the human constant domains CH1, CH2, and CH3 are derived from human IgG1. In some embodiments, the human CL domain is a human κ-type light chain domain, and the human hinge region and the human constant domains CH1, CH2, and CH3 are derived from human IgG1. In some embodiments, two Fc polypeptides are linked in the hinge region by intermolecular disulfide bonds. In some embodiments, a first Fc polypeptide and a second Fc polypeptide meet at an interface, one of the first and second Fc polypeptides comprising a club at the interface, and the other of the first and second Fc polypeptides comprising a mortar at the interface, wherein the club is localized into the mortar.In some embodiments, the first or second Fc polypeptide comprises a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries an amino acid mutation at position 389, wherein the original amino acid at position 389 has been mutated to introduce an amino acid with a larger side chain volume than the original amino acid; and wherein the other Fc polypeptide comprises a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries three amino acid mutations at positions 389, 391, and 438, wherein the original amino acids have been mutated to introduce amino acids with a smaller side chain volume than the original amino acid, wherein the amino acid numbering is according to Kabat's EU numbering scheme. In some embodiments, the original amino acids at positions 389, 391, and 438 are threonine, leucine, and tyrosine, respectively; and in the first or second Fc polypeptide, threonine at position 389 has been mutated to tryptophan; and wherein in the other Fc polypeptide, threonine at position 389 has been mutated to serine, leucine at position 391 has been mutated to alanine, and tyrosine at position 438 has been mutated to valine. In some embodiments, the human CL domain has the amino acid sequence shown in SEQ ID NO: 15, and the human CH1 domain has the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the first human Fc polypeptide has the amino acid sequence shown in SEQ ID NO: 17, and the second human Fc polypeptide has the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the anti-Met antibody fragment induces the shedding of the extracellular domain of Met upon binding to Met. In some embodiments, the Fc regions of the first and second Fc polypeptides contain mutants L234A, L235A, and P329A (according to the EU index), or mutants L234A, L235E, G237A, A330S, and P331S (according to the EU index). In some embodiments, the Fc regions of the first and second Fc polypeptides contain mutants L234A, L235A, and P329A (according to the EU index).

[0107] In some embodiments, this disclosure relates to isolated nucleic acids encoding anti-Met antibody fragments comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc polypeptides, and wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 3, 5 and 6; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs) having amino acid sequences as shown in SEQ ID NO: 8, 10, and 12. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain. The first polypeptide contains the amino acid sequence SEQ ID NO: 19, the second polypeptide contains the amino acid sequence SEQ ID NO: 20, and the third polypeptide contains the amino acid sequence SEQ ID NO: 18.

[0108] In some embodiments, this disclosure relates to isolated nucleic acids encoding anti-Met antibody fragments comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc polypeptides, and wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs), and wherein the first polypeptide has the amino acid sequence SEQ ID NO: 19; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs), and wherein the second polypeptide has the amino acid sequence SEQ ID NO: 20, and (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain and a human constant CH3 domain, and wherein the third polypeptide has the amino acid sequence SEQ ID NO: 18.

[0109] In some embodiments, this disclosure relates to isolated nucleic acids encoding anti-Met antibody fragments comprising a single antigen-binding arm and a silenced Fc region, wherein the Fc region comprises a complex of first and second Fc polypeptides, and wherein the antibody fragment comprises: (i) A first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity-determining regions (CDRs), and wherein the first polypeptide is encoded by the nucleic acid sequence SEQ ID NO: 21; (ii) A second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein the humanized VH domain contains three complementarity-determining regions (CDRs), and wherein the second polypeptide is encoded by the nucleic acid sequence SEQ ID NO: 22. (iii) A third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain and a human constant CH3 domain, and wherein the third polypeptide is encoded by a nucleic acid sequence SEQ ID NO: 23.

[0110] In some embodiments, this disclosure relates to compositions comprising two or more recombinant nucleic acids that collectively encode the anti-Met antibody fragment disclosed herein.

[0111] Medical Uses and Preparation Therapeutic compositions containing the active ingredient of this disclosure (i.e., humanized anti-Met antibody fragments) can be prepared in the form of aqueous solutions, lyophilized or other dried formulations using physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.; 1980). Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations used and include: buffers; antioxidants; preservatives; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids; monosaccharides, disaccharides and other carbohydrates; chelating agents; sugars; salt-forming anti-counterions; metal complexes and / or nonionic surfactants. The formulation may also contain other active compounds necessary for the specific indication being treated, preferably those compounds with complementary activities that do not adversely affect the therapeutic activity of hOA-DN30 alone or in combination with the extracellular portion of human Met. Such molecules exist appropriately in combinations of quantities that are effective for the intended purpose.

[0112] The active ingredient can also be encapsulated in microcapsules prepared using techniques disclosed, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.; 1980. Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing the active ingredient.

