Monoclonal antibodies against CLDN18.2 and their Fc-modified versions

Abstract

Optionally, the Fc region may be modified, and the antibody specifically binds to CLDN18.2 and A panel of monoclonal antibodies that do not specifically bind to DN18.1 is provided.

Description

Technical Field

[0001] This application claims priority to PCT Patent Application Nos. PCT / CN2021 / 097239 and PCT / CN2021 / 097240, filed on May 31, 2021, and PCT Patent Application Nos. PCT / CN2021 / 106783 and PCT / CN2021 / 106784, filed on July 16, 2021, the contents of which are incorporated herein by reference.

[0002] An antibody panel may optionally have a modified Fc region, specifically bind to CLDN18.2, and not specifically bind to CLDN18.1 is provided.

Background Art

[0003] Tight junction molecule Claudin 18 splice variant 2 (Claudin 18.2 (CLDN18.2)) is a member of the Claudin family of tight junction proteins. CLDN18.2 is a 27.8 kDa transmembrane protein containing four transmembrane domains with two small extracellular loops.

[0004] In normal tissues, except for the stomach, there is no detectable expression of CLDN18.2 by RT-PCR. Immunohistochemistry with a CLDN18.2 specific antibody reveals that the stomach is the only positive tissue.

[0005] CLDN18.2 is a highly selective gastric lineage antigen that is expressed exclusively in short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained during the process of malignant transformation and is therefore often presented on the surface of human gastric cancer cells. Moreover, this pan-tumor antigen is ectopically activated at significant levels in esophageal adenocarcinoma, pancreatic adenocarcinoma, and lung adenocarcinoma. The CLDN18.2 protein is also localized in lymph node metastases of gastric adenocarcinoma and, especially, in distal metastases to the ovary (so-called Krukenberg tumors).

Summary of the Invention

[0006] The present invention provides an anti-CLDN18.2 antibody.

[0007] The present invention provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, where the antibody (1) HVR-H1, HVR-H2, and HVR-H3 contained within VH as represented by SEQ ID NO: 1, and HVR-L1, HVR-L2, and HVR-L3 contained within VL as represented by SEQ ID NO: 2; (2) HVR-H1, HVR-H2, and HVR-H3 contained within VH as represented by SEQ ID NO: 3, and HVR-L1, HVR-L2, and HVR-L3 contained within VL as represented by SEQ ID NO: 4; (3) HVR-H1, HVR-H2, and HVR-H3 contained within VH as represented by SEQ ID NO: 5, and HVR-L1, HVR-L2, and HVR-L3 contained within VL as represented by SEQ ID NO: 6; or (4) HVR-H1, HVR-H2, and HVR-H3 contained within VH as represented by SEQ ID NO: 7, and HVR-L1, HVR-L2, and HVR-L3 contained within VL as represented by SEQ ID NO: 8, and for example, those as represented in FIGS. 1A, 1B, 1C, or 1D, and optionally may contain one or more mutations in the Fc region.

[0008] In one aspect, the antibody (1) HVR-H1 as represented by SEQ ID NO: 11, HVR-H2 as represented by SEQ ID NO: 12, HVR-H3 as represented by SEQ ID NO: 13, HVR-L1 as represented by SEQ ID NO: 14, HVR-L2 as represented by SEQ ID NO: 15, and HVR-L3 as represented by SEQ ID NO: 16; (2) HVR-H1 as represented by SEQ ID NO: 17, HVR-H2 as represented by SEQ ID NO: 18, HVR-H3 as represented by SEQ ID NO: 19, HVR-L1 as represented by SEQ ID NO: 20, HVR-L2 as represented by SEQ ID NO: 21, and HVR-L3 as represented by SEQ ID NO: 22; (3) HVR-H1 as represented by SEQ ID NO: 23, HVR-H2 as represented by SEQ ID NO: 24, HVR-H3 as represented by SEQ ID NO: 25, HVR-L1 as represented by SEQ ID NO: 26, HVR-L2 as represented by SEQ ID NO: 27, and HVR-L3 as represented by SEQ ID NO: 28; or (4) including HVR-H1 as represented by SEQ ID NO: 29, HVR-H2 as represented by SEQ ID NO: 30, HVR-H3 as represented by SEQ ID NO: 31, HVR-L1 as represented by SEQ ID NO: 32, HVR-L2 as represented by SEQ ID NO: 33, and HVR-L3 as represented by SEQ ID NO: 34.

[0009] In one aspect, the antibody is (1) HVR-H1 as represented by SEQ ID NO: 41, HVR-H2 as represented by SEQ ID NO: 42, HVR-H3 as represented by SEQ ID NO: 43, HVR-L1 as represented by SEQ ID NO: 44, HVR-L2 as represented by SEQ ID NO: 45, and HVR-L3 as represented by SEQ ID NO: 46; (2) HVR-H1 as represented by SEQ ID NO: 47, HVR-H2 as represented by SEQ ID NO: 48, HVR-H3 as represented by SEQ ID NO: 49, HVR-L1 as represented by SEQ ID NO: 50, HVR-L2 as represented by SEQ ID NO: 51, and HVR-L3 as represented by SEQ ID NO: 52; or (3) including HVR-H1 as represented by SEQ ID NO: 53, HVR-H2 as represented by SEQ ID NO: 54, HVR-H3 as represented by SEQ ID NO: 55, HVR-L1 as represented by SEQ ID NO: 56, HVR-L2 as represented by SEQ ID NO: 57, and HVR-L3 as represented by SEQ ID NO: 58.

[0010] The present invention further provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, wherein the antibody: (1) a VH comprising HVR-H1 as represented by SEQ ID NO: 11, HVR-H2 as represented by SEQ ID NO: 12, and HVR-H3 as represented by SEQ ID NO: 13, and a VL comprising HVR-L1 as represented by SEQ ID NO: 14, HVR-L2 as represented by SEQ ID NO: 15, and HVR-L3 as represented by SEQ ID NO: 16; (2) a VH comprising HVR-H1 as represented by SEQ ID NO: 17, HVR-H2 as represented by SEQ ID NO: 18, and HVR-H3 as represented by SEQ ID NO: 19, and a VL comprising HVR-L1 as represented by SEQ ID NO: 20, HVR-L2 as represented by SEQ ID NO: 21, and HVR-L3 as represented by SEQ ID NO: 22; (3) a VH comprising HVR-H1 as represented by SEQ ID NO: 23, HVR-H2 as represented by SEQ ID NO: 24, and HVR-H3 as represented by SEQ ID NO: 25, and a VL comprising HVR-L1 as represented by SEQ ID NO: 26, HVR-L2 as represented by SEQ ID NO: 27, and HVR-L3 as represented by SEQ ID NO: 28; or (4) a VH comprising HVR-H1 as represented by SEQ ID NO: 29, HVR-H2 as represented by SEQ ID NO: 30, and HVR-H3 as represented by SEQ ID NO: 31, and a VL comprising HVR-L1 as represented by SEQ ID NO: 32, HVR -L2 as represented by SEQ ID NO: 33, and HVR-L3 as represented by SEQ ID NO: 34, optionally comprising one or more mutations in the Fc region. Optionally, it may contain one or more mutations in the Fc region.

