Silenced antibody-based anti-MET construct for the treatment of tumors and metastases

Abstract

This disclosure relates to a novel silencing antibody-based therapeutic agent for the treatment of tumors and / or metastases. The therapeutic agent of this disclosure is monovalent and specific to MET.

Description

Technical Field

[0001] The present disclosure relates to a novel silenced antibody-based therapeutic agent for the treatment of tumors and / or metastases. The therapeutic agent of the present disclosure is monovalent and specific for MET.

Background Art

[0002] Cancer is a genetic disease in which somatic endogenous genes undergo mutations. Only a small number of genes known as oncogenes and tumor suppressor genes are altered in cancer cells and drive tumorigenesis. Activated oncogenes are promoters, and inactivated tumor suppressor genes are a lack of brakes on cancer cell growth. Following the discovery of mutant genes that cause cancer, a new concept regarding oncogenes, "oncogene addiction," emerged, indicating that cancer cells still depend on a single oncogenic protein for their sustained proliferation or survival, despite their numerous genetic changes. Therefore, new therapeutic interventions have been made to develop "targeted therapies" for curing cancer. In the last 20 years, pharmacological targeting of proteins encoded by mutant genes that cause cancer, via chemical drugs or antibodies, has represented the forefront tool for eliminating mutant cells and combating cancer diseases. Targeted therapies are expected to be more effective than conventional cytotoxic chemotherapy, often with fewer side effects. However, only patients who harbor a specific target gene altered in their tumors in the body are likely to benefit from targeted therapies. That is, personalized medicine is required in parallel to evaluate druggable genetic lesions through comprehensive genomic profiling of patient tumors.

[0003] The MET oncogene encodes a unique receptor tyrosine kinase with multifaceted functions. When genetic changes occur (by point mutations, gene fusions, translocations, and / or amplifications), MET initiates cellular transformation through its ability to activate invasive growth programs. In other words, MET gene lesions leading to constitutive MET kinase hyperactivation initiate and maintain a transformative phenotype ("MET toxicity").

[0004] MET gene lesions occur in the majority of solid tumors with an overall frequency 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, perimembrane domain, and catalytic domain). Very recently, next-generation sequencing has revealed exon 14 splicing site mutations in 3% of non-small cell lung cancers. 2 This results in exon skipping and deletion of the near-membrane region of the MET transcript, where the serine residue (Ser985) negatively regulates MET kinase activity. 3 The tyrosine residue (Tyr1003) is required for MET internalization and degradation. 4 .

[0005] In the "invasive proliferation" program induced by MET, the proliferative response is linked to migration, survival, extracellular matrix degradation, and induction of cell polarity. 5 These biological responses are sought by cells to adapt to adverse conditions and / or to escape and find a more favorable environment. In unfavorable contexts, Met is overexpressed via transcriptional upregulation in response to a variety of stimuli, including hypoxia, inflammatory cytokines, pro-angiogenic factors, mitogens, and even HGF itself. Finally, Met is overexpressed under conditions of radiation-induced DNA damage and contributes to resistance to radiotherapy by promoting the activation of DNA repair and avoiding programmed cell death in cancer cells.

[0006] Several Met-targeting molecules have been developed to selectively, robustly, and highly effectively swathe hyperactive Met signaling. These drugs include: HGF antagonists (either blocking antibodies or decoys), mAbs targeting the MET receptor, and chemotyrosine kinase inhibitors (TKIs). Anti-Met mAbs may represent a key step in the fight against MET-driven cancers. Recently, four anti-Met mAbs have entered early clinical trials: MetMab (onartuzumab, Roche), LY2875358 (emibetuzumab, Eli Lilly & Company), ARGX-111 (Argenx), and SAIT301 (Samsung) and Sym015 (Symphogen A / S), which are mixtures of two antibodies. These drugs act by competitively blocking HGF binding to MET (onartuzumab, ARGX-111) and / or downregulating MET (emibetuzumab, SAIT301, Sym015).

[0007] The mouse DN30 mAb (disclosed in WO2007 / 090807) is an IgG2A that binds to the extracellular domain of the human Met receptor and induces only some of the biological effects induced by Met. 6 This mAb partially activates receptor phosphorylation due to its bivalent nature, which allows for simultaneous binding to two different antigen molecules, resulting in stabilization of the receptor complex in a manner similar to that achieved by the native ligand. This undesirable partial agonist activity against Met was not observed with the monovalent DN30 Fab fragment (MvDN30). 7The conversion of the bivalent DN30 parent antibody to a monovalent Fab fragment unlocks the therapeutic potential of the DN30 anti-Met antibody, producing a complete antagonist molecule. However, the short half-life of Fab, due to its low molecular weight, is a significant limitation for its deployment in therapy. Therefore, we have developed a novel genetically modified molecule called DCD (Dual Constant Domain Fab), characterized by the duplication of constant domains present in DN30 Fab: DCD-1, in which the duplication is performed in tandem, and DCD-2 (disclosed in WO2014 / 108829), in which the light and heavy chain constant domains are alternately swapped. Both novel recombinant molecules exhibit biochemical properties equivalent to the original Fab in vitro and act as complete Met antagonists. In vivo, when administered systemically, the novel recombinant molecules reduce Met-toxic tumor growth. Although neither DCD-1 nor DCD-2 achieves the same behavior as the original mAb, they exhibit improved pharmacokinetic profiles compared to the original DN30 Fab. 8 .

[0008] WO2020 / 074459 discloses monovalent agents specific to Met. In these agents, one arm of the antibody is deleted by molecular genetic engineering, resulting in improved in vivo stability due to the activity of the Fc domain that binds 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) Mar 29;41(1): 112. Different one-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 silencing mutations in the Fc region. In addition to the effects commonly attributed to such mutations, such mutations also have important additional safety aspects. Deactivation of Fcγ receptor binding reduces the risk of residual cMET dimerization via crosslinking on the tumor cell surface mediated by immune cell Fcγ receptors. [Overview of the project]

[0009] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. This relates to anti-Met antibody fragments, including those mentioned above.

[0010] In certain embodiments, the humanized VL domain is fused with the human CL domain in the direction from the N-terminus to the C-terminus. In certain embodiments, the humanized VH domain is fused with the human CH1 domain in the direction from the N-terminus to the C-terminus, the human CH1 domain is fused with the human hinge region, the human hinge region is fused with the human CH2 domain, and the human CH2 domain is fused with the human CH3 domain in the direction from the N-terminus to the C-terminus. In certain embodiments, the human hinge region is fused with the CH2 domain in the direction from the N-terminus to the C-terminus, the CH2 domain is fused with the human CH3 domain, and the human hinge region is cleaved at the N-terminus.

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

[0012] In certain embodiments, the first Fc polypeptide and the second Fc polypeptide associate at an interface, with one of the first and second Fc polypeptides containing a knob at the interface and the other containing a hole at the interface, the knob being located within the hole. In certain embodiments, either the first or second Fc polypeptide contains a mutant CH3 constant domain, the mutant CH3 constant domain holds an amino acid mutation at position 389, where the original amino acid at position 389 is mutated to import an amino acid having a larger side-chain capacity than the original amino acid; the other Fc polypeptide contains a mutant CH3 constant domain, the mutant CH3 constant domain holds three amino acid mutations at positions 389, 391 and 438, where the original amino acid is mutated to import an amino acid having a smaller side-chain capacity than the original amino acid, and amino acid numbering follows Kabat's EU numbering scheme. In certain embodiments, the original amino acids at positions 389, 391, and 438 are threonine, leucine, and tyrosine, respectively; in the first or second Fc polypeptide, threonine at position 389 is mutated to tryptophan; in the other Fc polypeptide, threonine at position 389 is mutated to serine, leucine at position 391 is mutated to alanine, and tyrosine at position 438 is mutated to valine.

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

[0014] In certain embodiments, the anti-Met antibody fragment of this disclosure induces the detachment of the extracellular domain of Met when bound to Met.

[0015] In certain embodiments, the Fc regions of the first Fc polypeptide and the second Fc polypeptide include mutants L234A, L235A, and P329A (according to the EU index), or mutants L234A, L235E, G237A, A330S, and P331S (according to the EU index). In certain embodiments, the first FC polypeptide and the second Fc polypeptide include mutants L234A, L235A, and P329A (according to the EU index).

[0016] In certain embodiments, 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.

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

[0018] In certain embodiments, the disclosure relates to any of the above-described anti-Met antibody fragments for use in the treatment of tumors and / or metastases. In certain embodiments, the disclosure relates to any of the above-described anti-Met antibody fragments for use in the treatment of tumors and / or metastases in patients carrying genetic alterations of the MET gene. In certain embodiments, the disclosure relates to any of the above-described anti-Met antibody fragments for use in the treatment of tumors and / or metastases in patients carrying the wild-type MET gene.