[0113] The active ingredients (and adjuvants) of this disclosure are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and, if necessary, intralesional administration. The active ingredients of this invention can be suitably administered by pulsatile infusion, particularly using reduced doses of the active ingredients. Administration can be carried out by any suitable route, such as by injection, like intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term.

[0114] The active ingredient will be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical symptoms, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practicing physician. It is not mandatory, but optional, to formulate the active ingredient together with one or more agents currently used for the prevention or treatment of the condition in question. The effective amount of such other agents depends on the amount of active ingredient present in the formulation, the type of condition or treatment, and other factors discussed above. These are typically used at the same dosage and route of administration as used above, or at approximately 1 to 99 percent of the dosage used to date.

[0115] For the treatment of diseases, the appropriate dosage of the active ingredient depends on the type of disease to be treated, the severity and course of the disease, whether the active ingredient is applied for prevention or treatment, the patient's clinical history and response to the active ingredient of the present invention. All of the above factors should be taken into account and the decision should be made by the attending physician.

[0116] In one or more treatments, the anti-Met antibody fragments of this disclosure are appropriately administered to a patient. Depending on the type and severity of the disease, an antibody dose of about 1 mg / kg to 30 mg / kg is the initial candidate dose for administration to the patient, for example, by one or more single administrations or by continuous infusion. Depending on the factors described above, a typical daily dose range can be from about 1 µg / kg to 100 mg / kg or higher. For repeated administration over several days or longer, treatment continues until the desired suppression of disease symptoms is achieved, depending on the condition. An exemplary dose of the antibody fragment is in the range of about 0.05 mg / kg to about 20 mg / kg. Thus, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg can be administered to the patient. Such doses can be administered at intervals, for example, weekly or every three weeks (e.g., so that the patient receives about two to about twenty doses of the antibody, such as about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. An exemplary dosing regimen involves administering an initial loading dose of approximately 4 mg / kg, followed by a maintenance dose of approximately 2 mg / kg of antibody weekly. However, other dosing regimens may be used. Progression of this therapy can be easily monitored using routine techniques and assays.

[0117] In some embodiments, this disclosure relates to the use of the anti-Met antibody fragments disclosed herein in the treatment of tumors and / or metastases.

[0118] In some embodiments, this disclosure relates to the use of the anti-Met antibody fragments disclosed herein in the treatment of tumors and / or metastases in patients carrying genetic alterations to the MET gene.

[0119] In some embodiments, this disclosure relates to the use of the anti-Met antibody fragment disclosed herein in the treatment of tumors and / or metastases in patients carrying the wild-type MET gene.

[0120] In some embodiments, this disclosure relates to a method for preparing the anti-Met antibody fragment disclosed herein, the method comprising the steps of: (i) synthesizing cDNA sequences of a first, second, and third polypeptide constituting the anti-Met antibody fragment; (ii) inserting the three cDNA sequences into one or more plasmids suitable for expression in mammalian cell lines; (iii) transiently or stably co-transfecting mammalian cell lines with the plasmids; (iv) collecting the culture supernatant; and (v) purifying the anti-Met antibody fragment by affinity chromatography.

[0121] Example Example 1: Reasons and Selection for Possible Silent Mutations While Met is a proven target for treating tumors and metastases, there remains the possibility that a suitable targeting agent could trigger undesirable dimerization and activation of the c-Met receptor, thereby initiating a downstream immune cell-mediated signaling cascade. Therefore, it is inferred that eliminating Fcγ receptor binding could potentially prevent such undesirable downstream effects. This approach presupposes that the targeting agent is independent of Fc effector function. In this disclosure, an exemplary targeting agent of this kind has been selected to test this hypothesis. VERT001 is an anti-Met antibody fragment whose mode of action is independent of the Fc effector. It is hypothesized that a silenced version of VERT001 might be a suitable candidate. However, the engineering of antibodies and antibody fragments always carries the risk of not only introducing novel functions into the corresponding molecule but also potentially causing the molecule to lose other functions, such as potency, stability, or other properties, which could limit the use of the resulting derivatives for therapeutic development.

[0122] Derivatives of VERT001 were generated in which certain silent mutations were engineered into the Fc region of VERT001. VERT001 with the PA-LALA mutation produced VERT002. VERT001 with the AEASS mutation produced VERT004. VERT001, VERT002, and VERT004 were compared in the following examples.

[0123] The amino acid sequence of the binder is shown in the table below.

[0124] Table 1 VERT-002 has the following mutations in the CH2 domain: L234A, L235A, and P329A. VERT-004 has the following mutations in the CH2 domain: L234A, L235E, G237A, A330S, and P331S.

