Monoclonal antibodies against claudin 18.2 and fc-engineered versions thereof

Abstract

A panel of monoclonal antibodies is provided that specifically bind to CLDN18.2 and do not specifically bind to CLDN18.1, and optionally have an engineered Fc region.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to PCT patent applications filed on May 31, 2021, PCT / CN2021 / 097239 and PCT / CN2021 / 097240, and PCT patent applications filed on July 16, 2021, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] A set of monoclonal antibodies is provided that specifically bind to CLDN18.2 and not specifically bind to CLDN18.1, and optionally have an engineered Fc region. Background Technology

[0004] 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 and two small extracellular loops.

[0005] CLDN18.2 expression was undetectable by RT-PCR in normal tissues other than the stomach. Immunohistochemistry with a CLDN18.2-specific antibody revealed that the stomach was the only positive tissue.

[0006] CLDN18.2 is a highly selective gastric lineage antigen expressed only on short-lived differentiated gastric epithelial cells. CLDN18.2 is retained during malignant transformation and is therefore frequently displayed on the surface of human gastric cancer cells. Furthermore, 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, as well as in distant metastases, particularly in the ovary (so-called Krukenberg tumor). Summary of the Invention

[0007] This invention provides an antibody against CLDN18.2.

[0008] This invention provides an isolated monoclonal antibody (particularly Fc-engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises

[0009] (1) HVR-H1, HVR-H2 and HVR-H3 contained in VH as shown in SEQ ID NO:1, and HVR-L1, HVR-L2 and HVR-L3 contained in VL as shown in SEQ ID NO:2;

[0010] (2) HVR-H1, HVR-H2 and HVR-H3 contained in VH as shown in SEQ ID NO:3, and HVR-L1, HVR-L2 and HVR-L3 contained in VL as shown in SEQ ID NO:4;

[0011] (3) HVR-H1, HVR-H2 and HVR-H3 contained in VH as shown in SEQ ID NO:5, and HVR-L1, HVR-L2 and HVR-L3 contained in VL as shown in SEQ ID NO:6; or

[0012] (4) HVR-H1, HVR-H2 and HVR-H3 contained in VH as shown in SEQ ID NO:7, and HVR-L1, HVR-L2 and HVR-L3 contained in VL as shown in SEQ ID NO:8.

[0013] For example, such as Figure 1A , Figure 1B , Figure 1C or Figure 1D As shown, and

[0014] Optionally, it may include one or more mutations in the Fc region.

[0015] In one embodiment, the antibody comprises

[0016] (1) HVR-H1 as shown in SEQ ID NO:11, HVR-H2 as shown in SEQ ID NO:12, HVR-H3 as shown in SEQ ID NO:13, HVR-L1 as shown in SEQ ID NO:14, HVR-L2 as shown in SEQ ID NO:15 and HVR-L3 as shown in SEQ ID NO:16;

[0017] (2) HVR-H1 as shown in SEQ ID NO:17, HVR-H2 as shown in SEQ ID NO:18, HVR-H3 as shown in SEQ ID NO:19, HVR-L1 as shown in SEQ ID NO:20, HVR-L2 as shown in SEQ ID NO:21 and HVR-L3 as shown in SEQ ID NO:22;

[0018] (3) HVR-H1 as shown in SEQ ID NO:23, HVR-H2 as shown in SEQ ID NO:24, HVR-H3 as shown in SEQ ID NO:25, HVR-L1 as shown in SEQ ID NO:26, HVR-L2 as shown in SEQ ID NO:27, and HVR-L3 as shown in SEQ ID NO:28; or

[0019] (4) HVR-H1 as shown in SEQ ID NO:29, HVR-H2 as shown in SEQ ID NO:30, HVR-H3 as shown in SEQ ID NO:31, HVR-L1 as shown in SEQ ID NO:32, HVR-L2 as shown in SEQ ID NO:33 and HVR-L3 as shown in SEQ ID NO:34.

[0020] In one embodiment, the antibody comprises

[0021] (1) HVR-H1 as shown in SEQ ID NO:41, HVR-H2 as shown in SEQ ID NO:42, HVR-H3 as shown in SEQ ID NO:43, HVR-L1 as shown in SEQ ID NO:44, HVR-L2 as shown in SEQ ID NO:45 and HVR-L3 as shown in SEQ ID NO:46;

[0022] (2) HVR-H1 as shown in SEQ ID NO:47, HVR-H2 as shown in SEQ ID NO:48, HVR-H3 as shown in SEQ ID NO:49, HVR-L1 as shown in SEQ ID NO:50, HVR-L2 as shown in SEQ ID NO:51, and HVR-L3 as shown in SEQ ID NO:52; or

[0023] (3) HVR-H1 as shown in SEQ ID NO:53, HVR-H2 as shown in SEQ ID NO:54, HVR-H3 as shown in SEQ ID NO:55, HVR-L1 as shown in SEQ ID NO:56, HVR-L2 as shown in SEQ ID NO:57 and HVR-L3 as shown in SEQ ID NO:58.

[0024] The present invention further provides an isolated monoclonal antibody (particularly Fc-engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises

[0025] (1) A VH comprising HVR-H1 as shown in SEQ ID NO:11, HVR-H2 as shown in SEQ ID NO:12 and HVR-H3 as shown in SEQ ID NO:13, and a VL comprising HVR-L1 as shown in SEQ ID NO:14, HVR-L2 as shown in SEQ ID NO:15 and HVR-L3 as shown in SEQ ID NO:16;

[0026] (2) A VH comprising HVR-H1 as shown in SEQ ID NO:17, HVR-H2 as shown in SEQ ID NO:18 and HVR-H3 as shown in SEQ ID NO:19, and a VL comprising HVR-L1 as shown in SEQ ID NO:20, HVR-L2 as shown in SEQ ID NO:21 and HVR-L3 as shown in SEQ ID NO:22;

[0027] (3) A VH comprising HVR-H1 as shown in SEQ ID NO:23, HVR-H2 as shown in SEQ ID NO:24, and HVR-H3 as shown in SEQ ID NO:25, and a VL comprising HVR-L1 as shown in SEQ ID NO:26, HVR-L2 as shown in SEQ ID NO:27, and HVR-L3 as shown in SEQ ID NO:28; or

[0028] (4) A VH comprising HVR-H1 as shown in SEQ ID NO:29, HVR-H2 as shown in SEQ ID NO:30, and HVR-H3 as shown in SEQ ID NO:31, and a VL comprising HVR-L1 as shown in SEQ ID NO:32, HVR-L2 as shown in SEQ ID NO:33, and HVR-L3 as shown in SEQ ID NO:34, and

[0029] Optionally, it may include one or more mutations in the Fc region.

[0030] The present invention further provides an isolated monoclonal antibody (particularly Fc-engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises

[0031] (1) A VH comprising HVR-H1 as shown in SEQ ID NO:41, HVR-H2 as shown in SEQ ID NO:42 and HVR-H3 as shown in SEQ ID NO:43, and a VL comprising HVR-L1 as shown in SEQ ID NO:44, HVR-L2 as shown in SEQ ID NO:45 and HVR-L3 as shown in SEQ ID NO:46;

[0032] (2) A VH comprising HVR-H1 as shown in SEQ ID NO:47, HVR-H2 as shown in SEQ ID NO:48, and HVR-H3 as shown in SEQ ID NO:49, and a VL comprising HVR-L1 as shown in SEQ ID NO:50, HVR-L2 as shown in SEQ ID NO:51, and HVR-L3 as shown in SEQ ID NO:52; or

[0033] (3) A VH comprising HVR-H1 as shown in SEQ ID NO:53, HVR-H2 as shown in SEQ ID NO:54, and HVR-H3 as shown in SEQ ID NO:55, and a VL comprising HVR-L1 as shown in SEQ ID NO:56, HVR-L2 as shown in SEQ ID NO:57, and HVR-L3 as shown in SEQ ID NO:58, and

[0034] Optionally, it may include one or more mutations in the Fc region.