[0019] In certain embodiments, the present disclosure relates to a process for producing any of the above-mentioned anti-Met antibody fragments, comprising the steps: (i) synthesis of cDNA sequences of first, second, and third polypeptides constituting an anti-Met antibody fragment; (ii) insertion of the three cDNA sequences into one or more plasmids, wherein the plasmid is suitable for expression in a mammalian cell line; (iii) transient or stable co-transfection of a mammalian cell line using the plasmid; (iv) recovery of the culture supernatant; and (v) purification of the anti-Met antibody fragment by affinity chromatography. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1 shows the SCX chromatograms of the tested forms. All three forms showed similar distributions of charged molecular species. VERT-004 appears to be slightly acidic. [Figure 2] Figure 2 shows the RP chromatogram of the tested form. VERT-004 exhibits a somewhat different profile compared to VERT-001 and VERT-002, indicated by the presence of multiple peaks. [Figure 3] Figure 3 shows the mass spectrometry of each individual peak present in the RP chromatograms VERT-001, VERT-002, and VERT-004. [Figure 4] Figure 4 shows that the binding affinity of VERT001, VERT002, and VERT004 to c-Met ECD was measured in an ELISA assay. [Figure 5] Figure 5 shows the anti-proliferative activities of VERT-001, VERT-002, and VERT- against Hs746T cells (left) and EBC-1 cells (right). [Figure 6] Figure 6 shows the monomer, HMW, and LMW contents of VERT-001, VERT-002, and VERT-004 as determined by SEC analysis. RM = reference material fresh aliquot of VERT001. [Figure 7] Figure 7 shows the formation of charge variants as analyzed via strong cation exchange chromatography. RM = reference material fresh aliquot of VERT001. [Figure 8] Figure 8 shows 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 freshly thawed reference material. [Figure 9] Figure 9 shows the analysis of VERT-002 stressed at pH 3. The SEC chromatogram is shown on the left, and the SCX chromatogram is shown on the right. [Figure 10] Figure 10 shows the analysis of the anti-proliferative activities of stressed samples of VERT001, VERT002, and VERT004. [Figure 11] Female nude SCID mice were transplanted s.c. with Hs746T tumor cells on day 0 and treated with an isotype control, VERT-001, VERT-002, or VERT-004 over 28 days. Tumor volume (mm3) data are presented as mean ± standard error of the mean (SEM); 9 mice were included in each group. [Figure 12] Female nude SCID mice were transplanted s.c. with Hs746T tumor cells on day 0 and treated with an isotype control or the VERT-002 antibody. Tumor volume (mm3) data are presented as mean ± standard error of the mean (SEM); 9 mice were included in each group. A) Dose response to tumor growth inhibition; B) In vivo tumor growth at day 35. [Figure 13]Female nude Balb / c mice were sc-transplanted with EBC-1 tumor cells and treated with a 28-day chronic regimen using 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 treatment at days 1, 4, 8, 11, 15, 18, 22, and 25 after randomization; B) In vivo tumor growth at day 28; C) Levels of plasma-soluble MET ectodomain (sMET ECD) in response to various treatment conditions as shown. [Modes for carrying out the invention]

[0021] definition This disclosure relates to antibodies and antibody-based constructs that specifically bind to Met, as well as to the use of such constructs, particularly therapeutic uses, such as the treatment of tumors and metastases.

[0022] The terms "Met," "cMET," "cMet," and "MET" refer to the hepatocyte growth factor receptor, also known as HGFR or c-Met, a protein. Human Met has the following amino acid sequence (UniProt P08581):

[0023] The term “antibody,” as used herein, means a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, which interact with an antigen. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interrupted by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region 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 of the classical complement system (C1q). The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., Igd, IgG2, IgG3, IgG4, IgGA1, and IgGA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0024] The term "antibody fragment," as used herein, means one or more portions of an antibody that possess the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, or stabilizing spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; Fd fragments, which consist of VH and CH1 domains; Fv fragments, which consist of VL and VH domains from a single arm of the antibody; dAb fragments, which consist of a VH domain (Ward et al., (1989) Nature 341:544-546); and isolated 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 using recombinant methods by synthetic linkers, which allow them to be produced as a single protein chain that pairs the VL and VH regions together to form a monovalent molecule [known as 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 encompassed within the term “antibody fragments.” These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usability in the same manner as complete antibodies. Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs [see, for example, Hollinger and Hudson, (2005) Nature Biotechnology 23:1 126-1 136]. Antibody fragments can be transplanted into polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, describing fibronectin polypeptide monobodies).Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that form an antigen-binding site pair with a complementary light chain polypeptide [Zapata et al., (1995) Protein Eng. 8: 1057-1062; and U.S. Patent No. 5,641,870].

[0025] 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 [e.g., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 US 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]. See 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).

[0026] "Human antibody" or "human antibody fragment," as used herein, refers to an antibody and antibody fragment having a variable region in which both the framework region and the CDR region are derived from human sequences. Human antibodies can also be isolated from synthetic libraries or from transgenic mice (e.g., Xenomouse, OmniMouse, Harbour mouse, ATX-Gx mouse, Trianni mouse), insofar as each system produces an antibody having a variable region in which both the framework region and the CDR region are derived from human sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a sequence. Examples of human origin include antibodies containing human germline sequences, mutant versions of human germline sequences, or consensus framework sequences derived from human framework sequence analysis as described, for example, Knappik et al., (2000) J Mol Biol 296:57-86.

[0027] A “humanized antibody” or “humanized antibody fragment” is defined herein as an antibody molecule having a constant antibody region and a variable antibody region or a portion thereof derived from a human sequence, or where only the CDR is derived from another species. For example, a humanized antibody may be CDR-grafted, in which case the CDR of the variable domain is of non-human origin, but one or more frameworks of the variable domain are of human origin, and the constant domain (if present) is of human origin.

[0028] The terms “chimeric antibody” or “chimeric antibody fragment” are 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 corresponding to a sequence found in humans, and the variable antibody region (e.g., VH, VL, CDR, or FR region) is derived from a sequence found in a non-human animal, such as a mouse, rat, rabbit, or hamster.

[0029] The term "antigen-binding arm," as used herein, means a component of the antibody fragment of the present invention that has the ability to specifically bind to a target molecule of interest. The antigen-binding arm is a complex of variable domain sequences (VL and VH) containing the CDR and framework regions of the immunoglobulin light and heavy chains, and constant domain sequences (CL and CH) of the immunoglobulin light and heavy chains.

[0030] When used herein, the terms “hinge region,” “hinge array,” and their variations include meanings known in the art, as exemplified, for example, in Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999).

[0031] The term "cleaved hinge region," as used herein, means a polypeptide that contains a portion of a hinge sequence but not all of it. The cleaved hinge region is capable of binding to the "first" Fc polypeptide. If a wild-type hinge sequence is absent, the remaining sequence in the "second" Fc polypeptide will contain components capable of binding to the "first" Fc polypeptide. For example, such components may be modified residues or added cysteine ​​residues capable of forming disulfide bonds.

[0032] A “knob” refers to at least one amino acid side chain that protrudes from the interface of the first Fc polypeptide and may therefore be located in a compensatory hole in the adjacent interface (i.e., the interface of the second Fc polypeptide), thereby stabilizing the heteromultimer and thus making heteromultimer formation more likely than homomultimer formation, for example. Knobs 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 modified to encode a knob. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the first polypeptide is replaced by a nucleic acid encoding at least one “import” amino acid residue having a larger side chain capacity than the original amino acid residue. It will be understood that there can be two or more original residues and corresponding import residues. The upper limit on the number of original residues to be replaced is the total number of residues in the interface of the first polypeptide.

[0033] A “hole” refers to at least one amino acid side chain that recesses from the interface of the second Fc polypeptide and thus accommodates a corresponding knob on the adjacent interface of the first Fc polypeptide. Holes 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 second polypeptide is modified to encode a hole. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced by a nucleic acid encoding at least one “import” amino acid residue having a smaller side chain capacity than the original amino acid residue. It will be understood that there can be two or more original residues and corresponding import residues. The upper limit on the number of original residues to be replaced is the total number of residues in the interface of the second polypeptide.

[0034] The knob "may be positioned" within the hole, meaning that the spatial position of the knob and hole on the interface of the first and second Fc polypeptides, respectively, as well as the sizes of the knob and hole, are such that the knob can be positioned within the hole without significantly disrupting the normal association of the first and second polypeptides at the interface. Since the knob typically does not extend perpendicularly from the interface axis and has a preferred conformation, the alignment of the knob with the corresponding hole depends on modeling the knob / hole pair based on a three-dimensional structure, such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using techniques widely accepted in the art.

[0035] The term “isolated antibody” or “isolated antibody fragment” means an antibody or antibody fragment that substantially does not contain other antibodies or antibody fragments having different antigen specificities. Furthermore, an isolated antibody or antibody fragment may substantially not contain other cellular material and / or chemical substances. In other words, in some embodiments, the antibody provided is an isolated antibody separated from an antibody having different specificities. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. An isolated antibody that specifically binds to a target epitope, isoform, or variant may, however, exhibit cross-reactivity to other relevant antigens from other species (e.g., species homologs).

[0036] The terms “recombinant antibody” or “recombinant antibody fragment” as used herein include all antibodies or antibody fragments prepared, expressed, produced or isolated by means not found in nature. For example, antibodies isolated from host cells transformed to express the antibody, antibodies selected and isolated from a recombinant combinatorial human antibody library, and antibodies prepared, expressed, produced or isolated by any other means including splicing all or part of a human immunoglobulin gene sequence to another DNA sequence, or antibodies isolated from an animal (e.g., a mouse) or a hybridoma prepared from such an animal that is transgenic or transchromosomal with respect to the human immunoglobulin gene. Preferably, such recombinant antibodies have a variable region in which the framework and CDR region are derived from a human germline immunoglobulin sequence. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, if transgenic animals with respect to human Ig sequences are used, in vivo somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but are sequences that may not be naturally present in the human antibody germline repertoire in vivo. Recombinant antibodies may be monoclonal antibodies.