[0125] Example 2: General methods used in this study Example 2.1 Purity evaluation by size exclusion chromatography (SEC) Monomer content was assessed using HP-SEC analysis with an Agilent 1260 Infinity II. Liquid chromatography separation was performed using an Advance Bio SEC column (300A 2.7 μm, 4.6 * 300 mm). Chromatography was performed at room temperature (RT) at a flow rate of 0.35 ml / min. A 200 mM sodium phosphate buffer (pH 7.0) was used as the mobile phase. Samples were diluted to 1 mg / ml in a 200 mM sodium phosphate buffer (pH 7.0) before chromatography. Samples were centrifuged at 75,000 rcf for 5 min (except for samples subjected to 55°C and pH stress, which were filtered). 80 μl of supernatant was transferred to an HPLC vial with an inner tube. Manually integrated chromatograms were obtained using ChemStation software. MW standards were used for column quality control.

[0126] Example 2.2 Purity evaluation by strong cation exchange chromatography (SCX) Size variation spectra were analyzed using SCX chromatography with an Agilent 1260 Infinity II. Liquid chromatography separation was performed using an Agilent bio SCX NP1.7 SS column (4.6 * 50 mm). Chromatography was conducted at room temperature (RT) at a flow rate of 0.8 mL / min. Mobile phase A (loading) was 20 mM sodium phosphate buffer (pH 6.0), and mobile phase B (elution) was 20 mM sodium phosphate buffer (pH 6.0) + 0.5 M NaCl. The gradient applied was as follows: Table 2 For sample preparation, the solution was diluted in 20 mM sodium phosphate buffer at pH 6.0. Samples were centrifuged at 75000 rcf for 5 minutes (except for samples subjected to 55 °C and pH stress, which were filtered). 80 μl of the supernatant was transferred to an HPLC vial with an inner tube. Manually integrated chromatograms were obtained using ChemStation software.

[0127] Example 2.3 Purity evaluation by hydrophobic interaction chromatography (HIC) Hydrophobic interaction chromatography was performed to assess sample purity using an Agilent 1260 Infinity II liquid chromatography system. Liquid chromatography separation was performed using an Agilent bio HIC 4.6*100mm, 3.5 µm system. Chromatography was conducted at room temperature (RT) at a flow rate of 0.8 ml / min. Mobile phase A (loading) was 50 mM sodium phosphate buffer (pH 7.0) + 2 M (NH₄)₂SO₄, and mobile phase B (elution) was 50 mM sodium phosphate buffer (pH 7.0). The application gradient was as follows: Table 3 For sample preparation, the solution formulation was diluted to 1 mg / mL in 50 mM sodium phosphate buffer at pH 7.0. Samples were centrifuged at 75000 rcf for 5 min (except for samples subjected to 55 °C and pH stress, which were filtered). 80 μl of the supernatant was transferred to an HPLC vial with an inner tube. Manually integrated chromatograms were obtained using ChemStation software.

[0128] Example 2.4 Purity evaluation by reversed-phase UPLC-MS The purity of the samples was assessed by UPLC-MS analysis using a Waters Acquity Hclass+ system with a single quadrupole detector connected to a PDA and a Waters SQD2. Liquid chromatography separation was performed using a BEH column C4 300A, 2.1 x 50 mm.

[0129] For sample preparation, the solution formulation was diluted in water to a final volume of 0.5 mg / ml (100 μl). Data acquisition and processing were performed using MassLynx. The molecular weight of the protein was determined using Max Ent1. LC-MS parameters are shown in the table below.

[0130] Table 4 Example 2.5 Measurement of pyrolysis folding by differential scanning fluorescence Samples were characterized using Sypro Orange dye (SO) via differential scanning fluorometry (DSF). Briefly, protein samples were mixed with SO dye in PBS buffer, a temperature gradient was applied, and fluorescence was monitored as a function of temperature (°C). Under heating conditions, proteins begin to unfold and expose their hydrophobic core residues. SO interacts with this hydrophobic core, and an increase in fluorescence can be measured. 5000X Sypro Orange dye was diluted in PBS to a concentration of 10x. All samples were diluted in PBS buffer to a concentration of 2 mg / mL and then mixed with 10x SO at a 1:1 volume ratio to obtain a final concentration of 1 mg / mL sample and 5X SO in PBS. Each sample was measured three times.

[0131] Samples were transferred to 96-well PCR plates, and a temperature gradient from 25 °C to 95 °C was applied using a C1000 RT-PCR thermal cycler (Biorad), increasing by 0.5 °C every 10 seconds. The melting temperature (Tm) was determined using the first derivative of the curves, which provides information about the conformational stability of the protein samples. SO fluorescence at 25 °C relative to each sample (the starting point of the temperature increase) was used to provide qualitative information about the initial folding state.

[0132] Example 2.6 ELISA Binding Assay First, the microplates were coated. For this purpose, recombinant human cMET ECD His (SinoBiological PN: 10692-H08H) was diluted with PBS to a final concentration of 1 µg / ml in an F96 IMMUNOPLATE MAXISORP plate, with a final volume of 50 µl, and incubated overnight at 4 °C. The plate was then blocked to minimize nonspecific binding. The coating solution was removed, and 100 µl of blocking buffer (MSD blocking buffer) or 5% BSA in PBS was added to each well. The plate was incubated for 1 hour at room temperature on a rocker at 600 rpm. The plate was then washed three times with MSD washing buffer or PBS-0.05% P20.