[0035] The present invention further provides an isolated monoclonal antibody (particularly Fc-engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises

[0036] (1) VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2;

[0037] (2) VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4;

[0038] (3) VH as shown in SEQ ID NO:5 and VL as shown in SEQ ID NO:6; or

[0039] (4) VH as shown in SEQ ID NO:7 and VL as shown in SEQ ID NO:8, and

[0040] Optionally, it includes one or more mutations in the Fc region.

[0041] Optionally, the first two N-terminal amino acid residues of the VH are absent.

[0042] In one embodiment, the one or more mutations in the Fc region are one or more mutations that alter (e.g., increase or decrease) the binding to the Fc receptor and / or effector function (e.g., ADCC and / or CDC). In one embodiment, the one or more mutations in the Fc region are one or more substitutions selected from the following: L235V, F243L, R292P, Y300L, and P396L. In one embodiment, the one or more mutations in the Fc region are L235V, F243L, R292P, Y300L, and P396L.

[0043] The present invention further provides an isolated monoclonal antibody (particularly Fc-engineered) that specifically binds to human CLDN18.2, wherein the antibody:

[0044] i) Competing with an anti-CLDN18.2 antibody to bind to human CLDN18.2, said antibody comprising (1) VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2; (2) VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4; (3) VH as shown in SEQ ID NO:5 and VL as shown in SEQ ID NO:6; or (4) VH as shown in SEQ ID NO:7 and VL as shown in SEQ ID NO:8, and / or

[0045] ii) The antibody binds to the same epitope on human CLDN18.2, wherein the antibody comprises (1) VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2; (2) VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4; (3) VH as shown in SEQ ID NO:5 and VL as shown in SEQ ID NO:6; or (4) VH as shown in SEQ ID NO:7 and VL as shown in SEQ ID NO:8; and / or

[0046] iii) Mediating the ADCC of PBMCs on cells expressing human CLDN18.2 (e.g., 293T cells, CHO cells, CT26 cells, KATOIII cells, or NCI-N87 cells), for example, EC50 values ​​of approximately or less than 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. nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, 0.01nM, 0.009nM, 0.008nM, 0.007nM, 0.006nM, 0.005nM, 0.004nM, 0.003nM, 0.002nM, or 0.001nM, for example determined by LDH or FACS; and / or

[0047] iv) Does not mediate ADCC of PBMCs on cells expressing human CLDN18.1 (e.g., 293T cells, CHO cells, CT26 cells, KATOIII cells, or NCI-N87 cells); and / or

[0048] v) Mediates CDC on cells expressing human CLDN18.2 (e.g., 293T cells, CHO cells, CT26 cells, KATOIII cells, or NCI-N87 cells), for example, EC50 values ​​of approximately or less than 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 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, for example determined by LDH or FACS; and / or

[0049] vi) Does not mediate CDC in cells expressing human CLDN18.1 (e.g., 293T cells, CHO cells, CT26 cells, KATOIII cells, or NCI-N87 cells); and / or

[0050] vii) Binding to cells expressing human CLDN18.2 on their cell surface (e.g., 293T cells or CHO cells), for example, with Kd values ​​of approximately or less than 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; and / or

[0051] viii) It does not bind to cells expressing human CLDN18.1 on their cell surface (e.g., 293T cells or CHO cells); and / or

[0052] ix) specifically binds to human CLDN18.2, for example, with a Kd value of approximately or less than 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; and / or

[0053] x) does not specifically bind to human CLDN18.1.

[0054] In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is a mouse antibody, a chimeric antibody, or a humanized antibody.

[0055] In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is optionally selected from the following antigen-binding antibody fragments: Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, and biclonal antibody.

[0056] In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is a full-length antibody. In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention comprises, optionally, a human IgG (particularly IgG1) heavy chain constant region as shown in SEQ ID NO:9. In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention comprises, optionally, a mutated human IgG (particularly IgG1) heavy chain constant region as shown in SEQ ID NO:40. In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention comprises, optionally, a human κ light chain constant region as shown in SEQ ID NO:10. In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is a chimeric antibody (e.g., a mouse / human chimeric antibody). In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is Fc-engineered.

[0057] In one embodiment, the monoclonal antibody (mAb) or Fab fragment of the present invention has a crossover form (x-mAb or x-Fab) in which variable or (first) constant domains of the light and heavy chains are exchanged.

[0058] This invention provides an isolated nucleic acid encoding the monoclonal antibody of this invention. This invention provides a vector (e.g., a cloning vector or expression vector) comprising the nucleic acid of this invention. This invention provides a host cell comprising the nucleic acid of this invention or the vector of this invention. This invention provides a method for producing the monoclonal antibody of this invention, the method comprising culturing the host cell to produce the antibody. In one embodiment, the method further comprises recovering the antibody from the host cell or the cell culture.

[0059] This invention provides a composition comprising the monoclonal antibody of this invention. This invention also provides a pharmaceutical formulation comprising the monoclonal antibody of this invention and a pharmaceutically acceptable carrier.

[0060] This invention provides a monoclonal antibody of the present invention for use as a pharmaceutical agent. This invention provides a monoclonal antibody of the present invention for use in the treatment of cancer. This invention provides the use of the monoclonal antibody of the present invention in the manufacture of pharmaceutical agents. In one embodiment, the pharmaceutical agent is used to treat cancer. This invention provides a method for treating an individual suffering from cancer, the method comprising administering an effective amount of the monoclonal antibody of the present invention to the individual. Attached Figure Description

[0061] Figure 1A The amino acid sequence alignment of the VH and VL of the antibody of the present invention is shown, wherein the HVR sequence according to Kabat is highlighted by shading.

[0062] Figure 1B The amino acid sequence alignment of the VH and VL of the antibody of the present invention is shown, wherein the HVR sequence according to IMGT is highlighted by shading.

[0063] Figure 1C The amino acid sequence alignment of the VH and VL of the antibody of the present invention is shown, wherein the HVR sequence according to Chothia is highlighted by shading.

[0064] Figure 1D The amino acid sequence alignment of the VH and VL of the antibody of the present invention is shown, wherein the HVR sequence according to Contact is highlighted by shading.

[0065] Figure 2 The binding of the antibody of the present invention to cells expressing CLDN18.2 is shown.

[0066] Figure 3 The binding of the antibody of the present invention to cells expressing CLDN18.1 is shown.

[0067] Figure 4 The antibody-mediated ADCC of the present invention on cells expressing CLDN18.2 is shown.

[0068] Figure 5 The binding curve of the antibody of the present invention with huCLDN18.2 is shown.

[0069] Figure 6 The binding curves of the antibody of the present invention with cells expressing huCLDN18.2 are shown.

[0070] Figure 7 The results of ADCC assays for the antibodies of the present invention are shown.

[0071] Figure 8 The results of cell binding assays of the antibodies of the present invention are shown.

[0072] Figure 9 The results of the CDC assay for the antibody of the present invention are shown.

[0073] Figure 10 The results of ADCC assays for the antibodies of the present invention are shown.

[0074] Figure 11 The antibody-mediated CDC effect on CT26 expressing CLDN18.2, as determined by LDH assay, is shown.

[0075] Figure 12 The CDC effect of the antibody of the present invention on KATOIII expressing CLDN18.2, as determined by LDH assay, is shown.