[0011] The present invention further provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, wherein the antibody: (1) A VH comprising HVR-H1 as represented by SEQ ID NO: 41, HVR-H2 as represented by SEQ ID NO: 42, and HVR-H3 as represented by SEQ ID NO: 43, and a VL comprising HVR-L1 as represented by SEQ ID NO: 44, HVR-L2 as represented by SEQ ID NO: 45, and HVR-L3 as represented by SEQ ID NO: 46; (2) A VH comprising HVR-H1 as represented by SEQ ID NO: 47, HVR-H2 as represented by SEQ ID NO: 48, and HVR-H3 as represented by SEQ ID NO: 49, and a VL comprising HVR-L1 as represented by SEQ ID NO: 50, HVR-L2 as represented by SEQ ID NO: 51, and HVR-L3 as represented by SEQ ID NO: 52; or (3) A VH comprising HVR-H1 as represented by SEQ ID NO: 53, HVR-H2 as represented by SEQ ID NO: 54, and HVR-H3 as represented by SEQ ID NO: 55, and a VL comprising HVR-L1 as represented by SEQ ID NO: 56, HVR-L2 as represented by SEQ ID NO: 57, and HVR-L3 as represented by SEQ ID NO: 58, which optionally may contain one or more mutations in the Fc region. Optionally, it may contain one or more mutations in the Fc region.

[0012] The present invention further provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, where the antibody (1) comprises a VH as represented by SEQ ID NO: 1 and a VL as represented by SEQ ID NO: 2; (2) comprises a VH as represented by SEQ ID NO: 3 and a VL as represented by SEQ ID NO: 4; (3) comprises a VH as represented by SEQ ID NO: 5 and a VL as represented by SEQ ID NO: 6; or (4) comprises a VH as represented by SEQ ID NO: 7 and a VL as represented by SEQ ID NO: 8, which optionally may contain one or more mutations in the Fc region, and optionally may contain one or more mutations in the Fc region, and optionally the first two amino acid residues at the N-terminus of the VH are absent.

[0013] In one aspect, one or more mutations in the Fc region are one or more mutations that modify (e.g., increase or decrease) binding to Fc receptors and / or effector functions, such as ADCC and / or CDC. In one aspect, one or more mutations in the Fc region are one or more substitutions selected from the group consisting of L235V, F243L, R292P, Y300L, and P396L. In one aspect, one or more mutations in the Fc region are L235V, F243L, R292P, Y300L, and P396L.

[0014] The present invention further provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, wherein the antibody: i) with respect to binding to human CLDN18.2, (1) VH as represented by SEQ ID NO: 1 and VL as represented by SEQ ID NO: 2; (2) VH as represented by SEQ ID NO: 3 and VL as represented by SEQ ID NO: 4; (3) VH as represented by SEQ ID NO: 5 and VL as represented by SEQ ID NO: 6; or (4) competes with an anti-CLDN18.2 antibody comprising VH as represented by SEQ ID NO: 7 and VL as represented by SEQ ID NO: 8, and / or ii) (1) VH as represented by SEQ ID NO: 1 and VL as represented by SEQ ID NO: 2; (2) VH as represented by SEQ ID NO: 3 and VL as represented by SEQ ID NO: 4; (3) VH as represented by SEQ ID NO: 5 and VL as represented by SEQ ID NO: 6; or (4) binds to the same epitope of human CLDN18.2 as an anti-CLDN18.2 antibody comprising VH as represented by SEQ ID NO: 7 and VL as represented by SEQ ID NO: 8; and / or iii) The ADCC of PBMCs on cells expressing human CLDN18.2 (e.g., 293T cells or CHO cells or CT26 cells or KATOIII cells or NCI-N87 cells) is mediated by an EC50 value of 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, 0.009 nM, 0.008 nM, 0.007 nM, 0.006 nM, 0.005 nM, 0.004 nM, 0.003 nM, 0.002 nM, or 0.001 nM, in the vicinity of, or less than, that value, as determined, for example, by LDH or FACS; and / or iv) It does not mediate the ADCC of PBMCs on cells expressing human CLDN18.1 (e.g., 293T cells or CHO cells or CT26 cells or KATOIII cells or NCI-N87 cells); and / or v) The CDC on cells expressing human CLDN18.2 (e.g., 293T cells or CHO cells or CT26 cells or KATOIII cells or NCI-N87 cells) is mediated by an EC50 value of 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, 0.009 nM, 0.008 nM, 0.007 nM, 0.006 nM, 0.005 nM, 0.004 nM, 0.003 nM, 0.002 nM, or 0.001 nM, in the vicinity of, or less than, that value, as determined, for example, by LDH or FACS; and / or vi) It does not mediate the CDC on cells expressing human CLDN18.1 (e.g., 293T cells or CHO cells or CT26 cells or KATOIII cells or NCI-N87 cells); and / or vii) Those that bind to cells expressing human CLDN18.2 on the cell surface (e.g., 293T cells or CHO cells) at a Kd value of, for example, 50 pM, 45 pM, 40 pM, 38.6 pM, 35 pM, 30 pM, 25 pM, 20 pM, 15 pM, 13.1 pM, 10 pM, 9.5 pM, 9 pM, or 5 pM, in the vicinity thereof, or less than thereof; and / or viii) Those that do not bind to cells expressing human CLDN18.1 on the cell surface (e.g., 293T cells or CHO cells); and / or ix) Those that specifically bind to human CLDN18.2 at a Kd value of, for example, 10 nM, 9.5 nM, 9 nM, 8.5 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6.4 nM, 6 nM, 5.5 nM, 5 nM, 4.5 nM, 4 nM, 3.8 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.7 nM, 1.5 nM, or 1 nM, in the vicinity thereof, or less than thereof; and / or x) Those that do not specifically bind to human CLDN18.1.

[0015] In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is a mouse, chimeric, or humanized antibody.

[0016] In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is an antigen-binding antibody fragment, and optionally, may be selected from the group consisting of Fab fragment, Fab’ fragment, F(ab’)2 fragment, scFv fragment, and diabody.

[0017] In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is a full-length antibody. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention includes the heavy chain constant region of human IgG (especially IgG1), optionally as represented by SEQ ID NO: 9. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention includes the heavy chain constant region of mutant human IgG (especially IgG1), optionally as represented by SEQ ID NO: 40. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention includes the human kappa light chain constant region, optionally as represented by SEQ ID NO: 10. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is a chimeric antibody, for example, a mouse / human chimeric antibody. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is Fc-modified.

[0018] In one aspect, the monoclonal antibody (mAb) or Fab fragment of the present invention has a cross format (x-mAb or x-Fab), where either the variable domain or the (first) constant domain of the light chain and the heavy chain is exchanged.

[0019] The present invention provides an isolated nucleic acid encoding the monoclonal antibody of the present invention. The present invention provides a vector containing the nucleic acid of the present invention, for example, a cloning vector or an expression vector. The present invention provides a host cell containing the nucleic acid of the present invention or the vector of the present invention. The present invention provides a method for producing the monoclonal antibody of the present invention, which includes culturing the host cell so that the antibody is produced. In one aspect, the method further includes recovering the antibody from the host cell or the cell culture medium.