[0133] Next, 50 µl of diluted primary antibodies (VERT001, VERT002, and VERT-04; titrated in a separate plate using assay buffer = PBS 1x, 0.1% BSA w / v, 0.02% P20) were added to each well. Concentrations used were 400, 100, 25, 12.5, 6.25, 0.39, 0.098, 0.024, 0.0061, 0.0015, and 0.00038 μg / ml. All samples were prepared in duplicate. The plates were incubated at 600 rpm on a track shaker for 1 hour at room temperature, followed by washing three times with MSD wash buffer or PBS-0.05% P20. The diluted secondary antibody (goat anti-human IgG Fc secondary antibody, HRP 1:20000) was then added to each well (50 µl) and incubated on a rocker at 600 rpm for 1 hour at room temperature, followed by washing three times with MSD wash buffer or PBS-0.05% P20. Finally, for detection, TMB (3,3',5,5'-tetramethylbenzidine) (50 µl / well) was added and incubated for 1 to 5 minutes (until blue color appeared), followed by an equal volume of stop solution (2 M H2SO4). The absorbance was then measured at 450 nm using a microplate reader. A curve of absorbance at 450 nm relative to the primary antibody concentration was plotted, and the data were fitted to a 4PL formula to obtain the EC50.

[0134] Example 2.7 Proliferation Assay Hs746T cells were purchased from ATCC (catalog number HTB135) and passaged in Dulbecco's MEM (4.5 g / L glucose + GlutaMAX, Gibco 31966) supplemented with 1% sodium pyruvate (Gibco, 11360) and 10% heat-inactivated FBS (PAN Biotech P30-1909), incubated at 37 ℃ and 5% CO2. EBC-1 cells were purchased from JCRB (catalog number JCRB0820) and passaged in RPMI (+glutamine, + 25 mM HEPES, Gibco 22400) supplemented with 10% heat-inactivated FBS, incubated at 37 ℃ and 5% CO2.

[0135] Proliferation assays were performed on white 96-well plates (ThermoFisher 136101): cells were seeded on day 0 (5,000 Hs746T cells per well, 2,000 EBC-1 cells per well), the compound was added on day 1, and viability was measured on day 4. On day 1, the compound was diluted to a concentration of 2000 µg / ml, and 9 dose-response assays (1:4 serial dilutions) were prepared in culture medium, followed by cell addition (final starting concentration of 200 µg / ml). All samples were tested in duplicate. Viability was assessed using the CellTiter-Glo 2.0 luminescent cell viability assay kit (Promega) to measure cellular ATP content. CellTiter-Glo (1:10 dilution) was added, and the cells were co-incubated with the cell on a plate shaker at 600 rpm for 20 minutes in the dark. Luminescence was recorded using an EnVision multimode microplate reader (Perkin Elmer). On day 1, the viability of untreated cells was measured to quantify the background, and this value was subtracted from the viability results on day 4. Nonlinear regression was performed using a four-parameter (variable slope) curve implemented via GraphPad Prism (version 9.3.0) to determine the IC50, which was used to fit the dose-response data (after subtracting the background level from day 1).

[0136] Example 2.8 Stability Study For stability studies, six tubes containing 1 ml of the corresponding antibody were removed from a -80 °C freezer and thawed at room temperature under light-protected conditions. Then, each solution was aliquoted into 3R glass vials (400 µl each for VERT001 and VERT002, and 350 µl each for VERT004 due to its smaller initial volume) under a laminar flow hood, ensuring the solutions were sterile. The vials were then sealed with rubber stoppers and flip-off seals.

[0137] Each antibody was stored in three vials at 5 °C, three vials at 40 °C, and one vial at -80 °C. One vial was used for the T0 measurement. The time points were as follows: D0, week 1 (W1), W2, and W4. At each time point, a fresh aliquot of the corresponding antibody vial was removed from the -80 °C freezer and used as a standard in all characterization techniques. This allowed for evaluation of deviations and variations in the runs between time points.

[0138] Reserve 3 ml of each solution for forced degradation studies (pH and heat). Before the study began, samples were stored at 5 °C for 2 days.

[0139] Example 2.9 Forced Degradation Study Example 2.9.1 pH stability Samples were prepared as described in Example 2.8. The pH of each 850 µl solution was then adjusted to pH 3 with 1M HCl or to pH 9 with 1M NaOH. The vials were stored at room temperature protected from light for 5 days. Samples were taken and stored at -80 °C on D0 and D2. On D5, samples were stored directly at -80 °C. On the day of analysis, aliquoted samples were aseptically filtered to make them suitable for cell assays. Therefore, centrifugation was not used in the preparation of various LC samples.