[0076] Figure 13 The antibody-mediated ADCC effect of the present invention on KATOIII expressing CLDN18.2, as determined by LDH assay, is shown.

[0077] Figure 14 The antibody-mediated ADCC effect of the present invention on NCI-N87 expressing CLDN18.2, as determined by LDH assay, is shown. Detailed Implementation

[0078] I. Definition

[0079] The term “antibody” is used in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired antigen-binding activity.

[0080] An "antibody fragment" is a molecule that is distinct from the intact antibody, containing the portion of the intact antibody that binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0081] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0082] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. Based on the amino acid sequence of the constant domains, the light chains can be classified into one of two distinct types (called κ and λ).

[0083] "Effective functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with 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.

[0084] As used herein, the terms “engineer,” “engineered,” and “engineering” are considered to include any manipulation of the peptide backbone or post-translational modification of naturally occurring or recombinant peptides or fragments thereof. Engineering includes modifications to the amino acid sequence, modifications to glycosylation patterns, or modifications to the side chain groups of individual amino acids, as well as combinations of these methods.

[0085] The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends 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. In one embodiment, the anti-CLDN18.2 antibody as described herein is an IgG1 isotype and comprises the heavy chain constant region of SEQ ID NO:9 or SEQ ID NO:40. In one embodiment, it additionally comprises the C-terminal lysine (Lys447). Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0086] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0087] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document to refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing an Fc region.

[0088] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. The nucleic acid content of progeny cells may not be exactly the same as that of parent cells, but may contain mutations. This article includes mutant progeny with the same function or biological activity as screened or selected in the initial transformed cells.

[0089] A “humanized” antibody is a chimeric antibody comprising amino acid residues from a nonhuman HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all of at least one (and typically two) variable domains, wherein all or substantially all of the HVR (e.g., CDR) corresponds to the HVR of the nonhuman antibody, and all or substantially all of the FR corresponds to the FR of the human antibody. Optionally, the humanized antibody may comprise at least a portion of the antibody constant region derived from the human antibody. A “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has undergone humanization.

[0090] As used herein, the term "hypervariant region" or "HVR" refers to each region of an antibody variable domain that is sequence-hypervariant ("complementarity-determining region" or "CDR") and / or forms a structure-defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact"). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs as described herein include:

[0091] (a) Hypervariable rings present at the following 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));

[0092] (b) CDRs present at the following 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));

[0093] (c) Antigen contacts present at the following 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

[0094] (d) Combinations of (a), (b) and / or (c), including 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).

[0095] HVR residues can be found on websites (e.g., https: / / www.novopro.cn / tools / cdr.html Identification on )

[0096] Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered in this paper according to Kabat et al., ibid.

[0097] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies (i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, except for antibodies containing, for example, naturally occurring mutations or possible variants generated during the production of the monoclonal antibody formulation, such variants typically present in small quantities). In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0098] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using the VH or VL domains of the antibody binding said antigen to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0099] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the host cell genome that has already been introduced therein. Certain vectors are capable of guiding the expression of the nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0100] II. Exemplary Antibodies

[0101] This invention provides isolated monoclonal antibodies (particularly Fc-engineered) that specifically bind to human CLDN18.2, wherein the antibody comprises a VH containing HVR-H1 as shown in SEQ ID NO:11, HVR-H2 as shown in SEQ ID NO:12, and HVR-H3 as shown in SEQ ID NO:13, and a VL containing HVR-L1 as shown in SEQ ID NO:14, HVR-L2 as shown in SEQ ID NO:15, and HVR-L3 as shown in SEQ ID NO:16. In one embodiment, the antibody comprises a VH as shown in SEQ ID NO:1 and a VL as shown in SEQ ID NO:2. Optionally, the first two N-terminal amino acid residues of the VH are absent.

[0102] This invention provides isolated monoclonal antibodies (particularly Fc-engineered) that specifically bind to human CLDN18.2, wherein the antibodies comprise a VH containing HVR-H1 as shown in SEQ ID NO:17, HVR-H2 as shown in SEQ ID NO:18, and HVR-H3 as shown in SEQ ID NO:19, and a VL containing HVR-L1 as shown in SEQ ID NO:20, HVR-L2 as shown in SEQ ID NO:21, and HVR-L3 as shown in SEQ ID NO:22. This invention also provides isolated monoclonal antibodies (particularly Fc-engineered) that specifically bind to human CLDN18.2, wherein the antibodies comprise a VH containing HVR-H1 as shown in SEQ ID NO:41, HVR-H2 as shown in SEQ ID NO:42, and HVR-H3 as shown in SEQ ID NO:43, and a VL containing HVR-L1 as shown in SEQ ID NO:44, HVR-L2 as shown in SEQ ID NO:45, and HVR-L3 as shown in SEQ ID NO:46. This invention provides isolated monoclonal antibodies (particularly Fc-engineered) that specifically bind to human CLDN18.2, wherein the antibody comprises a VH containing HVR-H1 as shown in SEQ ID NO:47, HVR-H2 as shown in SEQ ID NO:48, and HVR-H3 as shown in SEQ ID NO:49, and a VL containing HVR-L1 as shown in SEQ ID NO:50, HVR-L2 as shown in SEQ ID NO:51, and HVR-L3 as shown in SEQ ID NO:52. This invention also provides isolated monoclonal antibodies (particularly Fc-engineered) that specifically bind to human CLDN18.2, wherein the antibody comprises a VH containing HVR-H1 as shown in SEQ ID NO:53, HVR-H2 as shown in SEQ ID NO:54, and HVR-H3 as shown in SEQ ID NO:55, and a VL containing HVR-L1 as shown in SEQ ID NO:56, HVR-L2 as shown in SEQ ID NO:57, and HVR-L3 as shown in SEQ ID NO:58. In one embodiment, the antibody comprises VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4. Optionally, the first two N-terminal amino acid residues of the VH are absent.

[0103] This invention provides isolated monoclonal antibodies (particularly Fc-engineered) that specifically bind to human CLDN18.2, wherein the antibody comprises a VH containing HVR-H1 as shown in SEQ ID NO:23, HVR-H2 as shown in SEQ ID NO:24, and HVR-H3 as shown in SEQ ID NO:25, and a VL containing HVR-L1 as shown in SEQ ID NO:26, HVR-L2 as shown in SEQ ID NO:27, and HVR-L3 as shown in SEQ ID NO:28. In one embodiment, the antibody comprises a VH as shown in SEQ ID NO:5 and a VL as shown in SEQ ID NO:6. Optionally, the first two N-terminal amino acid residues of the VH are absent.

[0104] This invention provides isolated monoclonal antibodies (particularly Fc-engineered) that specifically bind to human CLDN18.2, wherein the antibody comprises a VH containing HVR-H1 as shown in SEQ ID NO:29, HVR-H2 as shown in SEQ ID NO:30, and HVR-H3 as shown in SEQ ID NO:31, and a VL containing HVR-L1 as shown in SEQ ID NO:32, HVR-L2 as shown in SEQ ID NO:33, and HVR-L3 as shown in SEQ ID NO:34. In one embodiment, the antibody comprises a VH as shown in SEQ ID NO:7 and a VL as shown in SEQ ID NO:8. Optionally, the first two N-terminal amino acid residues of the VH are absent.

[0105] In one embodiment, the one or more mutations in the Fc region are one or more mutations that alter (e.g., increase or decrease) the binding to the Fc receptor and / or effector function (e.g., ADCC and / or CDC). In one embodiment, the one or more mutations in the Fc region are one or more substitutions selected from the following: L235V, F243L, R292P, Y300L, and P396L. In one embodiment, the one or more mutations in the Fc region are L235V, F243L, R292P, Y300L, and P396L.