[0037] When used herein, the term "monoclonal" typically has the meaning that is characteristic of the art, namely, an antibody or antibody fragment (or its corresponding functional fragment) resulting from a single clone of an antibody-producing cell that recognizes a single epitope on a bound antigen.

[0038] As used herein, an antibody that "specifically binds" is considered "specific to" or "specifically recognizes" an antigen, such as human Met, if such an antibody can distinguish the antigen from one or more reference antigens, since binding specificity is a relative rather than absolute property. For example, a standard ELISA assay or a standard flow cytometry assay can be performed. Scoring can be done by standard color development (e.g., secondary antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide) or by the binding affinity of a secondary antibody labeled with PE or another dye or marker. Reactions in a particular well are scored, for example, by optical density (OD) at 450 nm or by mean or median fluorescence intensity (MFI) in flow cytometry. A typical background (= negative reaction) may be 0.1 OD; a typical positive reaction may be 1 OD. Background and positive reaction MFIs are highly dependent on instrument settings. The positive / negative difference may be more than 10-fold. Typically, the determination of binding specificity is performed not with a single reference antigen, but with a set of about 3-5 unrelated antigens such as powdered milk, BSA, and transferrin. Regarding flow cytometry, various antigen-negative cells can be used. Antibodies that specifically bind to an antigen may, however, exhibit cross-reactivity to their respective ortholog antigens (e.g., species homologs) from other species. In certain embodiments, such cross-reactivity to ortholog antigens may even be desirable.

[0039] When used herein, an antibody exhibits or is cross-reactive when it binds to an orthologous antigen derived from another species. For example, when it binds to human Met and cynomolgus monkey Met, the antibody is cross-reactive.

[0040] As used herein, the term "affinity" refers to the strength of the interaction between a polypeptide and its target at a single site. Within each site, the binding domain of the polypeptide interacts with its target at multiple sites via weak, non-covalent forces; the more interactions that occur, the stronger the affinity.

[0041] The term "epitope" refers to any protein region that is specifically recognized by an antibody or an antibody fragment, or otherwise interacts with a molecule. Generally, an epitope is a chemically active surface population of a molecule, such as an amino acid, carbohydrate, or sugar side chain, and may generally possess specific three-dimensional structural properties as well as specific charge properties. As will be understood by those skilled in the art, practically anything to which an antibody can specifically bind can be an epitope.

[0042] The term "domain" or "protein domain" refers to a region of a protein polypeptide chain that forms a functional unit and / or independently forms a three-dimensional structure.

[0043] The “compositions” of this disclosure can be used for therapeutic or prophylactic applications. Accordingly, this disclosure includes pharmaceutical compositions containing antibodies or antibody fragments as disclosed herein and thus pharmaceutically acceptable carriers or excipients. In relevant embodiments, this disclosure provides methods for treating inflammatory diseases, autoimmune diseases, hematological malignancies and potentially other diseases. Such methods include administering an effective amount of a pharmaceutical composition containing antibodies or antibody fragments as described herein to a subject requiring it.

[0044] This disclosure provides a therapeutic method comprising administering a therapeutically effective dose of an antibody or antibody fragment as disclosed herein to a subject in need of such treatment. “Therapeutic dose” or “effective dose” as used herein means the amount of anti-Met antibody required to produce a desired biological response. According to this disclosure, a therapeutically effective dose is the amount of anti-Met antibody required to treat and / or prevent a disease.

[0045] "Administered" or "administered" can refer to, but are not limited to, the delivery of a drug in an injectable dosage form, such as intravenous, intramuscular, intradermal or subcutaneous routes, or via mucosal routes, for example, as an intranasal spray or inhaled aerosol, or as an orally administered solution, capsule or tablet. Preferably, administration is by an injectable dosage form.

[0046] As used herein, “treatment,” “to treat,” or “to treat” means a clinical intervention in an attempt to alter the natural course of a disease in a subject being treated, and may be carried out either for prevention or during the course of the clinicopathological disease. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, reducing symptoms, decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and achieving remission or an improved prognosis. In some embodiments, antibodies or antibody fragments according to this disclosure are used to delay the onset of the disease or to slow the progression of the disease.

[0047] "Prevention" means reducing the risk of contracting or developing a disease (i.e., preventing at least one of the clinical symptoms of a disease from developing in a subject who has been exposed to a disease-causing substance or who is susceptible to the disease prior to the onset of the disease). "Prevention" also means any method aimed at preventing the onset of a disease or its symptoms, or delaying the onset of a disease or its symptoms.

[0048] In this context, "subject" or "species" refers to any mammal, including rodents such as mice or rats, and primates such as crab-eating macaques (Macaca fascicularis), marmosets (Callithrix jacchus), rhesus macaques (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0049] The term "effector function" refers to the biological activity attributable to the Fc region of an antibody, which changes with antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and direct cell activation or direct cell inhibition.

[0050] Antibody-dependent cell-mediated cytotoxicity, or ADCC, is a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-carrying target cells and subsequently kill them using cytotoxins. NK cells, the primary cells mediating ADCC, express only FcyRIII, while monocytes / macrophages express FcyRI, FcyRII, and FcyRIII.

[0051] "Complement-dependent cell injury" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to the antibodies of this disclosure (of the appropriate subclass) bound to their congener antigens.

[0052] "Antibody-dependent cell phagocytosis" or "ADCP" refers to the mechanism by which antibody-coated target cells are removed through internalization by phagocytic cells such as macrophages or dendritic cells.

[0053] Throughout this specification, unless the context requires otherwise, the words "comprise," "have," and "include," as well as their respective variations such as "comprises," "has," "have," "includes," and "including," imply the inclusion of a specified element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0054] As used herein, the terms “genetically engineered” or “modified” refer to the manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant technology, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or a combination of some of these technologies). Preferably, antibodies or antibody fragments according to this disclosure are genetically engineered or modified to improve one or more properties such as antigen binding, stability, half-life, effector function, immunogenicity, safety, etc.

[0055] As used herein, "mutant" means a polypeptide that differs from a reference polypeptide by one or more modifications, such as amino acid substitution, insertion, or deletion. A mutant polypeptide typically retains most of the properties of the reference polypeptide, such as binding to a target antigen, but introduces novel additional features or properties, such as having a higher affinity for a target antigen compared to the reference polypeptide, or being a humanized version of the reference polypeptide.

[0056] The term "amino acid mutation," as used herein, encompasses amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications is possible, provided that the final construct retains the desired properties, such as reduced binding affinity to the Fc receptor. Amino acid sequence deletions and insertions include N and / or C-terminal deletions and insertions of amino acid residues. A specific amino acid mutation is an amino acid substitution. Amino acid substitutions include substitutions with amino acids that do not exist naturally or with naturally occurring amino acid derivatives of 20 standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods include site-directed mutagenesis, PCR, and gene synthesis. It should be considered that methods other than genetic manipulation, such as chemical modifications, to alter the side chain groups of amino acid residues may also be useful. Various notations may be used herein to indicate the same amino acid mutation. For example, a substitution of glycine to alanine at position 237 of the antibody Fc region can be represented as 237A, G237A, or Gly237Ala.

[0057] The term "EC50," as used herein, refers to the concentration of the antibody or antibody fragment that induces a response in an assay, midway between baseline and maximum. Therefore, it represents the antibody or ligand concentration at which 50% of the maximum effect is observed.

[0058] As used herein, the term "Ka" refers to the binding rate of a particular antibody-antigen interaction.

[0059] The term "Kd," as used herein, refers to the dissociation rate of a particular antibody-antigen interaction. The Kd value for an antibody can be determined using methods well established in the art.

[0060] The term "KD," as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and is expressed as a molar concentration. Preferred methods for determining the Kd of an antibody are, preferably, by using surface plasmon resonance with a biosensor system such as the Biacore system, or by using biolayer interferometry with an Octet BLI instrument.

[0061] The terms “inhibit” or “to inhibit,” “reduce” or “to reduce,” or “neutralize” or “to neutralize” mean a reduction or cessation of any phenotypic characteristic (such as binding or biological activity or function), or a reduction or cessation of the incidence, degree, or likelihood of occurrence of that characteristic. “Inhibition,” “reduction,” or “neutralization” does not need to be complete, as long as it is detectable using an appropriate assay. In some embodiments, “reduce,” “inhibit,” or “neutralize” means the ability to cause a reduction of 20% or more. In other embodiments, “reduce,” “inhibit,” or “neutralize” means the ability to cause a reduction of 50% or more. In yet another embodiment, “reduce,” “inhibit,” or “neutralize” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0062] As used herein, the term "antagonist" antibody means an antibody or antibody fragment that interacts with an antigen to partially or completely inhibit or neutralize the biological activity or function or any other phenotypic characteristics of the target antigen.

[0063] A "wild-type" protein is a version or variant of a protein as it appears in nature. The amino acid sequence of the Fc region of a wild-type protein, such as human IgG1 antibody, is the amino acid sequence of the naturally occurring protein. Due to allotypic differences, there can be more than one amino acid sequence for a wild-type protein. 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].

[0064] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally contains two constant domains, the CH2 domain and the CH3 domain. While the boundary of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is typically defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Various Fc modifications are commonly used. For a review, see, for example, Antibodies (2020) 9:64.