[0140] Example 2.9.2 Temperature stability Samples were prepared as described in Example 2.8. Each solution was then aliquoted 300 µl into Eppendorf tubes and incubated for 5 days at 55 °C in the dark. On the day of analysis, the samples were aseptically filtered to make them suitable for cell assays. Therefore, centrifugation was not used in the preparation of any of the LC samples.

[0141] Example 2.9.3 Freeze-thaw stability Samples were prepared as described in Example 2.8. Aliquots of each antibody were removed from -80 °C and stored at room temperature in the dark for 60-90 minutes. After complete thawing, the aliquots were stored again at -80 °C. A total of 3 freeze-thaw cycles were performed.

[0142] Example 2.10 Concentration Measurement Protein concentration was measured using a nanophotometer NP80 (Implen) via A280. The extinction coefficients (l / g*cm) for VERT001 and VERT004 were 1.48, and for VERT002, it was 1.49. 2 μl of sample was pipetted onto the sample window. A blank was performed using PBS buffer. The reported concentration is the average of two measurements.

[0143] Example 3: Biophysical Properties and Developability Assessment Example 3.1 Comparison of Forms VERT-001 (wild-type Fc), VERT-002 (PA-LALA), and VERT-004 (AEASS) were compared using multiple assays without exposure to any stress conditions.

[0144] Example 3.1.1 SEC Analysis In the SEC analysis, all forms showed high monomer content above 99%. For all three samples tested, the percentages of high molecular weight (HMW) and low molecular weight (LMW) components were substantially consistent. See Table 5.

[0145] Table 5 Example 3.1.2 SCX Analysis The SCX chromatogram is shown in Figure 1 In the SCX analysis, all three reagents showed similar distributions of charged species. VERT-004 appeared to be more acidic, possibly due to the L-to-E mutation. Table 6 shows the percentages of the major components identified by the SCX analysis, as well as the percentages of acidic and basic components.

[0146] Table 6 Example 3.1.3 HIC Analysis All three forms of HIC chromatograms appeared highly identical. All three forms showed relatively early retention times, indicating low hydrophobicity.

[0147] Example 3.1.4 Inverted UPLC-MS analysis On the RP chromatogram, VERT-004 showed a slightly different spectrum compared to VERT-001 and VERT-002, indicated by the presence of multiple peaks. See Figure 2 .

[0148] The mass of the major UV peak for each antibody was determined by MS and is summarized in the table below.

[0149] Table 7 Various forms of quality differences are highly consistent with their mutations.

[0150] ΔVERT-001-VERT-002 = 220 Da, corresponding to mutations PLL (SEQ ID NO: 24) to AAA (SEQ ID NO: 25): 2 x 110 Da.

[0151] ΔVERT-001 - VERT-004 = 15 Da, corresponding to the mutation LLGAP (SEQ ID NO: 26) to AEASS (SEQ ID NO: 27): 2 x 6 Da.

[0152] Detailed quality analysis was performed on each individual peak present in VERT-001, VERT-002, and VERT-004. The summary and tentative component assignment of VERT-004 are described in... Figure 3 VERT-001 and VERT-002 also contain small amounts of truncated HC and a small amount of unidentified components, corresponding to the MW – 10 kDa of the two antibodies at 90 and 89 kDa, respectively.

[0153] Example 3.1.5 Differential Scanning Fluorescence Method The thermal decomposition and folding curves of antibodies VERT-001, VERT-002, and VERT-004 were recorded using differential scanning fluorometry, and the melting temperature (Tm) and fluorescence observed at 25 °C (RFU t0) were determined. The Tm values ​​for VERT-001 and VERT-002 were 68 °C and 68.5 °C, respectively, while VERT-004 showed a lower Tm of 62.5 °C. Initial fluorescence for all Fc forms began at approximately 4000 RFU. The results are summarized in the table below.

[0154] Table 8 Example 3.1.6 Combination with human c-Met ECD The binding of VERT001, VERT002, and VERT004 to the ECD of c-Met was measured in the ELISA assay. The binding ELISA curves are shown... Figure 4 The measured EC50 values ​​are shown in the table below.

[0155] Table 9 The binding curves of all three binders, VERT001, VERT002, and VERT004, were similar. VERT002 showed the highest affinity.

[0156] Example 3.1.7 Affinity determination by BLI Affinity was determined by BLI, and the binding affinity of VERT-001, VERT-002, and VERT-004 was compared. The results are shown in Table 10.

[0157] Table 10 Of all three antibodies, VERT-002 showed the highest affinity.

[0158] Example 3.1.8 Proliferation The antiproliferative activities of VERT-001, VERT-002, and VERT-004 were measured in assays using Hs746T cells and EBC-1 cells, respectively. Results are shown in... Figure 5 The measured IC50 values ​​are shown in the table below.

[0159] Table 11 Example 3.2 Stability Study The sample was prepared as described in Example 2.8.