[0106] In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is a mouse antibody, a chimeric antibody, or a humanized antibody.

[0107] In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is optionally selected from the following antigen-binding antibody fragments: Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, and biclonal antibody.

[0108] In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is a full-length antibody. In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention comprises, optionally, a human IgG (particularly IgG1) heavy chain constant region as shown in SEQ ID NO:9. In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention comprises, optionally, a mutated human IgG (particularly IgG1) heavy chain constant region as shown in SEQ ID NO:40. In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention comprises, optionally, a human κ light chain constant region as shown in SEQ ID NO:10. In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is a chimeric antibody (e.g., a mouse / human chimeric antibody). In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the invention is Fc-engineered.

[0109] In one embodiment, the monoclonal antibody (mAb) or Fab fragment of the present invention has a crossover form (x-mAb or x-Fab) in which variable or (first) constant domains of the light and heavy chains are exchanged.

[0110] III. Recombination Methods and Compositions

[0111] Antibodies can be generated using recombinant methods and compositions, such as those described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0112] In the case of natural antibodies or fragments of natural antibodies, two nucleic acids are required: one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. One or more of these nucleic acids encode the amino acid sequence containing the VL of the antibody and / or the amino acid sequence containing the VH of the antibody (e.g., one or more light chains and / or heavy chains of the antibody). These nucleic acids may be expressed on the same expression vector or on different expression vectors.

[0113] In one implementation, an isolated nucleic acid is provided that encodes an antibody as used in the method reported herein.

[0114] In another embodiment, one or more vectors (e.g., expression vectors) containing one or more such nucleic acids are provided.

[0115] In another embodiment, a host cell containing one or more such nucleic acids is provided.

[0116] In one such implementation, the host cell contains (e.g., has been transformed):

[0117] (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising a VL containing the antibody and an amino acid sequence comprising a VH containing the antibody, or

[0118] (2) A first vector and a second vector, wherein the first vector contains a nucleic acid encoding an amino acid sequence of VL containing the antibody, and the second vector contains a nucleic acid encoding an amino acid sequence of VH containing the antibody.

[0119] In one embodiment, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, Sp2 / O cells). In one embodiment, a method for preparing an antibody is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding an antibody as provided above under conditions suitable for expressing the antibody, and optionally recovering the antibody from the host cell or a host cell culture medium.

[0120] For the recombinant production of antibodies, nucleic acids encoding antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of said antibodies), or generated by recombinant methods or obtained through chemical synthesis.

[0121] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be generated in bacteria, for example, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. Also see Charlton, KA, in: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in *Escherichia coli* (E. coli). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0122] Besides prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0123] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells.

[0124] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (which describe PLATNIBODIES for generating antibodies in transgenic plants). TM technology).

[0125] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth can be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 (COS-7) transformed with SV40; human embryonic kidney lines (such as 293 or 293 cells described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); young hamster kidney cells (BHK); mouse supporting cells (such as TM4 cells described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and, for example, in Mather, JP et al., Annals TRI cells, MRC 5 cells, and FS4 cells, as described in NYAcad.Sci. 383 (1982) 44-68. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines (such as Y0, NSO, and Sp2 / 0). For a review of some mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0126] IV. Measurement

[0127] The antibodies described herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activity using various assays known in the art.

[0128] Combining measurements with other measurements

[0129] On the one hand, the antigen-binding activity of the antibodies of the present invention can be tested, for example, by known methods such as ELISA and Western blotting.

[0130] On the other hand, competitive assays can be used to identify antibodies that competitively bind to CLDN18.2. In some embodiments, such competitive antibodies bind to the same epitope as the one bound by aCLDN18.2 (e.g., a linear epitope or a conformational epitope). Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996), “Epitope Mapping Protocols,” Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0131] In an exemplary competitive assay, immobilized CLDN18.2 is incubated in a solution containing a first labeled antibody and a second unlabeled antibody that binds to CLDN18.2, testing the ability of the second unlabeled antibody to competitively bind CLDN18.2 against the first antibody. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CLDN18.2 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing the first antibody to bind to CLDN18.2, excess unbound antibody is removed, and the amount of label associated with the immobilized CLDN18.2 is measured. If the amount of label associated with the immobilized CLDN18.2 in the test sample is significantly reduced relative to the control sample, it indicates that the second antibody competitively binds to CLDN18.2 against the first antibody. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0132] Activity assay

[0133] In one aspect, an assay is provided for identifying the biological activity of the anti-CLDN18.2 antibody. Biological activity may include, for example, the effect of the anti-CLDN18.2 antibody on ADCC of PBMCs on target cells expressing CLDN18.2. Antibodies exhibiting this biological activity in vivo and / or in vitro are also provided.

[0134] Example

[0135] After initial screening, four positive hybridoma cell lines from immunized mice were identified that specifically bound to cells expressing CLDN18.2 but not to cells expressing CLDN18.1.

[0136] Example 1: Cloning four CLDN18.2-specific monoclonal antibodies (mAbs) from mouse hybridoma cells.

[0137] This example illustrates how to clone the H and L chain genes of an antibody from mouse hybridoma cells to obtain a variable region sequence specific to CLDN18.2 for the production of chimeric antibodies.

[0138] Following standard procedures in the field, Quick-RNA was used. TM The Microprep kit (ZYMO Research, catalog number R1050) was used to isolate and purify RNA from hybridoma cells. First-strand cDNA was synthesized and used... RACE was performed using a 5' / 3' kit (Takara Bio USA, Inc., catalog number 634858) along with constant primers for IgG1 (SEQ ID NO:35), IgG2a (SEQ ID NO:36), and κ3' (SEQ ID NO:37). RACE DNA products were extracted using a NucelloSpin gel and a PCR Clean-Up kit (Takara, catalog number 740986.20). The linearized pRACE vector and the gel-purified PACE product in-fusion reaction mixture were transformed into Stellar competent cells (Clontech, catalog number 636766). Plasmid DNA was isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen, catalog number 27104) and sequenced using M13 sequencing primers (GENEWIZ). The final gene sequences of four pairs of heavy and light chains were obtained (data not shown), which are now identified as CLDN18.2 mAb (mAb1, mAb2, mAb3 and mAb4).

[0139] Example 2: Constructing chimeric antibodies by replacing mouse constant regions with human constant regions

[0140] This example illustrates the construction of chimeric antibodies by replacing the constant regions of four molecular clone mouse mAbs with constant regions derived from the human IgG1 heavy chain and κ light chain.

[0141] Plasmid pFUSE-CHIg-hG1 (InvivoGen, catalog number pfuse-hchg1) containing the constant region of the human IgG1 heavy chain (SEQ ID NO: 9) and plasmid pFUSE2-CLIg-hk (InvivoGen, catalog number pfuse2-hclk) containing the constant region of the human κ light chain (SEQ ID NO: 10) were digested with Hind III and Nhe I (for IgG1) or BsiWI (for κ) (all from NEB lab). Linearized plasmids were purified by gel purification using NucleoSpin gels and a PCR Clean-up kit (Takara, catalog number 740986.20). The coding sequences for the variable regions of the mouse heavy and light chains of mAb1, mAb2, mAb3, and mAb4 were amplified by PCR using HiFi HotStart (Kapa(Roche), KK2602) plasmids obtained in Example 1 along with specific primers (data not shown), and purified by gel purification using NucleoSpin gel and the PCRClean-up kit (Takara, catalog number 740986.20). Gibson was used. The linearized vector and insert were assembled using the HiFi One-Step Kit (SGI(VWR), catalog GA1100-50). The assembly reaction mixture was transformed into Stellar competent cells (Clontech, catalog 636766). Plasmid DNA was isolated from the transformants using the QIAprep Spin Miniprep Kit (Qiagen, catalog 27104) and subjected to Sanger sequencing (GENEWIZ). Four chimeric antibodies were constructed, each comprising a variable region from one of the four mouse mAbs generated in Example 1. The novel chimeric antibodies were designated YL-G1-19-01, YL-G1-19-02, YL-G1-19-03, and YL-G1-19-04.