[0065] When used herein in the context of a silencing antibody or an antibody containing a silencing mutation, the terms “silent” and “silencing” refer to a mutation in the Fc domain of such an antibody that partially or completely reduces its binding ability to one or more cell surface Fcγ receptors, thereby reducing or attenuating one or more Fc-mediated antibody effector functions, such as ADCC, ADCP, and CDC complement responses, and in some embodiments substantially completely withdrawing them [see, for example, Kang and Jung, Experimental and Molecular Medicine (2019) 51:138]. Silencing effector functions can be obtained by mutations in the Fc region of an antibody and have been described in the Art (e.g., Strohl, Biotechnology 20: 685-91 with respect to LALA and N297A; Baudino et al., J. Immunol. 181: 6664-69 with respect to 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 and 7,332,581; WO2004 / 056312, WO2021 / 234402, and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604]. Silencing mutations also include LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A), and PG-LALA (L234A / L235A / P329G) mutations (numbered according to the EU index), as well as AEASS mutations (L234A / L235E / G237A / A330S / P331S).

[0066] Embodiments of the Invention In the following description, numerous specific details are given to provide a complete understanding of the embodiments. The embodiments may be carried out without using one or more of these specific details, or using other methods, components, materials, etc. In other cases, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring the aspects of the embodiments.

[0067] This disclosure relates to novel therapeutic agents for the treatment of tumors and / or metastases.

[0068] It is estimated that over 200,000 patients develop "MET toxicity" annually, and MET inhibition may lead to disease remission. Given that mutations accumulate with age and the elderly population will increase over the next 20 years, the burden of cancer is expected to rise, having a significant impact on global healthcare resources for patient management. Genetic alterations causing "MET toxicity" have been found in gastric, esophageal, colorectal, and renal cancers, as well as lung cancer, melanoma, and brain tumors. Furthermore, selective MET gene lesions have been found as a mechanism for acquiring resistance to numerous other targeted therapies in colorectal cancer and non-small cell lung cancer (NSCLC).

[0069] The role of Met in metastasis has also been linked to Met's ability to help cells adapt to harsh environments. Met-driven metastatic ability depends not only on genetic and epigenetic changes, but also on parasecretion of HGF by tumor stromal tissue, which is composed of a wide variety of cell types, including fibroblasts, resident epithelial cells, pericytes, myofibroblasts, vascular and lymphatic endothelial cells, and invasive cells of the immune system.

[0070] Met has recently been recognized as a cancer-specific target for (i) personalized treatment of tumors with MET mutations / amplification ("MET-toxic"); (ii) prevention / reversal of primary and secondary resistance to other targeted cancer therapies driven by Met; and (iii) prevention / reversal of invasive / metastatic phenotypes driven by Met.

[0071] Monovalent antibodies are described, for example, in WO2005 / 063816 and Proc Natl Acad Sci USA (2013) 110, E2987. A "one-arm" anti-Met antibody, referred to as "hOA-DN30," is described in WO2020 / 074459. hOA-DN30 is a highly stable humanized monovalent antibody that blocks Met activation through depletion mechanisms, including (i) removal of Met from the cell surface by ectodomain "shedding"; (ii) sequestration of HGF ligand; (iii) inhibition of homodimerization or heterodimerization of Met receptors at the membrane; and (iv) stimulation of receptor degradation. This disclosure provides significant additional improvements to hOA-DN30. The introduction of silencing mutations not only produced antibodies with effects associated with each mutation, but also provided additional important safety aspects, and the withdrawal of the Fcγ receptor reduced the risk of residual MET dimerization via super-crosslinking mediated by immune cell Fcγ receptors on the tumor cell surface.

[0072] Anti-Met antibody fragment In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. This relates to anti-Met antibody fragments, including those mentioned above.

[0073] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, This relates to an anti-Met antibody fragment in which the humanized VL domain is fused to the human CL domain from the N-terminus to the C-terminus.

[0074] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, This relates to an anti-Met antibody fragment in which, from the N-terminus to the C-terminus, the humanized VH domain is fused to the human CH1 domain, the human CH1 domain is fused to the human hinge region, the human hinge region is fused to the human CH2 domain, and the human CH2 domain is fused to the human CH3 domain.

[0075] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, From the N-terminus to the C-terminus, the humanized VL domain is fused to the human CL domain. This relates to an anti-Met antibody fragment in which, from the N-terminus to the C-terminus, the humanized VH domain is fused to the human CH1 domain, the human CH1 domain is fused to the human hinge region, the human hinge region is fused to the human CH2 domain, and the human CH2 domain is fused to the human CH3 domain.

[0076] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, This relates to an anti-Met antibody fragment in which, from the N-terminus to the C-terminus, the human hinge region is fused to the CH2 domain, the CH2 domain is fused to the human CH3 domain, and the human hinge region is cleaved at the N-terminus.

[0077] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, From the N-terminus to the C-terminus, the humanized VL domain is fused to the human CL domain. From the N-terminus to the C-terminus, the humanized VH domain fuses with the human CH1 domain, the human CH1 domain fuses with the human hinge region, the human hinge region fuses with the human CH2 domain, and the human CH2 domain fuses with the human CH3 domain. This relates to an anti-Met antibody fragment in which, from the N-terminus to the C-terminus, the human hinge region is fused to the CH2 domain, the CH2 domain is fused to the human CH3 domain, and the human hinge region is cleaved at the N-terminus.

[0078] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The humanized VL domain relates to an anti-Met antibody fragment having the amino acid sequence described in SEQ ID NO: 13.

[0079] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The humanized VH domain relates to an anti-Met antibody fragment having the amino acid sequence described in SEQ ID NO: 14.

[0080] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The humanized VL domain has the amino acid sequence described in SEQ ID NO: 13. The humanized VH domain relates to an anti-Met antibody fragment having the amino acid sequence described in SEQ ID NO: 14.

[0081] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The human CL domain is a human light chain κ-type domain, and this relates to an anti-Met antibody fragment.

[0082] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, This relates to an anti-Met antibody fragment in which the human hinge region and human constant domains CH1, CH2, and CH3 are derived from human IgG1.

[0083] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The human CL domain is a human light chain κ-type domain. This relates to an anti-Met antibody fragment in which the human hinge region and human constant domains CH1, CH2, and CH3 are derived from human IgG1.

[0084] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The invention relates to an anti-Met antibody fragment in which two Fc polypeptides are linked via intermolecular disulfide bonds in the hinge region.

[0085] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The present invention relates to an anti-Met antibody fragment in which a first Fc polypeptide and a second Fc polypeptide associate at an interface, one of the first and second Fc polypeptides contains a knob at the interface, and the other of the first and second Fc polypeptides contains a hole at the interface, the knob may be located within the hole.

[0086] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, Either the first or second Fc polypeptide contains a mutant CH3 constant domain, which holds an amino acid mutation at position 389, where the original amino acid at position 389 has been mutated to import an amino acid with a larger side-chain capacity than the original amino acid; the other Fc polypeptide contains a mutant CH3 constant domain, which holds three amino acid mutations at positions 389, 391, and 438, where the original amino acid has been mutated to import an amino acid with a smaller side-chain capacity than the original amino acid, and amino acid numbering follows Kabat's EU numbering scheme. Regarding anti-Met antibody fragments.

[0087] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The original amino acids at positions 389, 391, and 438 are threonine, leucine, and tyrosine, respectively; in the first or second Fc polypeptide, threonine at position 389 is mutated to tryptophan; in the other Fc polypeptide, threonine at position 389 is mutated to serine, leucine at position 391 is mutated to alanine, and tyrosine at position 438 is mutated to valine, relating to an anti-Met antibody fragment.

[0088] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, This invention relates to an anti-Met antibody fragment in which the human CL domain has the amino acid sequence described in SEQ ID NO: 15, and the human CH1 domain has the amino acid sequence described in SEQ ID NO: 16.

[0089] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, This invention relates to an anti-Met antibody fragment in which the first human Fc polypeptide has the amino acid sequence described in SEQ ID NO: 17, and the second human Fc polypeptide has the amino acid sequence described in SEQ ID NO: 18.

[0090] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The anti-Met antibody fragment relates to an anti-Met antibody fragment that, upon binding to Met, induces the detachment of the extracellular domain of Met.

[0091] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The Fc regions of the first and second Fc polypeptides include mutations L234A, L235A, and P329A (according to the EU index), or mutations L234A, L235E, G237A, A330S, and P331S (according to the EU index). Regarding anti-Met antibody fragments.

[0092] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The Fc regions of the first FC polypeptide and the second Fc polypeptide contain mutations L234A, L235A, and P329A (according to the EU index). Regarding anti-Met antibody fragments.

[0093] In certain embodiments, the present 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The first polypeptide contains the amino acid sequence of SEQ ID NO: 19, the second polypeptide contains the amino acid sequence of SEQ ID NO: 20, and the third polypeptide contains the amino acid sequence of SEQ ID NO: 18. Regarding anti-Met antibody fragments.