[0160] Example 3.2.1 Visual inspection of stability samples Throughout the 4-week period, stability samples of all three antibody forms remained clear and free of particles at 5 °C and 37 °C. Concentrations remained constant during the 4-week period at 5 °C and 37 °C.

[0161] Example 3.2.2 Aggregate and fragment analysis using SEC The formation of monomers, HMW, and LMW during incubation was determined by SEC analysis. The results are shown in... Figure 6 .

[0162] For VERT002 (0.4%) and VERT004 (0.6%), a very slight monomer reduction associated with increased HMW was observed at 5 °C. At 37 °C, VERT-001 and VERT-002 exhibited similar slow to moderate monomer reduction kinetics, accompanied by aggregation (increased HMW) and fragmentation (increased LMW). Higher aggregation kinetics were observed for VERT004, as indicated by a rapid decrease in monomer content and an increase in the HMW component. The increase in LMW of VERT-004 followed a similar slope to that of VERT-001 and VERT-002.

[0163] Example 3.2.3 Charge Variant Analysis via SCX The formation of charge variants during incubation was analyzed by strong cation exchange chromatography. The results are shown in... Figure 7 At 5 °C, the charge variant spectra of all forms remained stable. When exposed to 37 °C, all forms showed the same trend in charge variant evolution, namely, an increase in acidic components over time, which may correspond to deamidation and a decrease in the main peak.

[0164] Example 3.2.4 Stability analysis by RP-HPLC Chemical stability was monitored during incubation using a RP with UV and MS detection. No major degradation was observed for any form. However, after 4 weeks at 37 °C, a small additional peak was detected in all antibodies (RT = 4.48 min), which may be attributed to fragmentation.

[0165] Example 3.2.5 Stability analysis using DSF The stability of all three antibodies after incubation at 5 °C and 37 °C for 4 weeks was investigated by DSF compared to freshly thawed reference material. The melting temperature of all samples was the same as that observed at time point zero (T0), regardless of storage conditions. All FC forms showed initial fluorescence of approximately 4000 RFU. The results are summarized in the table below.

[0166] Table 12 Example 3.2.6 Combination with human c-Met ECD In ELISA assays, the binding of VERT001, VERT002, and VERT004 to the ECD of c-Met was measured after incubation at 5 °C and 37 °C for 4 weeks, compared to freshly thawed reference materials. When samples were incubated at 37 °C for 4 weeks, the binding potency of VERT-001, VERT-002, and VERT-004 showed a slight decreasing trend compared to the assay standards. The same samples incubated at 5 °C for the same time appeared stable, and no decrease in binding potency was observed compared to the assay standards. Comparisons of sample binding to the reference material are shown in... Figure 8 .

[0167] Example 3.2.7 Proliferation The antiproliferative activities of VERT001, VERT002, and VERT004 after incubation at 5 °C and 37 °C for 4 weeks, compared to freshly thawed reference material, were compared in Hs746T and EBC-1 cells. Overall, the antiproliferative activities of all samples were within the expected range throughout the study period, but slight shifts in potency were observed at week 4 in both cell lines (higher IC50 in Hs746T and lower IC50 in EBC-1). The measured IC50 values ​​are shown in the table below.

[0168] Table 13 Example 3.3 Forced Degradation Study Example 3.3.1 Visual inspection of pH-stressed samples The pH-stressed samples remained clear and free of particles for 5 days. At D0 and D5, the concentrations of VERT002 and VERT004 decreased slightly at pH 3, possibly due to dilution during pH adjustment.

[0169] Example 3.3.2 Chemical degradation of pH-stressed samples by RP analysis pH stress at pH 3 and pH 9 did not result in any significant degradation of the antibodies, as indicated by the presence of a main peak corresponding to the correct intact mass and a constant peak height during D0 to D5. At pH 3, low-level degradation was observed for VERT-002 and VERT-004 after 5 days, manifested as a small elution peak preceding the intact antibody. The deconvolution intact mass (in Da) of the pH-stressed samples is shown in the table below.

[0170] Table 14 Example 3.3.3 Analysis of pH3-stressed samples by SCX and SEC At pH 3, all forms of protein strongly aggregated at D0. No peak was detected by SCX analysis of the sample. Since RP confirmed the presence of the protein, the reason for the undetectable SCX result is likely that strong aggregation prevented peak elution. An exemplary chromatogram (VERT002) is shown below. Figure 9 .

[0171] Example 3.3.4 Analysis of pH9 stress samples by SCX and SEC At pH 9, a slight increase in HMW was observed in all three antibodies. A small increase in the acidic component was also observed. No differences were observed among CERT001, VERT002, and VERT004.