[0142] Example 3: Confirmation of the specificity of these four chimeric monoclonal antibodies by surface staining

[0143] This example illustrates the testing of four chimeric monoclonal antibodies with CLDN18.2 binding specificity compared to the reference mAb (IMAB362, Ganymed).

[0144] Add FACS buffer (50 μL) to a solution with a density of 2 x 10⁻⁶. 6293T cells expressing CLDN18.2 and CLDN18.1 expressed at 50 μL / well were mixed with a series of dilutions (60.00, 20.00, 6.67, 2.22, 0.74, 0.25, 0.08 μg / mL in FACS buffer) of chimeric antibody, reference antibody, or IgG negative control in 96-well V plates and incubated on ice for 30 min. After washing with 200 μL / well of FACS buffer, the cells were resuspended in 30 μL / well of secondary antibody Alexa. Cells were incubated in 647 AffiniPure goat anti-human IgG (Fcγ fragment specific) (Jackson, catalog number 109-605-098) on ice for 20 min. After washing three times with 200 μL / well FACS buffer, cells were resuspended in 150 μL / well FACS buffer and FACS was performed using a BD LSR II flow cytometer (HTS). Data were analyzed using geometric mean and plotted using Prism GraphPad. Flow cytometry analysis showed that the four chimeric antibodies had higher specificity binding to cells expressing CLDN18.2 compared to the reference mAb (see [link to relevant documentation]). Figure 2 No binding was shown with cells expressing CLDN18.1 (see [link]). Figure 3 This indicates the high specificity of these four chimeric antibodies.

[0145] Example 4: The functional activity of these four chimeric antibodies was identified by ADCC assay.

[0146] This example illustrates the testing of ADCC-mediated killing activity of these four chimeric antibodies compared to the reference mAb (IMAB362, Ganymed).

[0147] 293T cells expressing CLDN18.2 and CLDN18.1 were used in eBioscience TM CFSE (Thermo, catalog number 65-0850-84) is marked with CFSE. The density in the cell separation medium (Cedarlane, catalog number CL5110) (50 μL) is 4 x 10⁻⁶. 5 CFSE-labeled cells at 100 μL / ml were mixed with a series of dilutions of chimeric antibody, reference antibody, or IgG negative control (20.00, 6.67, 2.22, 0.74, 0.25, 0.08, 0.03 μg / mL in the medium) in 96-well V plates and incubated in the dark at room temperature for 15 min. Then, cells were added at a density of 5 x 10⁻⁶ cells / mL. 6Cells / ml PBMC (50 μL) were incubated in the dark at 37°C for 2 hours. Cells were washed twice with PBS, and 100 μL of eBioscience was added. TM Can fix the active dye eFluor TM Working solution of AF660 (Thermo, catalog number 65-0864) was added to each well. The plate was incubated on ice in the dark for 30 min. After washing with PBS, cells were resuspended by adding 75 μL / well of PBS and 25 μL / well of 4% paraformaldehyde and subjected to FACS using a BD LSR II flow cytometer (HTS). Data were analyzed using the percentage of killing (CFSE / FVD-AF660 double-positive population divided by the CFSE-positive population) and plotted using PrismGraphPad. ADCC activity obtained by the FACS-based method indicated that the four chimeric antibodies could mediate ADCC activity against cells expressing CLDN18.2 (see [link to FACS]). Figure 4 ).

[0148] Example 5: Characterization of Fc-engineered chimeric antibody.

[0149] In addition, four Fc-engineered 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-engineered 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 compared to YL-G1-19-04) contain five substitutions in the Fc region: L235V, F243L, R292P, Y300L, and P396L (according to EU designation). The mutant constant regions of the human IgG1 heavy chain (including CH1, hinge, CH2, and CH3) are shown in SEQ ID NO:40.

[0150] This embodiment illustrates the characterization of these Fc-engineered chimeric antibodies.

[0151] 5.1: Antigen-antibody binding interaction.

[0152] This embodiment illustrates the characterization of antigen-antibody binding interactions of YL-G2-B, YL-G2-C, and YL-G2-D.

[0153] The in vitro bioactivity of YL-G2-B, YL-G2-C, and YL-G2-D was analyzed by monitoring the binding of recombinant huCLDN18.2-Fc and CHO cells overexpressing huCLDN18.2 using the KinExA 4000 system (KinExA, USA).

[0154] To determine affinity using the KinExA method, binding partner B (called the titrant) is serially diluted against a background of binding partner A (called the constant binding partner, CBP). This means that the concentration of CBP remains constant while the concentration of the titrant varies. Once these solutions reach equilibrium, the KinExA 4000 instrument can directly measure the amount of unbound or free binding partner of CBP remaining in the solution. Using Sapidyne software, the percentage of free CBP can be plotted against the total titrant concentration to generate a binding curve and determine affinity.

[0155] First, Kd was determined using an antibody and recombinant human CLDN18.2 purchased from Sino Biological (P / N: 20047-H02H). For equilibration experiments, the titrant (huCLDN18.2) was serially diluted five-fold against a CBP background. Two equilibration experiments were performed: one with a high concentration of 10 nM CBP (20 nM binding site) and a titrant serially diluted five-fold to 150 nM, and the other with a low concentration of 100 pM CBP (200 pM binding site) and a titrant serially diluted five-fold to 150 pM. Data were collected on a KinEx A4000 and analyzed using SAPIDYNE Instruments n-Curve analysis software version 4.4.26. Binding curves were obtained on... Figure 5 As shown in the image.

[0156] The binding affinity of the antibody to the surface protein of intact cells (i.e., CHO cells overexpressing huCLDN18.2) was also measured using a KinExA 4000 instrument. The antibody concentration was kept constant (CBP), and the concentration of whole cells containing the surface protein was varied (titrate). The concentration of whole cells containing the surface protein was diluted three-fold. The titrated cells were incubated with constant binding coupler (CBP). Once equilibration was achieved, the samples were centrifuged, the supernatant was recovered, and free CBP was detected using fluorescently labeled anti-CBP molecules. The binding curves were plotted at... Figure 6 As shown in the image.

[0157] To establish a more accurate Kd, two equilibrium curves were prepared and analyzed. One curve was prepared using a low concentration of 100 pM CBP (200 pM binding site) and the other using a three-fold dilution of 10 pM CBP. 6 Cells / mL, and a curve was plotted using 10 nM high concentration CBP (20 nM binding site) and 10 nM CBP at a three-fold dilution. 6Cells / mL. The amount of unbound CBP in solution was measured using a KinExA 4000. Analysis was performed using SAPidyne Instruments n-Curve analysis software version 4.4.26. A summary of the equilibrium dissociation constant Kd is shown in Table 1.

[0158] Table 1. Kd values ​​of YL-G2-B, YL-G2-C and YL-G2-D

[0159]

[0160] 5.2: Cell binding assay.

[0161] This example illustrates the cell binding assays for YL-G2-B, YL-G2-C, and YL-G2-D.