[0020] The present invention provides a composition containing the monoclonal antibody of the present invention. The present invention provides a pharmaceutical preparation containing the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.

[0021] The present invention provides the monoclonal antibody of the present invention for use as a medicament. The present invention provides the monoclonal antibody of the present invention for use in cancer treatment. The present invention provides the use of the monoclonal antibody of the present invention for the manufacture of a medicament. In one aspect, the medicament is used for cancer treatment. The present invention provides a method of treating an individual having cancer, comprising administering to the individual a therapeutically effective amount of the monoclonal antibody of the present invention.

Brief Description of the Drawings

[0022]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0023] I. Definitions

[0024] As used herein, the term "antibody" is used in the broadest sense and includes various antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity, but are not limited thereto.

[0025] The term "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0026] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species and the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0027] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major antibody classes, IgA, IgD, IgE, IgG, and IgM, several of which can be further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. Light chains can be assigned to one of two clearly different types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domains.

[0028] "Effector function" refers to biological activities that can result from the Fc region of an antibody and that differ depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0029] As used herein, the terms "modify, modified, modification" refer to any modification of the peptide backbone, or of a naturally occurring or recombinant polypeptide or its It is contemplated to include post-translational modifications of the fragment. Modifications include not only modifications of the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, but also combinations of these techniques.

[0030] The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and mutant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In one aspect, the anti-CLDN18.2 antibodies described herein are of the IgG1 isotype and include the heavy chain constant region of SEQ ID NO: 9 or SEQ ID NO: 40. In one aspect, it further includes the C-terminal lysine (Lys447). Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system, also called 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, and follows the EU numbering system.

[0031] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of the variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0032] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to the native antibody structure or having a heavy chain that includes an Fc region.

[0033] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" that include the primary transformed cell and progeny derived therefrom regardless of the number of passages. Progeny may not have exactly the same nucleic acid content as the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the original transformed cell are included herein.

[0034] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, and all or substantially all of the HVRs (e.g., CDRs) within the variable domains correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to the antibody that has been humanized.

[0035] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that has a sequence that is hypervariable ("complementarity-determining region" or "CDR") and / or forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact site"). Generally, an antibody comprises six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present in amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen - contacting regions present in amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996)); and (d) Combinations of (a), (b), and / or (c) that include HVR amino acid residues 46 - 56 (L2), 47 - 56 (L2), 48 - 56 (L2), 49 - 56 (L2), 26 - 35 (H1), 26 - 35b (H1), 49 - 65 (H2), 93 - 102 (H3), and 94 - 102 (H3) HVR residues can be identified at websites such as, for example, https: / / www.novopro.cn / tools / cdr.html.

[0036] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) within the variable domain are numbered herein according to Kabat et al. supra.

[0037] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies that may be present in minor amounts, such as those containing naturally occurring mutations or arising during the purification of the monoclonal antibody preparation. Typically, in contrast to polyclonal antibody preparations that contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the property of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be produced by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage-display methods, and methods that utilize transgenic animals containing all or part of the human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0038] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have a similar structure, each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. Further, an antibody that binds a particular antigen may be isolated by screening a library of VL domains or VH domains that bind the antigen, respectively, for a complementary VH domain or VL domain. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-6 28 (1991).

[0039] The term "vector" as used herein refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of an introduced host cell. A particular vector can direct the expression of a functionally linked nucleic acid. Such vectors are referred to herein as "expression vectors."

[0040] II. Exemplary Antibodies

[0041] The present invention provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, where the antibody comprises: a VH comprising HVR-H1 as represented by SEQ ID NO: 11, HVR-H2 as represented by SEQ ID NO: 12, and HVR-H3 as represented by SEQ ID NO: 13, and a VL comprising HVR-L1 as represented by SEQ ID NO: 14, HVR-L2 as represented by SEQ ID NO: 15, and HVR-L3 as represented by SEQ ID NO: 16. In one aspect, the antibody comprises a VH as represented by SEQ ID NO: 1 and a VL as represented by SEQ ID NO: 2. Optionally, the first two amino acid residues at the N-terminus of the VH are absent.

[0042] The present invention provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, where the antibody comprises: a VH comprising HVR-H1 as represented by SEQ ID NO: 17, HVR-H2 as represented by SEQ ID NO: 18, and HVR-H3 as represented by SEQ ID NO: 19, and a VL comprising HVR-L1 as represented by SEQ ID NO: 20, HVR-L2 as represented by SEQ ID NO: 21, and HVR-L3 as represented by SEQ ID NO: 22. In one aspect, the antibody comprises a VH as represented by SEQ ID NO: 3 and a VL as represented by SEQ ID NO: 4. Optionally, the first two amino acid residues at the N-terminus of the VH are absent.

[0043] The present invention provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, where the antibody comprises: a VH comprising HVR-H1 as represented by SEQ ID NO: 23, HVR-H2 as represented by SEQ ID NO: 24, and HVR-H3 as represented by SEQ ID NO: 25, and a VL comprising HVR-L1 as represented by SEQ ID NO: 26, HVR-L2 as represented by SEQ ID NO: 27, and HVR-L3 as represented by SEQ ID NO: 28. In one aspect, the antibody comprises a VH as represented by SEQ ID NO: 5 and a VL as represented by SEQ ID NO: 6. Optionally, the first two amino acid residues at the N-terminus of the VH are absent.

[0044] The present invention provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, wherein the antibody comprises: a VH comprising HVR-H1 as represented by SEQ ID NO: 29, HVR-H2 as represented by SEQ ID NO: 30, and HVR-H3 as represented by SEQ ID NO: 31, and a VL comprising HVR-L1 as represented by SEQ ID NO: 32, HVR-L2 as represented by SEQ ID NO: 33, and HVR-L3 as represented by SEQ ID NO: 34. The present invention provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, wherein the antibody comprises: a VH comprising HVR-H1 as represented by SEQ ID NO: 41, HVR-H2 as represented by SEQ ID NO: 42, and HVR-H3 as represented by SEQ ID NO: 43, and a VL comprising HVR-L1 as represented by SEQ ID NO: 44, HVR-L2 as represented by SEQ ID NO: 45, and HVR-L3 as represented by SEQ ID NO: 46. The present invention provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, wherein the antibody comprises: the arra A VH comprising HVR-H1 as represented by column number 47, HVR-H2 as represented by SEQ ID NO: 48, and HVR-H3 as represented by SEQ ID NO: 49, and a VL comprising HVR-L1 as represented by SEQ ID NO: 50, HVR-L2 as represented by SEQ ID NO: 51, and HVR-L3 as represented by SEQ ID NO: 52. The present invention provides an isolated monoclonal antibody that specifically binds to human CLDN18.2, particularly an Fc-modified one, wherein the antibody comprises: a VH comprising HVR-H1 as represented by SEQ ID NO: 53, HVR-H2 as represented by SEQ ID NO: 54, and HVR-H3 as represented by SEQ ID NO: 55, and a VL comprising HVR-L1 as represented by SEQ ID NO: 56, HVR-L2 as represented by SEQ ID NO: 57, and HVR-L3 as represented by SEQ ID NO: 58. In one aspect, the antibody comprises a VH as represented by SEQ ID NO: 7 and a VL as represented by SEQ ID NO: 8. Optionally, the first two amino acid residues at the N-terminus of the VH are absent.