[0172] Example 3.3.5 Analysis of Stressed Samples by DSF The effect of pH on the stability of VERT samples was analyzed by DSF. VERT-002 and VERT-004 incubated at pH 9 showed no visible change in initial fluorescence or Tm. When both samples were incubated at pH 3, very high initial fluorescence at t0 was detected, and Tm could not be determined. This indicates that VERT-002 and VERT-004 have lower stability (aggregation and / or misfolding) at acidic pH, while alkaline pH appears to have no effect on these samples in DSF. The observations at pH 3 correlated well with the SEC results showing aggregation. The results are summarized in the table below.

[0173] Table 15 Regarding temperature stress, incubation at 55 °C for 5 days showed all samples exhibited significantly higher initial fluorescence (RFU at t0) compared to the assay standards, possibly attributed to aggregation or misfolding events. All antibodies were unaffected by three freeze-thaw (F / T) cycles, as shown by the unaffected Tm and RFU at t0. Results are summarized in the table below.

[0174] Table 16 Example 3.3.6 Analysis of stress samples at 55 °C All antibodies were treated at 55 °C for 5 days. VERT001 and VERT002 formed some filaments at the end of the incubation period, while VERT004 showed particles. After filtration, only VERT004 showed a significant decrease in antibody concentration (4.4 mg / ml). Under these stress conditions, all forms showed increased aggregation (higher HMW) and fragmentation (higher LMW). See the table below.

[0175] Table 17 In RP chromatography analysis, some minor impurities / fragments were detected, which were found during elution of VERT-002 and VERT-004 (D5 55 ℃) at 4.50 min. Mass deconvolution suggested the presence of a fragment of approximately 14 kDa. VERT-001 was not analyzed due to limitations in the remaining sample volume. Interestingly, a reduction in initial fragments / impurities (truncated FC, LC, mismatched fragments) was observed for VERT-004 (and to a lesser extent for VERT-002). The 55 ℃ condition resulted in similar observations to those obtained after 4 weeks of incubation at 37 ℃.

[0176] Example 3.3.7 Analysis of samples subjected to freeze-thaw cycles VERT001, VERT002, and VERT004 underwent three freeze-thaw cycles (-80 °C). No changes were observed in IEX, SEC, or RP. The following table summarizes the percentages of monomer, HMW, and LMW determined by SEC analysis.

[0177] Table 18 Example 3.3.8 Analysis of antiproliferative activity of stressed samples The antiproliferative activity of all three antibodies, VERT001, VERT002, and VERT004, under different stress conditions was tested.

[0178] At pH 3, all antibodies completely lost their antiproliferative activity on Hs746T and EBC-1 cells, while at pH 9, the activity of stressed antibodies remained similar to that of the reference substance.

[0179] The antiproliferative activity of the binders subjected to 5 days of stress at 55 °C was tested only on Hs746T. The activities of VERT-001 and VERT-002 subjected to stress at 55 °C remained within the expected range, while VERT-004 lost its antiproliferative activity.

[0180] The results are shown in Figure 10 The IC50 values ​​for cell line Hs746T are shown in Table 18, and the IC50 values ​​for cell line EBC-1 are shown in Table 19.

[0181] Table 19 Table 20 The antiproliferative activity of VERT-001, VERT-002, and VERT-004 after three freeze-thaw (F / T) cycles did not show any deviation from the activity of the standard determined after F / T cycles.

[0182] Example 4: Pharmacological and therapeutic studies Example 4.1: Efficacy and pharmacology in the Hs746T xenograft model In a subcutaneous (sc) implantation model of Hs746T in female hairless SCID mice (MET exon 14 skipping and MET amplification), the in vivo antitumor potency of VERT-001 was compared with that of VERT-002 and VERT-004. All variants were tested at the same dose level, administered intravenously (iv) at 20 mg / kg twice weekly (BIW). Results are shown in Figure 11 All tested variants showed full and comparable potency at a dose of 20 mg / kg.

[0183] The dose-response effect of VERT-002 on in vivo antitumor efficacy was further evaluated in the Hs746T CDX model. VERT-002 was administered intravenously at doses of 10 and 20 mg / kg twice weekly and 20 mg / kg once weekly, while an isotype control was administered 20 mg / kg twice weekly for up to 4 weeks. Figure 12 VERT-002 demonstrated dose-dependent tumor growth inhibition (TGI), achieving complete tumor regression when administered at the highest dose of 20 mg / kg twice weekly. By day 35, 6 out of 9 mice from the highest dose group were tumor-free, reflecting a very low mean tumor volume level on day 35.

[0184] Mice treated with 20 mg / kg twice weekly were monitored for more than 30 days after the end of administration, with the majority of mice (6 / 9) in the treatment group remaining tumor-free. At the end of the administration period (day 32), the 3 / 9 mice with residual tumors were the only mice to experience tumor regrowth during the post-administration monitoring period.