[0162] Antibody binding was assessed using CHO cells expressing huCLDN18.2. For each sample, cells were loaded at 5 x 10⁻⁶ cells / day. 5 100 μl cells / well were seeded into the wells of a 96-well plate. Then, 100 μl of each serially diluted antibody was added to the cells, each dilution starting at 40 μg / ml and diluted 5-fold. Therefore, the final concentration of each antibody started at 20 μg / ml and was then serially diluted 5-fold. 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. The cells were analyzed by flow cytometry.

[0163] Using unstained cells as a reference for setting overall target cell gating and establishing a FITC-negative population allowed us to establish FITC-positive cell gating for each cell line. Additionally, the mean fluorescence intensity (MFI) of the entire cell population was calculated to secondary confirm the FITC-positive results. The ratio of gated positive cells to the total number of viable cells was considered the percentage of positive cells. Results in Figure 7 As shown in the image.

[0164] 5.3: Complement-dependent cytotoxicity (CDC) assay.

[0165] This example illustrates the complement-dependent cytotoxicity (CDC) assays for YL-G2-B, YL-G2-C, and YL-G2-D.

[0166] The target cells (i.e., CHO cells expressing huCLDN18.2) were washed once with DPBS. The cells were then loaded with 2 x 10⁻⁶ cells / mL. 4100 μL / well of cells were seeded into RPMI plates on a U-shaped substrate. Antibody was serially diluted 1:2 and co-incubated with cells at 50 μL / well for 15 minutes at room temperature. Then, 20% pooled serum was added at 50 μL / well to all wells, including spontaneous release and maximum release wells. The plates were incubated at 37°C for 3.5 hours. Forty-five minutes before the end of the incubation period, the plates were centrifuged at 1200 rpm for 5 minutes, and 20 μL of lysis buffer (CyQUANT) was added. TM LDH Cytotoxicity Assay Kit (catalog numbers C20300 and C20301) should only be added to the maximum release control wells containing target cells. Add 50 μL of supernatant along with 50 μL / well of reaction buffer (CyQUANT). TM Transfer the LDH cytotoxicity assay kit (catalog numbers C20300 and C20301) to a black-walled 96-well plate and add the reaction buffer to all wells (including the maximum release wells and spontaneous release wells). Incubate the plate in the dark for 30 minutes. At the end of the incubation, add 50 μL of stop solution (CyQUANT). TM LDH Cytotoxicity Assay Kit (catalog numbers C20300 and C20301) was added to all wells and gently mixed by tapping. OD was measured at 490 nm and 680 nm. Activity is expressed as a percentage of cytotoxicity: %Cytotoxicity = (Experimental value - Spontaneous release from target cells, without volume correction) / (Maximum release from target cells - Spontaneous release from target cells, with volume correction) * 100. Results are displayed in... Figure 8 As shown in the image.

[0167] 5.4: Internalization determination.

[0168] This example illustrates the internalization determination of YL-G2-B, YL-G2-C, and YL-G2-D.

[0169] In DMEM medium at 5 x 10 5 CHO cells expressing huCLDN18.2 were prepared at / mL. 100 μL of cells were seeded into each well of a U-bottom 96-well plate. Test and control antibodies were diluted to 40 μg / ml, and then each antibody was serially diluted 1:4 in culture medium. 50 μl of each diluted antibody was added to the cells per well. The plate was incubated at 37°C for 30 min. Then, 40 μg / ml of PEP-ZAP (a small Fc-binding peptide fused to a cytotoxic peptide, developed by AB Studio Inc.; see WO 2020 / 018732 A1) was added to each well to bring the final concentration of PEP-ZAP to 10 μg / ml. The plate was incubated at 37°C for 72 h. Finally, the cells were rotated and 100 μl of the supernatant was used to measure LDH. Results were presented in […]. Figure 9 As shown in the image.

[0170] 5.5: Antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

[0171] This example illustrates an antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

[0172] The ADCC function of the antibody was assessed using target cells (CHO cells expressing huCLDN18.2) and effector cells (NK 8837-F cells, ATCC PTA-8837). For each sample, target cells were prepared at 2 x 10⁻⁶ cells per cell. 4 50 μl of each cell was seeded into the wells of a 96-well plate in DMEM-F12 + 10% FBS medium. Then, 100 μl of each serially diluted 1:10 antibody was added to the cells. After 20 minutes, NK cells were seeded at 2 x 10⁻⁶ cells / well. 5 Cells were added at a concentration of 50 μl / cell to the plate, resulting in a target:effectant ratio of 1:10. Following this addition, the final concentration of each antibody was determined starting at 50 μg / ml, followed by 5, 0.5, and 0.05 μg / ml. The plates were incubated at 37°C in a CO2 incubator for 24 hours. Cells were then stained with 7AAD, washed twice, and resuspended in approximately 200 μl of FACS buffer. Cells were analyzed by flow cytometry.

[0173] Using unstained cells as a reference for setting overall target cell gating and establishing a 7AAD-negative population allows for the differentiation of 7AAD (dead cells) from live cells.

[0174] CT26 cells expressing huCLDN18.2 can also be used as target cells, and ADCC can also be determined via LDH.

[0175] A comparison of ADCC activity between YL-G1-02 and YL-G2-B (which have the same amino acid sequence except for the VLPLL substitution in the Fc region) is shown in [the original text]. Figure 10 As shown in the table. A summary of EC50 is shown in Table 2.

[0176] Table 2. ADCC of YL-G1-02 compared to YL-G2-B

[0177]

[0178] Example 6: Functional characterization of antibodies.

[0179] 6.1: Cells

[0180] CT26 CLDN18.2 cells (mouse colon cancer cells, Kyinno biotechnology, KC-1195) held in DMEM medium (Gibco, 31053-036) containing 10% FBS (ExCell Bio, catalog number FND500), KATOIII CLDN18.2 cells (human gastric cancer cells, Kyinno biotechnology, KC-1453) held in RPMI1640 medium (Gibco, 22400-089) containing 10% FBS, and NCI-N87 CLDN18.2 cells (human gastric cancer cells, Kyinno biotechnology, KC-1222) held in RPMI1640 medium (Kibco, 22400-089) containing 10% FBS were all tumor cells overexpressing human CLDN18.2, and they were used to determine the CDC and ADCC activities of the subject antibody.

[0181] 6.2: CDC determination

[0182] The CDC activity of the subject antibody was assessed by measuring changes in LDH levels released into the culture medium after cell lysis. CT26 CLDN18.2 cells or KATOIII CLDN18.2 cells were suspended at densities of 4E+05 cells / ml, 6E+05 cells / ml, or 1E+06 cells / ml in RPMI 1640 medium (Gibco, catalog number 11835-030) without phenol red and containing 1% FBS. The subject antibody was diluted with RPMI 1640 medium (1% FBS) without phenol red to 200, 50, 12.5, 3.13, 0.78, 0.195, 0.0488, 0.0122, 0.00305, 0.000763, and 0.000191 nM. Normal human serum complement (Quidel, catalog number A113) was diluted 1:50 with RPMI 1640 medium without phenol red containing 1% FBS. 50 μL of antibody dilution, 50 μL of normal human serum complement dilution, and 50 μL of tumor cell suspension were added to each well of a 96-well round-bottom microplate (Corning, catalog number 3799). Human IgG1 isotype antibody was included as a negative control. Reference antibody (IMAB362, Ganymed) was included as a positive control. The microplate was incubated at 37°C and 5% CO2 for 3–4 hours. After incubation, the release of LDH into the cell culture supernatant was detected according to the instructions provided with the LDH cytotoxicity assay kit (Roche, catalog number 11644793001). In short, the microplate (Eppendorf, model 5810R) was centrifuged at 1500 rpm for 5 minutes, and 70 μL of supernatant was removed from each well and transferred to a new well on the microplate. Then, 50 μL of LDH detection substrate was added to each well, and the microplate was incubated at room temperature for 0.5–2 hours. The optical density (OD) at 492 nm was measured using a SpectraMax M5e (Molecular Devices LLC), and the optical density (OD) at 690 nm was subtracted. 492 nm -OD 690 nm The CDC activity of the subject antibody was calculated using the percentage of specific cell lysis using the following formula:

[0183] Specific cell lysis (%) = (OD 抗体+补体+肿瘤细胞 -OD 补体+肿瘤细胞 )*100 / (OD 肿瘤细胞+Triton -OD 肿瘤细胞 ).