[0045] In one aspect, one or more mutations in the Fc region are one or more mutations that modify (e.g., increase or decrease) binding to Fc receptors and / or effector functions, such as ADCC and / or CDC. In one aspect, one or more mutations in the Fc region are one or more substitutions selected from the group consisting of L235V, F243L, R292P, Y300L, and P396L. In one aspect, one or more mutations in the Fc region are L235V, F243L, R292P, Y300L, and P396L.

[0046] In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is a mouse, chimeric, or humanized antibody.

[0047] In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is an antigen-binding antibody fragment, optionally selected from the group consisting of Fab fragment, Fab’ fragment, F(ab’)2 fragment, scFv fragment, and diabody.

[0048] In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is a full-length antibody. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention comprises a heavy chain constant region of human IgG (especially IgG1), optionally as represented by SEQ ID NO: 9. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention comprises a heavy chain constant region of mutant human IgG (especially IgG1), optionally as represented by SEQ ID NO: 40. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention comprises a human kappa light chain constant region, optionally as represented by SEQ ID NO: 10. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is a chimeric antibody, for example, a mouse / human chimeric antibody. In one aspect, the anti-CLDN18.2 monoclonal antibody of the present invention is Fc-modified.

[0049] In one aspect, the monoclonal antibody (mAb) or Fab fragment of the present invention has a crossed form (x-mAb or x-Fab), where either the variable domains or the (first) constant domains of the light and heavy chains are exchanged.

[0050] III. Recombinant Methods and Compositions

[0051] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0052] In the case of native antibodies or native antibody fragments, two nucleic acids are required, one for the light chain or its fragment and one for the heavy chain or its fragment. Such nucleic acids encode amino acid sequences containing the VL of the antibody and / or the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). These nucleic acids can be present on the same expression vector or on different expression vectors.

[0053] In one aspect, there is provided an isolated nucleic acid encoding an antibody used in the methods described herein.

[0054] In a further aspect, there is provided one or more vectors (e.g., expression vectors) containing such nucleic acids.

[0055] In a further aspect, there is provided a host cell containing such nucleic acids.

[0056] In such an aspect, the host cell contains (e.g., is transformed with) the following vectors: (1) a vector containing a nucleic acid encoding an amino acid sequence including the VL of the antibody and an amino acid sequence including the VH of the antibody, or (2) a first vector containing a nucleic acid encoding an amino acid sequence including the VL of the antibody and a second vector containing a nucleic acid encoding an amino acid sequence including the VH of the antibody.

[0057] In one aspect, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte cell line (e.g., Y0, NS0, Sp2 / 0 cells). In one aspect, there is provided a method for producing an antibody, the method including culturing a host cell containing a nucleic acid encoding the antibody provided above under conditions suitable for the expression of the antibody, and optionally recovering the antibody from the host cell or the host cell culture medium.

[0058] For the recombinant production of an antibody, for example, as described above, a nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody), or can be produced by recombinant methods or obtained by chemical synthesis.

[0059] Host cells suitable for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, particularly when glycosylation and Fc effector functions are not required, antibodies can be produced in bacteria. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (Also see Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which also describes the expression of antibodies in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and further purified.

[0060] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including those with "humanized" glycosylation pathways in fungal and yeast strains, resulting in the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0061] Host cells suitable for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified and can be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0062] Plant cell culture can be used as a host. For example, see U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES TM relating to antibody production in transgenic plants).

[0063] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cells transformed with SV40 (COS-7), human embryonic kidney cell lines (Graham F.L. et al., J. Gen Virol. 36: (1977) 59-74), the 293 cells or 293 cells described therein; baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells described in Mather J.P., Biol. Reprod. 23: (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT060562); for example, Mather J.P. et al., Annals N.Y. Acad. Sci. 383: (1982) 44-68), the TRI cells described therein; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines are DHFR-CHO cells (Urlaub Chinese hamster ovary (CHO) cells, including G. et al., Proc. Natl. Acad. Sci. USA 77:(1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A.M. Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ(2004), pp. 255-268.

[0064] IV. Assay

[0065] The antibodies provided herein can be identified and screened or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0066] Binding assays and other assays

[0067] In one aspect, the antibodies of the invention are tested for their antigen-binding activity by, for example, known methods such as ELISA, Western blot, etc.

[0068] In another aspect, a competition assay can be used to identify antibodies that compete with CLDN18.2 for binding to CLDN18.2. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by CLDN18.2. Detailed and exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) are provided.

[0069] In a typical competitive assay, immobilized CLDN18.2 is incubated in a solution containing a first labeled antibody that binds to CLDN18.2 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to CLDN18.2. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CLDN18.2 is incubated in a solution containing the labeled first antibody rather than the unlabeled second antibody. After incubation under conditions that permit binding of the first antibody to CLDN18.2, excess unbound antibody is removed and the amount of label bound to the immobilized CLDN18.2 is measured. If the amount of label bound to the immobilized CLDN18.2 is substantially decreased in the test sample compared to the control sample, this indicates that the second antibody is competing with the first antibody for binding to CLDN18.2. See Harlow and Lane (1988) Antibodies:A Laboratory Manual See ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0070] Assay of activity

[0071] In one aspect, the assay is provided to identify those anti-CLDN18.2 antibodies that have biological activity. The biological activity can include, for example, ADCC by PBMC against target cells expressing CLDN18.2 by the anti-CLDN18.2 antibody. Antibodies having such biological activity in vivo and / or in vitro are also provided.

Example

[0072] As a result of the first screening, four positive hybridoma cell lines derived from immunized mice were identified that specifically bind to CLDN18.2-expressing cells but do not bind to CLDN18.1-expressing cells.

[0073] Example 1: Cloning of Four CLDN18.2-Specific Monoclonal Antibodies (mAbs) from Mouse Hybridoma Cells

[0074] This example shows an example of cloning the genes of the H chain and L chain of an antibody from mouse hybridoma cells to obtain a variable region sequence specific to CLDN18.2 and preparing a chimeric antibody.

[0075] According to the conventional procedures in the art, Quick-RNA TM Microprep Kit (ZYMO Research, Cat. # R1050) was used to isolate and purify RNA from hybridoma cells. First-strand cDNA was synthesized, and SMARTer (R) RACE 5’ / 3’ kit (Takara Bio USA, Inc. Cat. # 634858) was used to perform RACE together with IgG1 3’ constant primer (SEQ ID NO: 35), IgG2a 3’ constant primer (SEQ ID NO: 36), and Kappa 3’ constant primer (SEQ ID NO: 37). The RACE DNA products were subjected to gel extraction using NuceloSpin Gel and PCR Clean-Up kit (Takara, Cat. # 740986.20). The in-fusion reaction mixture of the linearized pRACE vector and the gel-purified PACE products was transformed into Stellar competent cells (Clontech, Cat.# 636766). Plasmid DNA was separated from the transformants using QIAprep Spin Miniprep Kit (Qiagen, Cat. # 27104) and subjected to Sanger sequencing (GENEWIZ) using M13 sequencing primers. Four pairs of final gene sequences for the heavy and light chains were obtained (data not shown), which were determined as CLDN18.2 mAbs (mAb1, mAb2, mAb3, mAb4) as follows and were determined as CLDN18.2 mAbs (mAb1, mAb2, mAb3, mAb4) as follows.