[0185] Example 4.2: Efficacy and pharmacology in the EBC-1 xenograft model VERT-002 was further characterized in vivo in EBC-1 cells (carrying MET amplification) implanted subcutaneously in female Balb / c nude mice. VERT-002 was administered intravenously at doses of 5, 10, and 20 mg / kg twice weekly, and 20 mg / kg once weekly, while an isotype control was administered 20 mg / kg twice weekly. Figure 13Figures A and B). VERT-002 demonstrated a clear dose-dependent tumor reversion (TGI), with complete tumor regression observed at 20 mg / kg twice weekly, consistent with results from the Hs746T CDX model. Furthermore, VERT-002 induced a significant increase in soluble MET extracellular domain (sMET ECD) levels, which was dose-dependent and plateaued around day 6 of treatment. Figure 13 (Figure C). Interestingly, in response to VERT-002 administration, sMET ECD levels reflected the tumor regression curve, indicating that as the tumor shrank (tumor regression began around the same time point, day 7), the amount of shed ECD decreased. Similar dose-response results for TGI and sMET ECD were also observed in a chronic study (single-dose) of the EBC-1 model (data not shown).

Claims

1. An anti-Met antibody fragment comprising a single antigen binding arm and a silent Fc region, wherein the Fc region comprises a complex of a first and a second Fc polypeptide, wherein the antibody fragment comprises: (i) a first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain contains three complementarity determining regions (CDRs) having the amino acid sequences as set forth in SEQ ID NOs: 3, 5, and 6; (ii) a second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a hinge region, a human constant CH2 domain, and a human CH3 constant domain, wherein the humanized VH domain contains three complementarity determining regions (CDRs) having the amino acid sequences as set forth in SEQ ID NOs: 8, 10, and 12, and (iii) a third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human CH3 constant domain, wherein the Fc region of the first Fc polypeptide and the second Fc polypeptide comprises the mutations L234A, L235A, and P329A (according to EU index).

2. The anti-Met antibody fragment of claim 1, wherein the humanized VL domain is fused in N- to C-terminal direction to a human CL domain.

3. The anti-Met antibody fragment of claim 1 or 2, wherein in N- to C-terminal direction the humanized VH domain is fused to a human CH1 domain, which is fused to a human hinge region, which is fused to a human CH2 domain, which is fused to a human CH3 domain.

4. The anti-Met antibody fragment of any one of claims 1 to 3, wherein in N- to C-terminal direction the human hinge region is fused to a CH2 domain, which is fused to a human CH3 domain, wherein the human hinge region is truncated at the N-terminus.

5. The anti-Met antibody fragment of any one of claims 1 to 4, wherein the humanized VL domain has the amino acid sequence as set forth in SEQ ID NO:

13.

6. The anti-Met antibody fragment of any one of claims 1 to 5, wherein the humanized VH domain has the amino acid sequence as set forth in SEQ ID NO:

14.

7. The anti-Met antibody fragment of any one of claims 1 to 6, wherein the first and the second Fc polypeptides meet at an interface, and one of the first and the second Fc polypeptides comprises a knob at the interface and the other of the first and the second Fc polypeptides comprises a hole at the interface, wherein the knob is positionable into the hole, wherein the first or the second Fc polypeptide comprises a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries an amino acid mutation at position 389, wherein the original amino acid at position 389 has been mutated to introduce an amino acid with a larger side chain volume than the original amino acid; and wherein the other Fc polypeptide comprises a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries three amino acid mutations at positions 389, 391 and 438, wherein the original amino acids have been mutated to introduce amino acids with a smaller side chain volume than the original amino acids, wherein the amino acid numbering is according to the EU numbering scheme of Kabat.

8. The anti-Met antibody fragment of claim 7, wherein the original amino acids at positions 389, 391 and 438 are threonine, leucine and tyrosine, respectively; and wherein in the first or the second Fc polypeptide, the threonine at position 389 has been mutated to a tryptophan; and wherein in the other Fc polypeptide, the threonine at position 389 has been mutated to a serine, the leucine at position 391 has been mutated to an alanine and the tyrosine at position 438 has been mutated to a valine.

9. The anti-Met antibody fragment of any one of claims 1 to 8, wherein the human CL domain has the amino acid sequence of SEQ ID NO: 15 and the human CH1 domain has the amino acid sequence of SEQ ID NO:

16.

10. The anti-Met antibody fragment of any one of claims 1 to 9, wherein the first human Fc polypeptide has the amino acid sequence of SEQ ID NO: 17 and the second human Fc polypeptide has the amino acid sequence of SEQ ID NO:

18.

11. The anti-Met antibody fragment of any one of claims 1 to 10, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 19, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20 and the third polypeptide comprises the amino acid sequence of SEQ ID NO:

18.

12. An isolated nucleic acid encoding the anti-Met antibody fragment of any one of claims 1 to 11.

13. A composition comprising two or more recombinant nucleic acids collectively encoding the anti-Met antibody fragment of any one of claims 1 to 11.

14. Use of the anti-Met antibody fragment of any one of claims 1 to 11 for the treatment of a tumor and / or metastasis.

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