[0094] nucleic acid In certain embodiments, the present disclosure provides an isolated nucleic acid encoding 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. including, The present invention relates to isolated nucleic acids. In certain embodiments, the humanized VL domain is fused to the human CL domain in the N-terminus to C-terminus direction. In certain embodiments, the humanized VH domain is fused to the human CH1 domain in the N-terminus to C-terminus direction, the human CH1 domain is fused to the human hinge region, the human hinge region is fused to the human CH2 domain, and the human CH2 domain is fused to the human CH3 domain. In certain embodiments, the human hinge region is fused to the CH2 domain in the N-terminus to C-terminus direction, the CH2 domain is fused to the human CH3 domain, and the human hinge region is cleaved at the N-terminus. In certain embodiments, the humanized VL domain fuses with the human CL domain in the N-terminus-C-terminus direction, the humanized VH domain fuses with the human CH1 domain in the N-terminus-C-terminus direction, the human CH1 domain fuses with the human hinge region, the human hinge region fuses with the human CH2 domain, the human CH2 domain fuses with the human CH3 domain in the N-terminus-C-terminus direction, the human hinge region fuses with the CH2 domain, the CH2 domain fuses with the human CH3 domain, and the human hinge region is cleaved at the N-terminus. In certain embodiments, the humanized VL domain has the amino acid sequence described in SEQ ID NO: 13. In certain embodiments, the humanized VH domain has the amino acid sequence described in SEQ ID NO: 14. In certain embodiments, the humanized VL domain has the amino acid sequence described in SEQ ID NO: 13, and the humanized VH domain has the amino acid sequence described in SEQ ID NO: 14. In certain embodiments, the humanized CL domain is a human light chain κ-type domain. In certain embodiments, the human hinge region and the human constant domains CH1, CH2, and CH3 are derived from human IgG1. In certain embodiments, the human CL domain is a human light chain κ-type domain, and the human hinge region and the human constant domains CH1, CH2, and CH3 are derived from human IgG1. In certain embodiments, the two Fc polypeptides are linked via intermolecular disulfide bonds at the hinge region.In certain embodiments, the first Fc polypeptide and the second Fc polypeptide associate at an interface, with one of the first and second Fc polypeptides containing a knob at the interface and the other containing a hole at the interface, the knob being located within the hole. In certain embodiments, either the first or second Fc polypeptide contains a mutant CH3 constant domain, the mutant CH3 constant domain holds an amino acid mutation at position 389, where the original amino acid at position 389 is mutated to import an amino acid having a larger side-chain capacity than the original amino acid; the other Fc polypeptide contains a mutant CH3 constant domain, the mutant CH3 constant domain holds three amino acid mutations at positions 389, 391 and 438, where the original amino acid is mutated to import an amino acid having a smaller side-chain capacity than the original amino acid, and amino acid numbering follows Kabat's EU numbering scheme. In certain embodiments, the original amino acids at positions 389, 391, and 438 are threonine, leucine, and tyrosine, respectively; in the first or second Fc polypeptide, threonine at position 389 is mutated to tryptophan; in the other Fc polypeptide, threonine at position 389 is mutated to serine, leucine at position 391 is mutated to alanine, and tyrosine at position 438 is mutated to valine. In certain embodiments, the human CL domain has the amino acid sequence described in SEQ ID NO: 15, and the human CH1 domain has the amino acid sequence described in SEQ ID NO: 16. In certain embodiments, the first human Fc polypeptide has the amino acid sequence described in SEQ ID NO: 17, and the second human Fc polypeptide has the amino acid sequence described in SEQ ID NO: 18. In certain embodiments, the anti-Met antibody fragment induces the detachment of the extracellular domain of Met when bound to Met. In certain embodiments, the Fc regions of the first and second Fc polypeptides include mutations L234A, L235A, and P329A (according to the EU index), or mutations L234A, L235E, G237A, A330S, and P331S (according to the EU index).In certain embodiments, the Fc regions of the first FC polypeptide and the second Fc polypeptide include mutations L234A, L235A, and P329A (according to the EU index).

[0095] In certain embodiments, the present disclosure provides an isolated nucleic acid encoding 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The first polypeptide contains the amino acid sequence of SEQ ID NO: 19, the second polypeptide contains the amino acid sequence of SEQ ID NO: 20, and the third polypeptide contains the amino acid sequence of SEQ ID NO: 18. Regarding isolated nucleic acids.

[0096] In certain embodiments, the present disclosure provides an isolated nucleic acid encoding 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs), and the first polypeptide has the amino acid sequence of 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, the humanized VH domain comprises three complementarity-determining regions (CDRs), and the second polypeptide has the amino acid sequence of 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 the third polypeptide has the amino acid sequence of SEQ ID NO: 18. including, Regarding isolated nucleic acids.

[0097] In certain embodiments, the present disclosure provides an isolated nucleic acid encoding 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs), and the first polypeptide is encoded by the nucleic acid sequence of Sequence 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, the humanized VH domain comprises three complementarity-determining regions (CDRs), and the second polypeptide is encoded by the nucleic acid sequence of Sequence ID No. 22, 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 the third polypeptide is encoded by the nucleic acid sequence of Sequence ID No. 23. including, Regarding isolated nucleic acids.

[0098] In certain embodiments, this disclosure relates to a composition comprising two or more recombinant nucleic acids that collectively encode the anti-Met antibody fragments disclosed herein.

[0099] Medical use and manufacture The therapeutic compositions comprising the active ingredient of this disclosure, i.e., humanized anti-Met antibody fragment a, may be prepared in the form of aqueous solution, lyophilized, or other dry formulations using physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the doses and concentrations used and include buffers; antioxidants; preservatives; low molecular weight (less than about 10 residues) polypeptides; 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 counterions; metal complexes and / or nonionic surfactants. The formulations may also contain other active compounds, preferably, having complementary activity that does not adversely affect the therapeutic activity of hOA-DN30, either alone or in combination with the extracellular components of human Met, as required for the specific indication of the therapeutic target. Such molecules preferably exist in combination in amounts effective for the intended purpose.

[0100] The active ingredient can also be encapsulated in microcapsules prepared using techniques disclosed, in particular, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Sustained-release preparations can be prepared. A preferred example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing the active ingredient.

[0101] The active ingredients (and adjunctive therapeutic agents) of this disclosure are administered by any preferred means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, where desired for local treatment, intralesional administration. The active ingredients of the present invention can preferably be administered by pulse infusion, particularly with a tapering dose of the active ingredient. Dosage may be by injection, such as intravenous or subcutaneous injection, by any preferred route, depending in part whether the administration is short-term or chronic.

[0102] The active ingredient will be formulated, administered, and given in a manner consistent with good medical practice. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the physician. The active ingredient does not need to be formulated together with one or more drugs currently used to prevent or treat the disorder in question, but may be. The effective dose of such other drugs depends on the amount of the active ingredient (present in the formulation), the type of disorder or treatment, and other factors discussed above. These are commonly used with the same doses and routes of administration as used herein, or at approximately 1 to 99 percent of the doses used herein.

[0103] With regard to the treatment of diseases, the appropriate dosage of the active ingredient depends on the type of disease being treated, the severity and course of the disease, whether the active ingredient is administered for preventive or therapeutic purposes, the patient's clinical history, and the response to the active ingredient of the present invention, and is at the discretion of the attending physician.

[0104] The anti-Met antibody fragments of this disclosure are suitably administered to patients as a single dose or over a series of treatments. Depending on the type and severity of the disease, approximately 1 mg / kg to 30 mg / kg of antibody is an initial candidate dose for administration to a patient, whether, for example, by a single or multiple separate doses or by continuous infusion. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg or more, depending on the factors described above. With regard to repeated administration over several days or more, treatment is continued, depending on the condition, until the desired suppression of disease symptoms occurs. One exemplary dose of the antibody fragment would be in the range of approximately 0.05 mg / kg to approximately 20 mg / kg. Thus, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to a patient. Such doses can be administered intermittently, for example, every week or every three weeks (e.g., so that the patient receives approximately 2 to approximately 20 doses of antibody, e.g., approximately 6 doses). An initial higher loading dose may be administered, followed by one or more lower doses. An exemplary dosing regimen includes an initial loading dose of approximately 4 mg / kg, followed by a weekly maintenance dose of approximately 2 mg / kg of antibody. However, other dosing regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays.

[0105] In certain embodiments, this disclosure relates to anti-Met antibody fragments disclosed herein for use in the treatment of tumors and / or metastases.

[0106] In certain embodiments, this disclosure relates to anti-Met antibody fragments disclosed herein for use in the treatment of tumors and / or metastases in patients who harbor genetic alterations of the MET gene.

[0107] In certain embodiments, this disclosure relates to anti-Met antibody fragments disclosed herein for use in the treatment of tumors and / or metastases in patients carrying the wild-type MET gene.

[0108] In certain embodiments, the present disclosure relates to a process for producing an anti-Met antibody fragment as disclosed herein, comprising the steps: (i) synthesis of cDNA sequences of first, second, and third polypeptides constituting an anti-Met antibody fragment; (ii) insertion of the three cDNA sequences into one or more plasmids, wherein the plasmid is suitable for expression in a mammalian cell line; (iii) transient or stable co-transfection of a mammalian cell line using the plasmid; (iv) recovery of the culture supernatant; and (v) purification of the anti-Met antibody fragment by affinity chromatography.

[0109] Example 1: Rational and Selection of Possible Silencing Mutations While Met is a validated target for the treatment of tumors and metastases, there is still a possibility that each targeted agent may induce undesirable dimerization and activation of the c-Met receptor, thereby initiating a downstream immune cell-mediated signaling cascade. Therefore, it was justified that dissociating Fcγ receptor binding could potentially prevent such undesirable downstream effects. A requirement for such an approach is a targeted agent that is independent of Fc effector function. In this disclosure, one such exemplary targeted agent was selected to test this hypothesis. VERT001 is an anti-Met antibody fragment with an Fc effector-independent mode of action. It was hypothesized that a silent version of VERT001 could be a suitable candidate. However, genetic engineering of antibodies and antibody fragments always carries the risk that not only will novel functions be introduced into the respective molecules, but the molecules may also lose other functions, such as efficacy, stability, or other properties that could limit the usefulness of the resulting derivatives for therapeutic development.