[0076] Example 2: Construction of Chimeric Antibodies with Mouse Constant Regions Replaced by Human Constant Regions

[0077] This example shows an example of constructing a chimeric antibody by replacing the constant regions of four molecularly cloned mouse mAbs with those from human IgG1 heavy chain and kappa light chain.

[0078] Plasmid pFUSE-CHIg-hG1 (InvivoGen, Cat# pfuse-hchg1) (SEQ ID NO: 9) containing the constant region of human IgG1 heavy chain and plasmid pFUSE2-CLIg-hk (InvivoGen, Cat # pfuse2-hclk) (SEQ ID NO: 10) containing the constant region of human kappa light chain were digested with Hind III and Nhe I (for IgG1) or BsiW I (for kappa) (all from NEB lab). The linearized plasmids were purified by gel purification using NucleoSpin Gel and PCR Clean-up kit (Takara, Cat. # 740986.20). The coding sequences of the mouse heavy and light chain variable regions of mAb1, mAb2, mAb3, and mAb4 were PCR amplified with specific primers using HiFi HotStart (Kapa (Roche), KK2602) from the plasmids obtained in Example 1 (data not shown) and purified by gel purification using NucleoSpin Gel and PCR Clean-up kit (Takara, Cat.# 740986.20). The linearized vector and the insert fragment were Gibson Assembly (R) HiFi 1 Step Kit (SGI (VWR), Cat. # It was ligated using (GA1100-50). The assembly reaction mixture was transformed into Stellar competent cells (Clontech, Cat.# 636766). Plasmid DNA was isolated from the transformants using the QIAprep Spin Miniprep Kit (Qiagen, Cat. # 27104) and subjected to Sanger sequencing (GENEWIZ). Four chimeric antibodies containing the variable region of one of the four mouse mAbs prepared in Example 1 were constructed. The new chimeric antibodies were named YL-G1-19-01, YL-G1-19-02, YL-G1-19-03, and YL-G1-19-04.

[0079] Example 3: Confirmation of the Specificity of Four Chimeric Monoclonal Antibodies by Surface Staining

[0080] This example is an example of testing the binding specificity of four chimeric monoclonal antibodies to CLDN18.2 by comparing them with a reference mAb (IMAB362, Ganymed).

[0081] CLDN18.2-expressing 293T cells and CLDN18.1-expressing 293T cells (50 μL) at a density of 2×10 6 cells / ml were mixed with a dilution series of chimeric antibodies, reference antibodies, or IgG negative control (50 μL) (60.00, 20.00, 6.67, 2.22, 0.74, 0.25, 0.08 μg / mL, in FACS buffer) in a 96-well V-bottom plate and incubated on ice for 30 minutes. After washing with 200 μL / well of FACS buffer, 30 μL / well of the secondary antibody Alexa Fluor (R) 647 AffiniPure Goat Anti-Human The cells were resuspended in IgG, Fcγ fragment specific (Jackson, Cat. #109-605-098) and incubated on ice for 20 minutes. After washing three times with 200 μL / well of FACS buffer, the cells were resuspended in 150 μL / well of FACS buffer and subjected to FACS using a BD LSR II flow cytometer (HTS). Geometric mean was used for data analysis and the plots were created using Prism GraphPad. As a result of flow cytometry analysis, four chimeric antibodies were shown to have high specific binding to CLDN18.2-expressing cells compared to the reference mAb (see Figure 2). No binding to CLDN18.1-expressing cells was observed (see Figure 3), suggesting high specificity of the four chimeric antibodies.

[0082] Example 4: Functional confirmation of four chimeric antibodies by ADCC assay

[0083] This example is an example of testing the ADCC-mediated cytotoxic activity of these four chimeric antibodies in comparison with a reference mAb (IMAB362, Ganymed).

[0084] CLDN18.2-expressing 293T cells and CLDN18.1-expressing 293T cells were labeled with CFSE using eBioscience TM CFSE (Thermo, Cat. #65-0850-84). Lympholyte (R) Cell Separ ation Media (Cedarlane, Cat. # CL5110) (50 μL) containing CFSE-labeled cells at a density of 4 x 10 5 cells / ml were mixed with serial dilutions (20.00, 6.67, 2.22, 0.74, 0.25, 0.08, 0.03 μg / mL in medium) of the chimeric antibody, reference antibody, or IgG negative control (100 μL) in a 96-well V-bottom plate and incubated in the dark at room temperature for 15 minutes. Next, 5×10 6PBMC (50 μL) with a density of cells / ml was added, and the plate was incubated at 37 °C in the dark for 2 hours. The cells were washed twice with PBS, and eBioscience TM Fixable Viability Dye eFluor TM 660 (Thermo, Cat. # 65-0864) working solution was added at 100 μL per well. The plate was incubated on ice in the dark for 30 minutes. After washing with PBS, 75 μL / well of PBS and 25 μL / well of 4% paraformaldehyde were added to resuspend the cells, and they were subjected to FACS using a BD LSR II Flow Cytometer (HTS). The injury rate (CFSE-positive population vs. CFSE / FVD-AF660 double-positive population) was used for data analysis, and the plots were created using Prism GraphPad. The ADCC activity by the FACS-based method showed that the four chimeric antibodies could mediate the ADCC activity against CLDN18.2-expressing cells (see Figure 4).

[0085] Example 5: Characterization of Fc-Modified Chimeric Antibodies

[0086] Furthermore, four Fc-modified chimeric antibodies were constructed. Compared with the first-generation initial chimeric antibodies YL-G1-19-01, YL-G1-19-02, YL-G1-19-03, and YL-G1-19-04, the second-generation Fc-modified chimeric antibodies YL-G2-A, YL-G2-B, YL-G2-C, and YL-G2-D (in YL-G2-D, the first two N-terminal amino acid residues of the VH domain are absent) contain five substitutions in the Fc region, namely L235V, F243L, R292P, Y300L, and P396L, according to the EU numbering. The mutant constant region of the human IgG1 heavy chain (including CH1, hinge, CH2, and CH3) is described in SEQ ID NO: 40.

[0087] In this example, the characterization of these Fc-modified chimeric antibodies is described.

[0088] 5.1: Antigen-antibody binding interaction

[0089] This example shows the characterization of the antigen-antibody binding interactions of YL-G2-B, YL-G2-C, and YL-G2-D.

[0090] The in vitro bioactivities of YL-G2-B, YL-G2-C, and YL-G2-D were analyzed by monitoring their binding to recombinant huCLDN18.2-Fc and huCLDN18.2-overexpressing CHO cells using the KinExA 4000 system (KinExA, US). ed.