[0184] The data was analyzed using four-parameter nonlinear regression analysis with GraphPad Prism 7 software, and EC was calculated and obtained. 50 value.

[0185] 6.3: ADCC Measurement

[0186] The ADCC activity of the subject antibody was assessed by measuring changes in LDH levels released into the culture medium after cell lysis. NCI-N87 CLDN18.2 cells or KATOIII CLDN18.2 cells were suspended at a density of 6E+05 cells / mL in phenol red-free RPMI 1640 medium. The subject antibody was diluted with 1% FBS-free RPMI 1640 medium 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 (Saily, from volunteer #XC11057W) were suspended at a density of 1.2E+07 cells / mL in 1% FBS-free RPMI 1640 medium. Add 50 μL of antibody diluent, 50 μL of human PBMC cell suspension, and 50 μL of tumor cell suspension to each well of a 96-well round-bottom microplate. Human IgG1 isotype was included as a negative control. Reference mAb (IMAB362, Ganymed) was included as a positive control. Incubate the microplate in an incubator set to 37°C and 5% CO2 for 4–6 hours. After incubation, detect the release of LDH into the cell culture supernatant according to the instructions provided with the LDH cytotoxicity assay kit as described above. Calculate the ADCC activity of the subject antibody using the percentage of specific cell lysis using the following formula:

[0187] Specific cell lysis (%) = (OD 抗体+PBMC+肿瘤细胞 -OD PBMC+肿瘤细胞 )*100 / (OD 肿瘤细胞+Triton -OD 肿瘤细胞 ).

[0188] The data was analyzed using four-parameter nonlinear regression analysis with GraphPad Prism 7 software, and EC was calculated and obtained. 50 value.

[0189] 6.4: CDC effect on CT26 CLDN18.2 cells obtained by LDH assay

[0190] like Figure 11 As shown in Table 3, the two batches of YL-G2-B exhibited comparable CDC effects on CT26 CLDN18.2 cells. Figure 11 (See Figure A). YL-G1-19-02, YL-G2-B, YL-G1-19-03, YL-G2-C, YL-G1-19-04, and YL-G2-D all exhibited a stronger CDC effect on CT26 CLDN18.2 cells than the positive control. Figure 11 ).

[0191] Table 3: CDC effect on CT26 CLDN18.2 cells

[0192]

[0193]

[0194] 6.5: CDC effect on KATOIII CLDN18.2 cells obtained by LDH assay

[0195] like Figure 12 As shown in Table 4, the two batches of YL-G2-B exhibited comparable CDC effects on CT26 CLDN18.2 cells. Figure 12 (See Figure A). YL-G1-19-02, YL-G2-B, YL-G1-19-03, YL-G2-C, YL-G1-19-04, and YL-G2-D all exhibited a stronger CDC effect on KATOIII CLDN18.2 cells than the positive control and the positive control. Figure 12 ).

[0196] Table 4: CDC effect on KATOIII CLDN18.2 cells

[0197]

[0198] 6.6: ADCC effect on KATOIII CLDN18.2 cells obtained by LDH assay

[0199] like Figure 13 As shown in Table 5, the two batches of YL-G2-B exhibited comparable ADCC effects on KATOIII CLDN18.2 cells. Figure 13 (Figure A). Compared with the positive control (EC30 ... 50 Compared to (=0.12nM, 0.22nM or 0.27nM), YL-G2-B (different batches of EC) 50 =0.028nM or 0.018nM), YL-G2-C (EC 50=0.019nM) and YL-G2-D(EC 50 =0.021 nM) showed a stronger ADCC effect on KATOIII CLDN18.2 cells (lower EC50). 50 ), and YL-G1-19-02 (EC 50 =0.16nM), YL-G1-19-03 (EC 50 =0.21nM) and YL-G1-19-04 (EC 50 =0.14 nM) showed comparable ADCC effects on KATOIII CLDN18.2 cells ( Figure 13 ).

[0200] Table 5: ADCC effect on KATOIII CLDN18.2 cells

[0201]

[0202]

[0203] 6.7: ADCC effect on NCI-N87 CLDN18.2 cells obtained by LDH assay

[0204] like Figure 14 As shown in Table 6, the two batches of YL-G2-B exhibited comparable ADCC effects on NCI-N87 CLDN18.2 cells. Figure 14 (Figure A). Compared with the positive control (EC30 ... 50 Compared to (0.057nM, 0.082nM, or 0.10nM), YL-G2-B (different batches of EC) 50 =0.0067nM or 0.012nM), YL-G2-C (EC 50 =0.0078nM) and YL-G2-D(EC 50 =0.0089 nM) showed a stronger ADCC effect on KATOIII CLDN18.2 cells (lower EC50). 50 ), and YL-G1-19-02 (EC 50 =0.072nM), YL-G1-19-03 (EC 50 =0.13nM) and YL-G1-19-04 (EC 50 =0.067 nM) showed comparable ADCC effects on KATOIII CLDN18.2 cells ( Figure 14 ).

[0205] Table 6: ADCC effect on NCI-N87 CLDN18.2 cells

[0206]

[0207] 6.8: SPR

[0208] According to the USP 43 immunological test method—surface plasmon resonance <1105> (USP 43IMMUNOLOGICALTEST METHODS--SURFACE PLASMON RESONANCE <1105> The binding affinity of the subject antibody was determined via SPR using the Human Antibody Capture Kit Type 2 (Cytiva, catalog number 29234600) (CP, 2020 edition, Part IV, General Rules, 3429 Immunochemical Methods (IV) Surface Plasmon Resonance). In short, the anti-human IgG (Fc) antibody was diluted to 25 μg / mL with immobilization buffer and injected at a flow rate of 10 μL / min onto the S-Series Sensor CM5 chip (Cytiva, catalog number BR100530) for 6 minutes to achieve approximately 7000–14000 response units (RU) of conjugated secondary antibody. The subject antibody was then diluted to 5 μg / mL with running buffer and injected at a flow rate of 10 μL / min to achieve approximately 200 RU of conjugated primary antibody. For kinetic measurements, His-labeled human dendritic protein 18.2 serially diluted (0.195–50 nM) was injected at a flow rate of 30 μL / min, and binding was monitored on a Biacore 8K (Cytiva) at 120 sec for association and 300 sec for dissociation. The association rate (ka) and dissociation rate (kd) were calculated using a simple one-to-one binding model by simultaneously fitting association and dissociation sensor maps. The equilibrium dissociation constant (K) was also determined. D The ratio kd / ka is calculated. The results are shown in Table 7 below.