[0110] Derivatives of VERT001 were created by genetically modifying the Fc region of VERT001 with specific silencing mutations. VERT001 with the PA-LALA mutation produced VERT002. VERT001 with the AEASS mutation produced VERT004. VERT001, VERT002, and VERT004 are compared in the following examples.

[0111] The amino acid sequence of the binder is shown in the table below.

[0112] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0113] 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.

[0114] Example 2: General method used in this study Example 2.1 Purity evaluation by size exclusion chromatography (SEC) Monomer content was evaluated by HP-SEC analysis using an Agilent 1260 infinity II. Liquid chromatography separation was performed through 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. 200 mM sodium phosphate buffer pH 7.0 was used as the mobile phase. Prior to chromatography, the sample was diluted to 1 mg / mL in 200 mM sodium phosphate buffer pH 7.0. The sample was centrifuged at 75000 rcf for 5 minutes (except for samples subjected to 55°C and pH stress; these were filtered). 80 μL of the supernatant was transferred to an HPLC vial with an insert. Chromatograms were manually integrated using ChemStation software. MW standards were used as column quality controls.

[0115] Example 2.2 Purity evaluation by strong cation exchange chromatography (SCX) Size variant profiles were analyzed by SCX chromatography using 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 performed at room temperature (RT) at a flow rate of 0.8 mL / min. 20 mM sodium phosphate buffer pH 6.0 was used as mobile phase A (load), and 20 mM sodium phosphate buffer pH 6.0 + 0.5 M NaCl was used as mobile phase B (elution). The applied gradient was as follows:

[0116] [Table 2]

[0117] For sample preparation, the solution formulation was diluted to 20 mM sodium phosphate buffer, pH 6.0. The samples were centrifuged at 75,000 rcf for 5 minutes (except for samples subjected to 55°C and pH stress; these were filtered). 80 μL of the supernatant was transferred to an HPLC vial with an insert. Chromatograms were manually integrated using ChemStation software.

[0118] Example 2.3 Purity evaluation by hydrophobic interaction chromatography (HIC) The purity of the samples was evaluated by hydrophobic interaction chromatography using an Agilent 1260 infinity II. Liquid chromatography separation was performed through an Agilent bio HIC 4.6 × 100 mm, 3.5 μm. Chromatography was performed at a flow rate of 0.8 mL / min at room temperature (RT). 50 mM sodium phosphate buffer pH 7.0 + 2 M (NH4)2SO4 was used as mobile phase A (load), and 50 mM sodium phosphate buffer pH 7.0 was used as mobile phase B (elution). The applied gradient was as follows:

[0119] [Table 3]

[0120] For sample preparation, the solution formulation was diluted to 1 mg / mL in 50 mM sodium phosphate buffer, pH 7.0. The samples were centrifuged at 75,000 rcf for 5 minutes (except for samples subjected to 55°C and pH stress; these were filtered). 80 μL of the supernatant was transferred to an HPLC vial with an insert. Chromatograms were manually integrated using ChemStation software.

[0121] Example 2.4 Purity evaluation by reverse-phase ULC-MS Sample purity was evaluated by ULC-MS analysis using a Waters Acquity H class+ system coupled to a single quadrupole detector from PDA and Waters SQD2. Liquid chromatography separation was performed through a BEH column C4 300A, 2.1 × 50 mm.

[0122] For sample preparation, the solution formulation was diluted in water to 0.5 mg / mL (final volume 100 μL). Mass lynx was used for data acquisition and processing. Max Ent1 was used to determine the molecular mass of the protein. LC-MS parameters are shown in the table below.

[0123] [Table 4]

[0124] Example 2.5 Measurement of thermal unfolding via differential scanning fluorescence measurement. The samples were characterized by differential scanning fluorescence (DSF) using Sypro Orange dye (SO). Briefly, protein samples were mixed with SO dye in PBS buffer, followed by the application of a temperature gradient, and fluorescence was monitored as a function of temperature (°C). Under heating, the protein begins to unfold, exposing hydrophobic core residues that interact with SO, allowing for the measurement of increased fluorescence. 5000× of Sypro Orange dye was diluted to a concentration of 10× in PBS. All samples were diluted to a concentration of 2 mg / mL in PBS buffer, followed by a 1:1 volumetric mixing with 10× SO to obtain final concentrations of 1 mg / mL of sample and 5× SO in PBS. Each sample was measured using triple replicates.

[0125] Samples were transferred to 96-well PCR plates, and a temperature gradient from 25°C to 95°C was applied using an RT-PCR thermocycler C1000 (Biorad), with a 0.5°C increase every 10 seconds. The first derivative of the curve was used to determine the melting temperature (Tm), which provides information about the conformational stability of the protein samples. 25°C (the starting point of the temperature gradient), compared to each sample, was used to provide qualitative information about the initial folding state.

[0126] Example 2.6 ELISA binding assay First, the microplates were coated. To do this, recombinant human cMET ECD His (SinoBiological PN:10692-H08H) was diluted to a final concentration of 1 μg / mL in PBS in an F96 IMMUNOPLATE MAXISORP plate to a final volume of 50 μL and incubated overnight at 4°C. Subsequently, the plates were blocked to minimize nonspecific binding. The coating solution was removed, and then 100 μL of blocking buffer (MSD blocking buffer) or 5% BSA in PBS was added to each well, and the plates were incubated on a 600 rpm orbital shaker at room temperature for 1 hour. The blocking buffer was then washed three times with MSD washing buffer or PBS-0.05% P20.

[0127] Next, 50 μL of diluted primary antibodies (VERT001, VERT002, and VERT-04; titrated in a separate plate using assay buffer = PBS 1×, 0.1% BSA w / v, 0.02% P20) were added to each well. The 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 double replicates. The plates were incubated on a 600 rpm orbital shaker at room temperature for 1 hour, and then washed three times with MSD wash buffer or PBS-0.05% P20. Next, diluted secondary antibody (goat anti-human IgG Fc secondary antibody, HRP 1:20000) was added to each well (50 μL), incubated at room temperature for 1 hour on a 600 rpm orbital shaker, and then washed three times with MSD washing buffer or PBS-0.05% P20. Finally, for detection, TMB (3,3',5,5'-tetramethylbenzidine) was added (50 μL / well), incubated for 1-5 minutes (until blue color appeared), and then an equal volume of stop solution (2 M H2SO4) was added. Subsequently, the optical density was measured at 450 nm using a plate reader. The absorbance at 450 nm was plotted against the concentration of the primary antibody, and the data was fitted to the 4PL equation to obtain EC50.

[0128] Example 2.7 Growth assay Hs746T cells were purchased from ATCC (catalog number HTB135), partially cultured in Dulbecco's MEM (4.5g / L glucose + GlutaMAX, Gibco 31966) supplemented with 1% sodium pyruvate (Gibco, 11360) and 10% heat-inactivated FBS (PAN Biotech P30-1909), and incubated at 37°C and 5% CO2. EBC-1 cells were purchased from JCRB (catalog number JCRB0820), partially cultured in RPMI (+ glutamine, + 25mM HEPES, Gibco 22400) containing 10% heat-inactivated FBS, and incubated at 37°C and 5% CO2.

[0129] Cell plating on day 0 (5,000 cells per well for Hs746T cells and 2,000 cells per well for EBC-1 cells) was used for a proliferation assay on a white 96-well assay plate (ThermoFisher 136101). The compound was added on day 1, and viability was examined on day 4. On day 1, the compound was diluted to a concentration of 2000 μg / mL, and a 9-point dose-response (1:4 dilution series) was prepared in culture medium and then added to the cells (final starting concentration of the dose-response, 200 μg / mL). All samples were tested in double replicates. Viability was evaluated using the CellTiter-Glo 2.0 luminescent cell viability assay kit (Promega), which measures the ATP content of cells. CellTiter-Glo was added (1:10 dilution) and incubated with cells for 20 minutes in the dark on a 600 rpm plate shaker. Luminescence was recorded using an EnVision multimode plate 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 result on day 4. The IC50 was determined by nonlinear regression using 4-parameter (variable gradient) curve fitting implemented in GraphPad Prism (version 9.3.0), and the dose-response data (after subtracting the day 1 background level) was fitted.

[0130] Example 2.8 Stability Study For stability studies, 6 × 1 mL tubes containing each antibody solution were removed from a -80°C freezer and allowed to thaw at room temperature while protected from light. Subsequently, each solution was aliquoted into 3R glass vials under a laminar flow hood to maintain sterility (400 μL per vial for VERT001 and VERT002, and 350 μL for VERT004 due to its relatively low starting volume). The vials were then stoppered with rubber stoppers and sealed using flip-off seals.

[0131] Three vials were stored at 5°C, three vials at 40°C, and one vial at -80°C per antibody. One vial was used for T0 measurement. The time points were as follows: D0, week 1 (W1), W2, and W4. At each time point, a fresh aliquot from each antibody vial was removed from the -80°C freezer to be used as the assay standard in all characterization techniques. This allowed for evaluation of inter-experimental deviations and variability between time points.