[0091] To measure the affinity using the KinExA method, serial dilutions of binding partner B (known as the titrant) were performed in the background of binding partner A (constant binding partner, known as CBP). That is, CBP was kept at a constant concentration, and the titrant concentration was varied. When these solutions reached equilibrium, the KinExA 4000 instrument could directly measure the amount of CBP, the unbound or free binding partner remaining in the solution. Using software from Sapidyne, the dissociation rate of CBP could be plotted against the total concentration of the titrant to create a binding curve and determine the affinity.

[0092] The Kd was first measured using an antibody purchased from Sino Biological and recombinant human CLDN18.2 (P / N: 20047-H02H). In the equilibrium experiment, the titrant (huCLDN18.2) was serially diluted 5-fold in the background of CBP. Two equilibrium experiments were performed. One was adding a 5-fold serial dilution of 150 nM titrant to 10 nM high-concentration CBP (20 nM binding sites), and the other was adding a 5-fold serial dilution of 150 nM titrant to 100 pM low-concentration CBP (200 pM binding sites). The data were collected by KinExA 4000 and analyzed using Sapidyne Instruments n-Curve Analysis Software version 4.4.26. The binding curves are shown in Figure 5.

[0093] The KinExA 4000 instrument was also used to measure the binding affinity of an antibody to the surface proteins of intact cells, i.e., CHO cells overexpressing huCLDN18.2. The antibody concentration was kept constant (CBP), and the concentration of total cells containing the surface protein was varied (titrated). The concentration of total cells containing the surface protein was diluted threefold. The titrated cells were incubated with a constant binding partner (CBP). When equilibrium was reached, the samples were centrifuged, the supernatant was collected, and free CBP was detected with a fluorescently labeled anti-CBP molecule. The binding curve is shown in Figure 6.

[0094] To set a more accurate Kd, two equilibrium curves were created and analyzed. One was a curve with low concentrations of CBP at 100 pM (binding sites 200 pM) and 10 6 cells / mL diluted threefold, and the other was a curve with high concentrations of CBP at 10 nM (binding sites 20 nM) and 10 6 cells / mL diluted threefold. The KinExA 4000 measured the amount of unbound CBP in solution. The analysis was performed using Sapidyne Instruments n-Curve Analysis Software version 4.4.26. The equilibrium dissociation constant Kd is summarized in Table 1.

[0095]

Table 1

[0096] 5.2: Cell Binding Assay

[0097] In this example, the cell binding assays for YL-G2-B, YL-G2-C, and YL-G2-D are described.

[0098] To evaluate antibody binding, CHO cells expressing huCLDN18.2 were used. For each sample, 5 × 10 5Cells were seeded at [number] cells. Next, 100 μl of each serially diluted antibody was added to the cells, starting each dilution at 40 μg / ml and diluting 5-fold. Thus, the final concentration of each antibody started at 20 μg / ml and serial 5-fold dilutions were performed. After 1 hour, the cells were washed twice and 100 μl of GAH-FITC (1:200 dilution) was added to each well. After 30 minutes, the cells were washed twice and resuspended in 120 μl of FACS buffer. These cells were analyzed by flow cytometry.

[0099] Using unstained cells as a reference, gating of the entire population of target cells was set, and by establishing the FITC-negative population, the FITC-positive cell gate for each cell line could be established. Furthermore, to secondarily verify the FITC-positive results, the mean fluorescence intensity (MFI) of the entire cell population was calculated. The ratio of the number of gated positive cells to the total number of live cells was taken as the percentage of positive cells. The results are shown in Figure 7.

[0100] 5.3: Complement-dependent cytotoxicity (CDC) assay

[0101] In this example, the complement-dependent cytotoxicity (CDC) assay for YL-G2-B, YL-G2-C, and YL-G2-D is described.

[0102] Target cells (i.e., CHO cells expressing huCLDN18.2) were washed once with DPBS. Then, [number] cells in RPMI were seeded in a U-bottom plate at 100 μL / well. The antibody was serially diluted 1:2 and incubated with the cells at room temperature for 15 minutes at 50 μl / well. Next, 50 μl / well of 20% pooled serum was added to all wells including the natural release and maximum release wells. The plate was incubated in a 37°C incubator for 3.5 hours. 45 minutes before the end of incubation, the plate was centrifuged at 1200 rpm for 5 minutes, and 20 μL of Lysis buffer (CyQUANT 4 was added only to the maximum release control well containing the target cells. TMThe LDH Cytotoxicity Assay Kit, Cat.# C20300 and C20301) was added. 50 μL of the supernatant was transferred to a black-walled 96-well plate, and Reaction buffer (CyQUANT TM The LDH Cytotoxicity Assay Kit, Cat.# C20300 and C20301) was also added to all wells, the maximum release wells, and the spontaneous release wells. The plate was incubated in the dark for 30 minutes. After the incubation, 50 μL of Stop solution (CyQUANT TM The LDH Cytotoxicity Assay Kit, Cat.# C20300 and C20301) was added to all wells and gently mixed by tapping. The OD was measured at 490 nM and 680 nM. The activity is the cytotoxicity %. Cytotoxicity % = (experimental value - target cell spontaneous, without volume correction) / (maximum release of target cells - target cell spontaneous, with volume correction) * 100. The results are shown in Figure 8.

[0103] 5.4: Internalization Assay

[0104] In this example, the internalization assays of YL-G2-B, YL-G2-C, and YL-G2-D are described.

[0105] 5×10 in DMEM medium 5CHO cells expressing huCLDN18.2 at / ml were prepared. 100 μL of cells were seeded into each well of a U-bottom 96-well plate. The test antibody and control antibody were diluted to 40 μg / ml, and each antibody was serially diluted 1:4 in the culture medium. Each diluted antibody was added to the cells at 50 μl / well. The plate was incubated at 37 °C for 30 minutes. Next, 40 μg / ml of PEP-ZAP (a small Fc-binding peptide fused with a cytotoxic peptide, developer: AB Studio Inc.; see WO 2020 / 018732 A1) was added to each well at 50 μl / well to give a final concentration of PEP-ZAP of 10 μg / ml. The plate was incubated at 37 °C for 72 hours. Finally, the cells were spun down and 100 μl of the supernatant was taken for LDH measurement. The results are shown in Figure 9.

[0106] 5.5: Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay

[0107] In this example, the antibody-dependent cell-mediated cytotoxicity (ADCC) assay is described.

[0108] To evaluate the ADCC function of the antibody, target cells (CHO cells expressing huCLDN18.2) and effector cells (NK 8837-F cells, ATCC PTA-8837) were used. The target cells were seeded in DMEM-F12 + 10% FBS medium at 2 × 10 4 per 50 μl in one well of a 96-well plate for each sample. Next, 100 μl of each antibody serially diluted 1:10 was added to the cells. After 20 minutes, NK cells were added onto the plate at 2 × 10 5 per 50 μl such that the target:effector ratio was 1:10. After this addition, the final concentration of each antibody started at 50 μg / ml and continued at 5, 0.5, 0.05 μg / ml. This plate was placed in a 37 °C, CO2 incubator for 24 hours. Next, the cells were stained with 7AAD, washed twice, and resuspended in approximately 200 μl of FACS buffer. These cells were analyzed by flow cytometry.