[0209] Table 7

[0210] Theme Antibody Capture Level (RU) <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (M)]]> Rmax(RU) <![CDATA[Chi 2 (RU 2 )]]> YL-G1-19-02 162.4 6.73E+05 1.41E-03 2.09E-09 181.1 7.15 YL-G1-19-03 163.1 4.75E+05 5.67E-04 1.19E-09 167.9 1.26 YL-G1-19-04 168.6 6.28E+05 7.80E-04 1.24E-09 175.1 3.30 YL-G2-B 168.7 6.86E+05 1.40E-03 2.05E-09 187.5 9.14 YL-G2-C 160.5 5.32E+05 5.91E-04 1.11E-09 177.7 3.85 YL-G2-D 169.4 7.07E+05 7.22E-04 1.02E-09 187.2 3.67

[0211] sequence list

[0212]

[0213]

[0214]

Claims

1. A monoclonal antibody that specifically binds to CLDN18.2, said monoclonal antibody comprising: (1) HVR-H1 as shown in SEQ ID NO: 17, HVR-H2 as shown in SEQ ID NO: 18, HVR-H3 as shown in SEQ ID NO: 19, HVR-L1 as shown in SEQ ID NO: 20, HVR-L2 as shown in SEQ ID NO: 21 and HVR-L3 as shown in SEQ ID NO: 22, according to the IMGT numbering method; (2) HVR-H1 as shown in SEQ ID NO: 41, HVR-H2 as shown in SEQ ID NO: 42, HVR-H3 as shown in SEQ ID NO: 43, HVR-L1 as shown in SEQ ID NO: 44, HVR-L2 as shown in SEQ ID NO: 45 and HVR-L3 as shown in SEQ ID NO: 46, according to the Kabat numbering method; (3) HVR-H1 as shown in SEQ ID NO: 47, HVR-H2 as shown in SEQ ID NO: 48, HVR-H3 as shown in SEQ ID NO: 49, HVR-L1 as shown in SEQ ID NO: 50, HVR-L2 as shown in SEQ ID NO: 51, and HVR-L3 as shown in SEQ ID NO: 52, according to the Chothia numbering system; or (4) HVR-H1 as shown in SEQ ID NO: 53, HVR-H2 as shown in SEQ ID NO: 54, HVR-H3 as shown in SEQ ID NO: 55, HVR-L1 as shown in SEQ ID NO: 56, HVR-L2 as shown in SEQ ID NO: 57 and HVR-L3 as shown in SEQ ID NO: 58, according to the Contact numbering method.

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody comprises: VH as shown in SEQ ID NO: 3 and VL as shown in SEQ ID NO:

4.

3. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody is a mouse antibody.

4. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody is a chimeric antibody.

5. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a humanized antibody.

6. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody is a full-length antibody.

7. The monoclonal antibody according to claim 6, wherein the monoclonal antibody comprises a constant region of the human IgG heavy chain and / or a constant region of the human κ light chain.

8. The monoclonal antibody of claim 7, wherein the monoclonal antibody comprises the human IgG1 heavy chain constant region.

9. The monoclonal antibody of claim 7, wherein the monoclonal antibody comprises the human IgG1 heavy chain constant region as shown in SEQ ID NO: 9 and / or the human κ light chain constant region as shown in SEQ ID NO:

10.

10. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody is an antigen-binding antibody fragment.

11. The monoclonal antibody of claim 10, wherein the monoclonal antibody is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, and biantibody.

12. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody is isolated.

13. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody is a naked antibody.

14. An Fc-engineered monoclonal antibody that specifically binds to CLDN18.2, wherein the monoclonal antibody comprises: (1) HVR-H1 as shown in SEQ ID NO: 17, HVR-H2 as shown in SEQ ID NO: 18, HVR-H3 as shown in SEQ ID NO: 19, HVR-L1 as shown in SEQ ID NO: 20, HVR-L2 as shown in SEQ ID NO: 21 and HVR-L3 as shown in SEQ ID NO: 22, according to the IMGT numbering method; (2) HVR-H1 as shown in SEQ ID NO: 41, HVR-H2 as shown in SEQ ID NO: 42, HVR-H3 as shown in SEQ ID NO: 43, HVR-L1 as shown in SEQ ID NO: 44, HVR-L2 as shown in SEQ ID NO: 45 and HVR-L3 as shown in SEQ ID NO: 46, according to the Kabat numbering method; (3) HVR-H1 as shown in SEQ ID NO: 47, HVR-H2 as shown in SEQ ID NO: 48, HVR-H3 as shown in SEQ ID NO: 49, HVR-L1 as shown in SEQ ID NO: 50, HVR-L2 as shown in SEQ ID NO: 51, and HVR-L3 as shown in SEQ ID NO: 52, according to the Chothia numbering system; or (4) HVR-H1 as shown in SEQ ID NO: 53, HVR-H2 as shown in SEQ ID NO: 54, HVR-H3 as shown in SEQ ID NO: 55, HVR-L1 as shown in SEQ ID NO: 56, HVR-L2 as shown in SEQ ID NO: 57, and HVR-L3 as shown in SEQ ID NO: 58, according to the Contact numbering method, and It contains one or more mutations in the Fc region.

15. The monoclonal antibody of claim 14, wherein the monoclonal antibody comprises: VH as shown in SEQ ID NO: 3 and VL as shown in SEQ ID NO:

4.

16. The monoclonal antibody of claim 14, wherein the monoclonal antibody comprises: VH as shown in SEQ ID NO: 3 and VL as shown in SEQ ID NO: 4, The first two N-terminal amino acid residues of the VH are absent.

17. The monoclonal antibody according to any one of claims 14 to 16, wherein the monoclonal antibody is a chimeric antibody.

18. The monoclonal antibody of claim 14, wherein the monoclonal antibody is a humanized antibody.

19. The monoclonal antibody according to any one of claims 14 to 16, wherein the one or more mutations in the Fc region are one or more mutations that increase or decrease binding to the Fc receptor.

20. The monoclonal antibody according to any one of claims 14 to 16, wherein the one or more mutations in the Fc region are one or more mutations that increase or decrease effector function.

21. The monoclonal antibody according to any one of claims 14 to 16, wherein the one or more mutations in the Fc region are one or more mutations that increase or decrease ADCC.

22. The monoclonal antibody according to any one of claims 14 to 16, wherein the one or more mutations in the Fc region are one or more mutations that increase or decrease CDC.

23. The monoclonal antibody according to any one of claims 14 to 16, wherein the one or more mutations in the Fc region are one or more substitutions selected from the following: L235V, F243L, R292P, Y300L and P396L.

24. The monoclonal antibody according to any one of claims 14 to 16, wherein the monoclonal antibody comprises Human IgG heavy chain constant region, and / or Human κ light chain constant region.

25. The monoclonal antibody of claim 24, wherein the monoclonal antibody comprises... Human IgG1 heavy chain constant region.

26. The monoclonal antibody of claim 24, wherein the monoclonal antibody comprises... As shown in SEQ ID NO: 40, the human IgG1 heavy chain constant region, and / or As shown in SEQ ID NO: 10, the human κ light chain constant region.

27. The monoclonal antibody according to any one of claims 14 to 16, wherein the monoclonal antibody is isolated.

28. The monoclonal antibody according to any one of claims 14 to 16, wherein the monoclonal antibody is a naked antibody.

29. An isolated nucleic acid, said isolated nucleic acid encoding a monoclonal antibody according to any one of claims 1 to 28.

30. A vector comprising the nucleic acid according to claim 29.

31. A host cell comprising the nucleic acid according to claim 29 or the vector according to claim 30, wherein the host cell is not an animal or plant species.

32. A method for producing a monoclonal antibody according to any one of claims 1 to 28, the method comprising producing the antibody by culturing a host cell according to claim 31, and optionally recovering the antibody from the host cell or the cell culture.

33. A composition comprising a monoclonal antibody according to any one of claims 1 to 28.

Images