[0132] 3 mL of each solution was held for forced decomposition studies (pH and heat). Samples were held at 5°C for 2 days prior to the start of the study.

[0133] Example 2.9 Forced Decomposition Study Example 2.9.1 pH Stability Samples were prepared as described in Example 2.8. 850 μL of each solution was pH adjusted to 3 with 1 M HCl or to 9 with 1 M NaOH. The vials were kept at RT for 5 days, protected from light. Samples were taken on D0 and D2 and stored at -80°C. On D5, the samples were immediately stored at -80°C. On the day of analysis, aliquots were sterile filtered to ensure suitability for the cell assay. Therefore, centrifugation was not applied to the various LC sample preparations.

[0134] Example 2.9.2 Temperature Stability Samples were prepared as described in Example 2.8. Subsequently, 300 μL of each solution was aliquoted into Eppendorf tubes and placed in an Eppendorf thermoblock set to 55°C for 5 days without shaking, protected from light. On the day of analysis, the samples were sterile filtered to ensure suitability for the cell assay. Therefore, centrifugation was not applied to the various LC sample preparations.

[0135] Example 2.9.3 Freeze-thaw stability Samples were prepared as described in Example 2.8. Aliquots for each antibody were removed from -80°C and allowed to stand at RT for 60–90 minutes while protected from light. Once fully thawed, the aliquots were stored again at -80°C. A total of three F / T (freeze / thaw) cycles were performed.

[0136] Example 2.10 Concentration Measurement Protein concentrations were measured using a nanophotomer NP80 (Implen) with an A280 filter. The decay coefficient (L / g × cm) was 1.48 for VERT001 and VERT004, and 1.49 for VERT002. 2 μL of sample was pipetted onto the sample window. PBS buffer was used for blanking. The reported concentrations are the average of two measurements.

[0137] Example 3: Evaluation of biophysical properties and potential applications Example 3.1 Comparison of Forms VERT-001 (wild-type Fc), VERT-002 (PA-LALA), and VERT-004 (AEASS) were compared using various assays without exposure to any stress conditions.

[0138] Example 3.1.1 SEC Analysis SEC analysis revealed high monomer content exceeding 99% for all forms. The percentages of high molecular weight (HMW) and low molecular weight (LMW) molecular species were essentially identical for all three samples tested. See Table 5.

[0139] [Table 5]

[0140] Example 3.1.2 SCX Analysis The SCX chromatograms are shown in Figure 1. SCX analysis revealed that all three types of chromatograms showed similar distributions of charged molecular species. VERT-004 appears slightly acidic, likely due to a mutation from L to E. Table 6 shows the percentages of major molecular species and acidic and basic molecular species as determined by SCX analysis.

[0141] [Table 6]

[0142] Example 3.1.3 HIC Analysis The HIC chromatograms of all three forms appear highly identical. All three forms exhibit relatively short retention times, which suggests low hydrophobicity.

[0143] Example 3.1.4 Reverse-phase ULC-MS analysis On the RP chromatogram, VERT-004 exhibits a somewhat different profile compared to VERT-001 and VERT-002, which are indicated by the presence of multiple peaks. See Figure 2.

[0144] The masses of the major UV peaks were determined for each antibody by MS, and a summary is shown in the table below.

[0145] [Table 7]

[0146] The mass differences between the various forms correspond well to their mutations: ΔVERT-001-VERT-002 = 220 Da, corresponding to mutation PLL (sequence number 24) to AAA (sequence number 25): 2 × 110 Da. ΔVERT-001-VERT-004 = 15Da, corresponding to the mutation LLGAP (sequence number 26) to AEASS (sequence number 27): 2 × 6Da.

[0147] Detailed mass spectrometry was performed on each individual peak present in VERT-001, VERT-002, and VERT-004. An overview and provisional molecular species assignment for VERT-004 are depicted in Figure 3. VERT-001 and VERT-002 also contain small levels of cleavage-type HC, as well as small proportions of unspecified molecular species at 90 and 89 kDa, corresponding to MW-10 kDa for both antibodies, respectively.

[0148] Example 3.1.5 Differential Scanning Fluorescence Measurement Method Thermal unfolding curves for antibodies VERT-001, VERT-002, and VERT-004 were recorded via differential scanning fluorescence (SCFL) to determine the melting temperature (Tm) and fluorescence observed at 25°C (RFU t0). The Tm for VERT-001 and VERT-002 were 68°C and 68.5°C, respectively, while VERT-004 showed a relatively low Tm of 62.5°C. All Fc forms started at approximately 4000 RFU in initial fluorescence. The results are summarized in the table below.

[0149] [Table 8]

[0150] Example 3.1.6 Binding to human c-Met ECD The binding affinity of VERT001, VERT002, and VERT004 to c-Met ECD was measured using an ELISA assay. The binding affinity ELISA curve is shown in Figure 4. The determined EC50 values ​​are shown in the table below.

[0151] [Table 9]

[0152] The binding curves for all three types of binders, VERT001, VERT002, and VERT004, were similar. VERT002 showed the highest affinity.

[0153] Example 3.1.7 Affinity determination by BLI The binding affinities of VERT-001, VERT-002, and VERT-004 were compared by affinity determination via BLI. The results are shown in Table 10.

[0154] [Table 10]

[0155] Of the three types of antibodies, VERT-002 showed the highest affinity.

[0156] Example 3.1.8 Proliferation The antiproliferative activity of VERT-001, VERT-002, and VERT-004 was measured using Hs746T cells and EBC-1 cells, respectively. The results are shown in Figure 5. The determined IC50 values ​​are shown in the table below.

[0157] [Table 11]

[0158] Example 3.2 Stability Study The samples were prepared as described in Example 2.8.

[0159] Example 3.2.1 Visual inspection of stability samples Stability samples of all three antibody formulations at 5°C and 37°C remained visually clear and particle-free throughout the entire 4-week period. Concentrations remained unchanged at both 5°C and 37°C over the 4-week period.

[0160] Example 3.2.2 Aggregate and fragment analysis via SEC Monomer formation, HMW, and LMW content were determined over incubation time via SEC analysis. The results are shown in Figure 6.

[0161] A very slight monomer decrease associated with an increase in HMW is observed for VERT002 (0.4%) and VERT004 (0.6%) at 5°C. At 37°C, VERT-001 and VERT-002 exhibit similar slow to medium-rate kinetics of monomer decrease accompanied by aggregation (increased HMW) and fragmentation (increased LMW). A higher aggregation kinetic is observed for VERT004, as indicated by the rapid decrease in monomer content and the increase in HMW molecular species. The increase in LMW for VERT-004 follows a similar gradient to that for VERT-001 and VERT-002.

[0162] Example 3.2.3 Analysis of charge variants via SCX The formation of charge mutants over incubation time was analyzed via strong cation exchange chromatography. The results are shown in Figure 7. At 5°C, the charge mutant profiles remained stable for all forms. When exposed to 37°C, all forms showed the same trend in charge mutant evolution, accompanied by an increase in acidic molecular species over time, which may correspond to deamidation and reduction at the major peak.

[0163] Example 3.2.4 Stability analysis via RP-HPLC Chemical stability was monitored over incubation time by RP using UV and MS detection. No significant degradation was observed in any form. However, a small additional peak (RT=4.48 min) was detected for all antibodies after 4 weeks at 37°C, which was likely due to fragmentation.

[0164] Example 3.2.5 Stability analysis via DSF The stability of all three antibodies after 4 weeks of incubation at 5°C and 37°C, as studied by DSF, was compared to a newly thawed reference material. The thawing temperature for all samples was the same as that observed at time zero (T0), regardless of storage conditions. All FC forms exhibited initial fluorescence of approximately 4000 RFU. The results are summarized in the table below.

[0165] [Table 12]

[0166] Example 3.2.6 Binding to human c-Met ECD The binding affinity of VERT001, VERT002, and VERT004 to c-Met ECD after 4 weeks of incubation at 5°C and 37°C, as measured in an ELISA assay, was compared to a newly thawed reference material. When samples were incubated at 37°C for 4 weeks, a slight decrease in binding efficacy was observed for VERT-001, VERT-002, and VERT-004 compared to the assay standard. The same samples incubated at 5°C for the same period appeared stable, and no decrease in binding efficacy was observed compared to the assay standard. A comparison of the binding affinity of the samples to the reference material is shown in Figure 8.

[0167] Example 3.2.7 Proliferation The antiproliferative activity of VERT001, VERT002, and VERT004 after 4 weeks of incubation at 5°C and 37°C was compared to a newly thawed reference material in Hs746T and EBC-1 cells. Overall, the antiproliferative activity of all samples was within the range expected throughout the study, although a slight shift in potency was observed at 4 weeks in both cell lines (relatively high IC50 in Hs746T and relatively low IC50 in EBC-1). The determined IC50s are shown in the table below.

[0168] [Table 13]

[0169] Example 3.3 Forced Decomposition Study Example 3.3.1 Visual inspection of pH stressed samples All pH-stressed samples remained clear and particle-free over the five days. Concentrations decreased slightly at pH 3 for VERT002 and VERT004 at D0 and D5, which may have been due to dilution during pH adjustment.