[0109] By setting the gating for the entire target cells with unstained cells as a reference and establishing the 7AAD-negative population, it was possible to distinguish 7AAD (dead cells) from live cells.

[0110] Also, CT26 cells expressing huCLDN18.2 may be used as target cells, and ADCC may be determined by LDH.

[0111] The comparison of the ADCC activities of YL-G1-02 and YL-G2-B (having the same amino acid sequence except for the VLPLL substitution in the Fc region) is shown in Fig. 10. The EC50 is summarized in Table 2.

[0112]

Table 2

[0113] Example 6: Functional Characterization of Antibodies

[0114] 6.1: Cells

[0115] CT26 CLDN18 .2 cells (mouse colon cancer cells, Kyinno biotechnology, Cat. No. KC-1195), maintained in DMEM medium (Gibco, Cat. No. 31053-036) containing 10% FBS . KATOIII CLDN18.2 cells (human gastric cancer cells, maintained in RPMI1640 medium (Gibco, Cat. No. Cat.145) containing 10% FBS, Kyinno Biotechnology, Cat. No. KC-1453), and RPMI1640 medium (Gibco, Cat. No. 22400-089 The NCI-N87 CLDN18.2 cells (human gastric cancer cells, Kyinno biotechnology, Cat. No. KC-1222) maintained in

[0116] 6.2: CDC assay

[0117] The CDC activity of the target antibody was evaluated by measuring the change in the LDH level released into the culture medium after cell lysis. CT26 CLDN18.2 cells or KATOIII CL DN18.2 cells were suspended in RPMI1640 medium without phenol red containing 1% FBS (Gibco, Cat. No. 11835-030) at a density of 4E+05 cells / ml or 6E+05 or 1E+06 cells / ml, respectively. The target antibody was diluted in RPMI1640 medium without phenol red containing 1% FBS to 200, 50, 12.5, 3.13, 0. 78, 0.195, 0.0488, 0.0122, 0.00305, 0.000763, 0.000191 nM. Normal human serum complement (Quidel, Cat. No. A113) was diluted 1:50 in RPMI1640 medium without phenol red containing 1% FBS. To each well of a round-bottom 96-well microplate (Corning, Cat. No. 3799), 50 μL of antibody dilution, 50 μL of normal human serum complement dilution, and 50 μL of tumor cell suspension were added. As a negative control, a human IgG1 isotype antibody was added. A reference antibody (IMAB362, Ganymed) was added as a positive control. The microplate was incubated in an incubator set at 37 °C and 5% CO2 for 3 - 4 hours. After incubation, the release of LDH into the supernatant of the cell culture was detected according to the instructions attached to the LDH cytotoxicity assay kit (Roche, Cat. No. 11644793001). Briefly, the microplate was centrifuged at 1500 rpm for 5 minutes (Eppendorf, model 5810R), and 70 μL was taken from each well The supernatant was taken and transferred to the wells on a new microplate. Next, 50 μL of the LDH detection substrate was added to each well, and the microplate was incubated at room temperature for 0.5 - 2 hours. . Using SpectraMax M5e (Molecular Devices LLC) , the optical density (OD) at 492 nm was detected and the optical density at 690 nm was subtracted (OD492nm - OD690nm). The CDC activity of the target antibody was calculated by the rate of specific cell lysis using the following formula: Specific cell lysis (%) = (OD 抗体+補体+腫瘍細胞 - OD 補体+腫瘍細胞 ) * 10 0 / (OD 腫瘍細胞+Triton - OD 腫瘍細胞 ).

[0118] Data were analyzed by non - linear regression including four parameters using GraphPad Prism 7 software, and the EC 50 value was calculated.

[0119] 6.3: ADCC assay

[0120] The ADCC activity of the target antibody was evaluated by measuring the change in the amount of LDH released into the culture medium after cell lysis. NCI - N87 CLDN18.2 cells or KATOIII CLDN18.2 cells were suspended in RPMI1640 medium without phenol red at a density of 6E+05 cells / mL. The target antibody was diluted in RPM I1640 medium without phenol red containing 1% FBS to 20, 4, 0.8, 0.16, 0.032, 0.0064, 1.28E - 03, 2.56E - 04, 5.12E - 05, 1.02E - 05, 2.05E - 06, 4.10E - 07 , 8.19E - 08, 1.64E - 08, 3.28E - 09, 6.55E - 10, 1.31E - 10, 2.62E - 11 and 5.24E - 12 nM. Fresh human PBMC cells (from Saily, volunteer #XC11057W) were used in RPMI1640 medium without phenol red containing 1% FBS It was suspended in RPMI 1640 medium without additives at a density of 1.2E+07 cells / mL. 50 μL of antibody dilution solution, 50 μL of human PBMC cell suspension, and 50 μL of tumor cell suspension were added to each well of a round-bottom 96-well microplate. As a negative control, human IgG1 isotype antibody was added. The reference mAb (IMAB362, Ganymed) was added as a positive control. The microplate was incubated in an incubator set at 37 °C and 5% CO2 for 4 to 6 hours. After incubation, the release of LDH into the supernatant of the cell culture was detected according to the instructions attached to the above LDH cytotoxicity assay kit. The ADCC activity of the target antibody was calculated by the rate of specific cell lysis using the following formula: Specific cell lysis (%) = (OD 抗体+PBMC+腫瘍細胞 - OD PBMC+腫瘍細胞 ) * 100 / (OD 腫瘍細胞+Triton - OD 腫瘍細胞 ).

[0121] Data was analyzed by non-linear regression including four parameters using GraphPad Prism 7 software, and the EC 50 value was calculated.

[0122] 6.4: CDC effect on CT26 CLDN18.2 cells by LDH assay

[0123] As shown in Figure 11 and Table 3, two batches of YL-G2-B showed equivalent CDC effects on CT26 CLDN 18.2 cells (Figure 11, panel A). YL-G1-19-02, YL-G2-B, YL-G1-19-03, YL-G2-C, YL-G1-19-04, and YL-G2-D all showed stronger CDC effects on CT26 CLDN18.2 cells than the positive control (Figure 11).

[0124]

Table 3

[0125] 6.5: CDC effect on KATOIII CLDN18.2 cells by LDH assay

[0126] As shown in Figure 12 and Table 4, two batches of YL-G2-B showed equivalent CDC effects on CT26 CLDN 18.2 cells (Figure 12, panel A). Any of YL-G1-19-02, YL-G2-B, YL-G1-19-03, YL-G2-C, YL-G1-19-04, and YL-G2-D showed a stronger CDC effect than the positive control on KATOIII CLDN18.2 cells (Figure 12).