[0170] Example 3.3.2 Analysis of chemical degradation of pH-stressed samples using RP pH stress at pH 3 and pH 9 did not result in any significant degradation of the antibody, as indicated by the presence of a major peak corresponding to a proper intact mass, and also by a consistent peak height between D0 and D5. Low levels of degradation can be observed at pH 3 after 5 days for VERT-002 and VERT-004, as suggested by the presence of a small peak eluted before the intact antibody. The deconvoluted intact mass (at Da) for the pH-stressed samples is shown in the table below.

[0171] [Table 14]

[0172] Example 3.3.3 Analysis of samples stressed at pH 3 with SCX and SEC At pH 3, all forms already strongly aggregate at D0. SCX analysis of the sample did not detect any peaks. Since RP confirmed the presence of the protein, the absence of detection by SCX may be due to strong aggregation preventing peak elution. An exemplary chromatogram (VERT002) is shown in Figure 9.

[0173] Example 3.3.4 Analysis of samples stressed at pH 9 using SCX and SEC At pH 9, a slight increase in HMW was observed for all three types of antibodies. A small increase was also observed in the acidic molecular species. No differences were observed among CERT001, VERT002, and VERT004.

[0174] Example 3.3.5 Analysis of stress-treated samples using DSF The effect of pH on VERT sample stability was analyzed by DSF. VERT-002 and VERT-004 incubated at pH 9 showed no visual changes 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 relatively low stability (aggregation and / or misfolding) of VERT-002 and VERT-004 at acidic pH, but basic pH does not appear to affect their samples in DSF. Observations at pH 3 correlate well with SEC results showing aggregation. The results are summarized in the table below.

[0175] [Table 15]

[0176] Regarding temperature stress, incubation at 55°C for 5 days resulted in significantly higher initial fluorescence (RFU at t0) for all samples compared to the assay standard, likely due to aggregation or misfolding events. As can be observed from the unaffected Tm and RFU at t0, none of the antibodies were affected by the three freeze / thaw (F / T) cycles. The results are summarized in the table below.

[0177] [Table 16]

[0178] Example 3.3.6 Analysis of a sample subjected to stress at 55°C All antibodies were subjected to 55°C for 5 days. VERT001 and VERT002 had some fibrous material formed at the end of the incubation period, while VERT004 had particles. After filtration, only VERT004 showed a significant decrease in antibody concentration (4.4 mg / mL). Under these stress conditions, all forms showed increased aggregates (relatively high HMW) and fragmentation (relatively high LMW). See the table below.

[0179] [Table 17]

[0180] RP chromatography analysis revealed some minor impurities / fragments, which eluted immediately before 4.50 minutes for VERT-002 and VERT-004 at 55°C, D5. Mass deconvolution suggested fragments of approximately 14 kDa. VERT-001 could not be analyzed due to the limited remaining sample volume. Interestingly, a decrease in initial fragments / impurities (cleaved FC, LC, mispaired fragments) was observed for VERT-004 (and was also too low for VERT-002). The 55°C condition yielded similar observations to a 4-week incubation at 37°C.

[0181] Example 3.3.7 Analysis of samples subjected to freeze / thaw cycles VERT001, VERT002, and VERT004 were subjected to three freeze (-80°C) / thaw (RT) cycles. No changes were observed in IEX, SEC, or RP. The following table summarizes the monomer, HMW, and LMW percentages as determined by SEC analysis.

[0182] [Table 18]

[0183] Example 3.3.8 Analysis of antiproliferative activity of stress-induced samples Three types of antibodies, VERT001, VERT002, and VERT004, that were stressed under different conditions were tested for their antiproliferative activity.

[0184] At pH 3, all antibodies completely lost their antiproliferative activity against Hs746 T cells and EBC-1 cells, but at pH 9, the activity of the stress-induced antibodies remained similar to that of the reference substance.

[0185] The antiproliferative activity of binders stressed at 55°C for 5 days was tested against Hs746T only. The activity of VERT-001 and VERT-002, stressed at 55°C, remained within the expected range, but VERT-004 lost its antiproliferative activity.

[0186] The results are shown in Figure 10. The IC50 determined for the cell line Hs746T is shown in Table 18, and the IC50 determined for the cell line EBC-1 is shown in Table 19.

[0187] [Table 19]

[0188]

Table 20

[0189] The anti - proliferative activities of VERT - 001, VERT - 002 and VERT - 004 after 3 freeze / thaw (F / T) cycles showed no deviation from the activity of the assay standard after the F / T cycles.

[0190] Example 4: Pharmacological and efficacy studies Example 4.1 Efficacy and pharmacology in the Hs746T xenograft model The in - vivo anti - tumor efficacy of VERT - 001 was compared with VERT - 002 and VERT - 004 in the Hs746T model (MET exon 14 skipping and MET amplification) subcutaneously (s.c.) transplanted into female hairless SCID mice. All variants were tested at the same dose level of 20 mg / kg administered intravenously (i.v.) twice a week (BIW). The results are shown in Figure 11. All variants tested were sufficient and showed equivalent efficacy at 20 mg / kg.

[0191] The dose - response regarding the anti - tumor efficacy of VERT - 002 in vivo was further evaluated in the Hs746T CDX model. VERT - 002 was administered i.v. twice a week at doses of 10 and 20 mg / kg and once a week at 20 mg / kg for up to 4 weeks, while the isotype control was administered at 20 mg / kg twice a week (Figure 12). VERT - 002 showed dose - dependent tumor growth inhibition (TGI) including complete tumor regression when administered at the highest dose of 20 mg / kg twice a week. As reflected by the very low mean tumor volume levels on day 35, 6 out of 9 mice from the highest dose group had no tumors by day 35.

[0192] Mice treated twice weekly with 20 mg / kg were monitored for 30 days after dosing was completed, at which time the majority (6 / 9 mice) of the treatment group remained tumor - free. The 3 / 9 mice that had residual tumors at the end of the dosing period (day 32) were the only mice that had tumor regrowth during the post - administration monitoring.

[0193] Example 4.2 Efficacy and Pharmacology in the EBC - 1 Xenograft Model VERT - 002 was further profiled in vivo using EBC - 1 cells (bearing MET amplification) s.c. transplanted into female Balb / c nude mice. VERT - 002 was administered i.v. at doses of 5, 10, and 20 mg / kg twice weekly and 20 mg / kg once weekly, while an isotype control was administered at 20 mg / kg twice weekly (Figure 13, panels A and B). VERT - 002 showed a clear dose - dependent TGI, including complete tumor regression seen at 20 mg / kg twice weekly, which was consistent with the results from the Hs746T CDX model. In addition, VERT - 002 induced a marked increase in the level of soluble MET ectodomain (sMET ECD), which was dose - dependent and reached a plateau around day 6 of treatment (Figure 13, panel C). Interestingly, in response to VERT - 002 dosing, the sMET ECD levels closely resembled the tumor regression profile, indicating that less ECD is reduced when the tumor shrinks (tumor regression occurs around the same time point on day 7). Similar results regarding the VERT - 002 dose response for TGI and sMET ECD were also seen in a chronic study (single dose) in the EBC - 1 model (data not shown).

Claims

1. 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 polypeptides, and the antibody fragment is (i) A first polypeptide comprising a humanized light chain variable (VL) domain and one human light chain constant (CL) domain, wherein the humanized VL domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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, and the humanized VH domain comprises three complementarity-determining regions (CDRs) having the amino acid sequences described 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 constant CH3 domain. Includes, The anti-Met antibody fragment wherein the Fc regions of the first FC polypeptide and the second Fc polypeptide contain mutations L234A, L235A, and P329A (according to the EU index).

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

3. The anti-Met antibody fragment according to claim 1 or 2, wherein, in the direction from the N-terminus to the C-terminus, the humanized VH domain is fused to the human CH1 domain, the human CH1 domain is fused to the human hinge region, the human hinge region is fused to the human CH2 domain, and the human CH2 domain is fused to the human CH3 domain.

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

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

13.

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

14.

7. An anti-Met antibody fragment according to any one of claims 1 to 6, wherein a first Fc polypeptide and a second Fc polypeptide associate at an interface, one of the first and second Fc polypeptides includes a knob at the interface, and the other of the first and second Fc polypeptides includes a hole at the interface, the knob may be located within the hole, either the first or second Fc polypeptide includes a mutant CH3 constant domain, the mutant CH3 constant domain holds an amino acid mutation at position 389, the original amino acid at position 389 is mutated to import an amino acid having a larger side-chain capacity than the original amino acid; the other Fc polypeptide includes a mutant CH3 constant domain, the mutant CH3 constant domain holds three amino acid mutations at positions 389, 391 and 438, the original amino acid is mutated to import an amino acid having a smaller side-chain capacity than the original amino acid, and amino acid numbering follows Kabat's EU numbering scheme.

8. The original amino acids at positions 389, 391, and 438 are threonine, leucine, and tyrosine, respectively; in the first or second Fc polypeptide, threonine at position 389 is mutated to tryptophan; in the other Fc polypeptide, threonine at position 389 is mutated to serine, leucine at position 391 is mutated to alanine, and tyrosine at position 438 is mutated to valine, according to claim 7.

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

16.

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

18.

11. The anti-Met antibody fragment according to 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 an anti-Met antibody fragment according to any one of claims 1 to 11.

13. A composition comprising two or more recombinant nucleic acids that collectively encode the anti-Met antibody fragments described in any one of claims 1 to 11.

14. An anti-Met antibody fragment according to any one of claims 1 to 11, for use in the treatment of tumors and / or metastases.

Images