[0127] [Table 4]

[0128] 6.6: ADCC effect on KATOIII CLDN18.2 cells by LDH assay

[0129] As shown in Figure 13 and Table 5, two batches of YL-G2-B showed equivalent ADCC effects on KATOIII C LDN18.2 cells (Figure 13, panel A). YL-G2-B (EC50 = 0.028 nM or 0.018 nM in different batches), YL-G2-C (EC50 = 0.019 nM), and YL-G2-D (EC50 = 0.021 nM) showed stronger ADCC effects (lower EC 50 ), and YL-G1-19-02 (EC 50 = 0.1 6 nM), YL-G1-19-03 (EC 50 = 0.21 nM), YL-G1-19-04 ( EC50 = 0.14 nM) showed equivalent ADCC effects on KATOIII CLDN18.2 cells compared to the positive control (EC50 = 0.12 nM, 0.22 nM, or 0.27 nM in three runs) (Figure 13).

[0130]

Table 5

[0131] 6.7: ADCC effect on NCI-N87 CLDN18.2 cells by LDH assay Effect

[0132] As shown in Figure 14 and Table 6, two batches of YL-G2-B showed equivalent ADCC effects on NCI-N87 CLDN18.2 cells (Figure 14, panel A). YL-G2-B (EC50 = 0.0067 nM or 0.012 nM in different batches), YL-G2-C (EC50 = 0.0078 nM), and YL-G2-D (EC50 = 0.0089 nM) showed stronger ADCC effects (lower EC50), while YL-G1-19-02 (EC50 = 0.072 nM), YL-G1-19-03 (EC50 = 0.13 nM), YL-G1-19-0 4 (EC50 = 0.067 nM) showed equivalent ADCC effects on KATOIII CLDN18.2 cells compared to the positive control (EC50 = 0.057 nM, 0.082 nM, or 0.10 nM in three runs) (Figure 14).

[0133]

Table 6

[0134] 6.8: SPR

[0135] The binding affinity of the target antibody was determined by USP 43 IMMUNOLOGICAL TEST METHODS -- SURFACE PLASMON RESONANCE <1105> and C P, 2020 edition, Part IV, General Rules, 3429 IMMUNOCHEMISTRY, NON-LABELING IMMUNOCHEMICAL METHODS (IV) SURFACE PLASMON RESONANCE Therefore, it was measured by SPR using a human antibody capture kit, type 2 (Cytiva, Cat. No. 29234600). Briefly, the anti-human IgG (Fc) antibody was diluted to 25 μg / mL with immobilization buffer and injected into a Series S Sensor CM5 chip (Cytiva, Cat No. BR100530) at a flow rate of 10 μL / min for 6 minutes, and the cup The ring secondary antibody had a response of approximately 7000 - 14000 response units (RU). Next, the target antibody was diluted to 5 μg / mL with running buffer and injected at a flow rate of 10 μL / min, and the coupling primary antibody had a response of approximately 200 RU. For kinetic measurements, a two-fold serial dilution (0.195 - 50 nM) of His-tagged human Claudin-18.2 was injected at a flow rate of 30 μL / min, and Bi acore 8K (Cytiva) was used to monitor binding for 120 seconds and dissociation for 300 seconds. The association rate (ka) and dissociation rate (kd) were calculated by simultaneously fitting the sensorgrams of binding and dissociation using a simple 1:1 binding model. The equilibrium dissociation constant (KD) was calculated as the kd / ka ratio. The results are shown in Table 7 below.

[0136]

Table 7

[0137] Sequence Listing

Table 8

[0138] Sequence Listing (continued)

Table 9

[0139] Sequence Listing (continued)

Table 10

[0140] Sequence Listing (continued)

Table 11

[0141] Sequence Listing (continued)

Table 12

Claims

**Claim 1** An Fc-modified monoclonal antibody that specifically binds to CLDN18.2, comprising HVR-H1, HVR-H2, and HVR-H3 contained within VH as set forth in SEQ ID NO: 7, and HVR-L1, HVR-L2, and HVR-L3 contained within VL as set forth in SEQ ID NO: 8, and comprising mutations of L235V, F243L, R292P, Y300L, and P396L in the human IgG1 Fc region, a monoclonal antibody. **Claim 2** (1) HVR-H1 as set forth in SEQ ID NO: 29, HVR-H2 as set forth in SEQ ID NO: 30, HVR-H3 as set forth in SEQ ID NO: 31, HVR-L1 as set forth in SEQ ID NO: 32, HVR-L2 as set forth in SEQ ID NO: 33, and HVR-L3 as set forth in SEQ ID NO: 34; (2) HVR-H1 as set forth in SEQ ID NO: 41, HVR-H2 as set forth in SEQ ID NO: 42, HVR-H3 as set forth in SEQ ID NO: 43, HVR-L1 as set forth in SEQ ID NO: 44, HVR-L2 as set forth in SEQ ID NO: 45, and HVR-L3 as set forth in SEQ ID NO: 46; (3) HVR-H1 as set forth in SEQ ID NO: 47, HVR-H2 as set forth in SEQ ID NO: 48, HVR-H3 as set forth in SEQ ID NO: 49, HVR-L1 as set forth in SEQ ID NO: 50, HVR-L2 as set forth in SEQ ID NO: 51, and HVR-L3 as set forth in SEQ ID NO: 52; or (4) The monoclonal antibody according to claim 1, comprising HVR-H1 as set forth in SEQ ID NO: 53, HVR-H2 as set forth in SEQ ID NO: 54, HVR-H3 as set forth in SEQ ID NO: 55, HVR-L1 as set forth in SEQ ID NO: 56, HVR-L2 as set forth in SEQ ID NO: 57, and HVR-L3 as set forth in SEQ ID NO:

58. **Claim 3** The monoclonal antibody according to claim 1, comprising VH as set forth in SEQ ID NO: 7 and VL as set forth in SEQ ID NO: 8, and optionally, the first two amino acid residues at the N-terminus of VH are absent. **Claim 4** The monoclonal antibody according to any one of claims 1 to 3, which is a chimeric antibody or a humanized antibody. **Claim 5** The monoclonal antibody according to any one of claims 1 to 3, wherein the mutation in the Fc region is a mutation that increases CDC. **Claim 6** The monoclonal antibody according to any one of claims 1 to 3, comprising the human IgG1 heavy chain constant region represented by SEQ ID NO: 40 and the human kappa light chain constant region represented by SEQ ID NO:

10.

7. The monoclonal antibody according to any one of claims 1 to 3, wherein the monoclonal antibody is isolated, naked, and / or conjugated.

8. An isolated nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 3.

9. A vector comprising the nucleic acid of claim 8.

10. A host cell comprising the vector of claim 9.

11. A method for producing a monoclonal antibody, comprising culturing the host cell according to claim 10 so that the antibody is produced, and optionally recovering the antibody from the host cell or the cell culture medium.

12. A composition comprising the monoclonal antibody according to any one of claims 1 to 3.

13. An isolated nucleic acid encoding the monoclonal antibody according to claim 6.

14. A vector comprising the nucleic acid of claim 13.

15. A host cell comprising the vector of claim 14.

16. A method for producing a monoclonal antibody, comprising culturing the host cell according to claim 15 so that the antibody is produced, and optionally recovering the antibody from the host cell or the cell culture medium.

17. A composition comprising the monoclonal antibody according to claim 6.

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