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 Application Nos. PCT / CN2021 / 097239 and PCT / CN2021 / 097240, filed May 31, 2021, and PCT Patent Application Nos. PCT / CN2021 / 106783 and PCT / CN2021 / 106784, filed July 16, 2021, the contents of which are incorporated by reference in their entirety. TECHNICAL FIELD

[0003] A set 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. BACKGROUND

[0004] The 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 comprising four transmembrane domains with two small extracellular loops.

[0005] In normal tissues other than the stomach, expression of CLDN18.2 could not be detected by RT-PCR. Immunohistochemistry with CLDN18.2-specific antibodies revealed the stomach as the only positive tissue.

[0006] CLDN18.2 is a highly selective gastric lineage antigen that is expressed only on short-lived, differentiated gastric epithelial cells. CLDN18.2 is maintained during malignant transformation and is therefore frequently displayed on the surface of human gastric cancer cells. In addition, this pan-tumor antigen is ectopically activated at significant levels in esophageal adenocarcinoma, pancreatic adenocarcinoma, and lung adenocarcinoma. CLDN18.2 protein is also localized in lymph node metastases of gastric cancer adenocarcinoma, as well as in distant metastases, especially into the ovary (so-called Krukenberg tumors). SUMMARY

[0007] The present invention provides anti-CLDN18.2 antibodies.

[0008] The present invention provides an isolated monoclonal antibody (in particular, Fc engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises

[0009] (1) HVR-H1, HVR-H2, and HVR-H3 contained in a VH as shown in SEQ ID NO: 1, and HVR-L1, HVR-L2, and HVR-L3 contained in a VL as shown in SEQ ID NO: 2; (2) HVR-H1, HVR-H2, and HVR-H3 contained in a VH as shown in SEQ ID NO: 3, and HVR-L1, HVR-L2, and HVR-L3 contained in a VL as shown in SEQ ID NO: 4;

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

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

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

[0013] e.g., as shown in Table 1, and Figure 1A , Figure 1B , Figure 1C or Figure 1D and

[0014] optionally comprising one or more mutations in the Fc region.

[0015] In one embodiment, the antibody comprises

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

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

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

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

[0020] In one embodiment, the antibody comprises

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

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

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

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

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

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

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

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

[0029] optionally comprising one or more mutations in the Fc region.

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

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

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

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

[0034] optionally comprising one or more mutations in the Fc region.

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

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

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

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

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

[0040] optionally comprising one or more mutations in the Fc region,

[0041] optionally wherein 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 substitutions selected from 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 application further provides an isolated monoclonal antibody (in particular, Fc engineered) that specifically binds to human CLDN18.2, wherein the antibody:

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

[0045] ii) binds to the same epitope on human CLDN18.2 as an anti-CLDN18.2 antibody comprising (1) a VH as depicted in SEQ ID NO: 1 and a VL as depicted in SEQ ID NO: 2; (2) a VH as depicted in SEQ ID NO: 3 and a VL as depicted in SEQ ID NO: 4; (3) a VH as depicted in SEQ ID NO: 5 and a VL as depicted in SEQ ID NO: 6; or (4) a VH as depicted in SEQ ID NO: 7 and a VL as depicted in SEQ ID NO: 8; and / or

[0046] iii) mediates ADCC of PBMCs against cells expressing human CLDN18.2 (e.g., 293T cells or CHO cells or CT26 cells or KATO III cells or NCI-N87 cells), for example with an EC50 value of, about, 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, 0.2 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 as determined via LDH or FACS; and / or

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

[0048] v) mediates CDC against cells expressing human CLDN18.2 (e.g., 293T cells or CHO cells or CT26 cells or KATO III cells or NCI-N87 cells), for example with an EC50 value of, about, 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, 0.2 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 as determined via LDH or FACS; and / or

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

[0050] vii) binds to a cell (e.g., 293T cell or CHO cell) expressing human CLDN18.2 on the cell surface, e.g., with a Kd value of, about, 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) does not bind to a cell (e.g., 293T cell or CHO cell) expressing human CLDN18.1 on the cell surface; and / or

[0052] ix) specifically binds to human CLDN18.2, e.g., with a Kd value of, about, 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 application is a murine antibody, a chimeric antibody, or a humanized antibody.

[0055] In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the application is an antigen-binding antibody fragment, optionally selected from the group consisting of a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFv fragment, and a diabody.

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

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

[0058] The application provides an isolated nucleic acid encoding a monoclonal antibody of the application. The application provides a vector (e.g., a cloning vector or an expression vector) comprising a nucleic acid of the application. The application provides a host cell comprising a nucleic acid of the application or a vector of the application. The application provides a method of producing a monoclonal antibody of the application, comprising culturing the host cell so that the antibody is produced. In one embodiment, the method further comprises recovering the antibody from the host cell or the cell culture.

[0059] The application provides a composition comprising a monoclonal antibody of the application. The application provides a pharmaceutical formulation comprising a monoclonal antibody of the application and a pharmaceutically acceptable carrier.

[0060] The application provides a monoclonal antibody of the application for use as a medicament. The application provides a monoclonal antibody of the application for use in treating cancer. The application provides use of a monoclonal antibody of the application in the manufacture of a medicament. In one embodiment, the medicament is for treating cancer. The application provides a method of treating an individual having cancer, comprising administering to the individual an effective amount of a monoclonal antibody of the application. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1A An alignment of the amino acid sequences of the VH and VL of the antibodies of the application is shown, with the HVR sequences according to Chothia highlighted by shading.

[0062] Figure 1B An alignment of the amino acid sequences of the VH and VL of the antibodies of the application is shown, with the HVR sequences according to IMGT highlighted by shading.

[0063] Figure 1C An alignment of the amino acid sequences of the VH and VL of the antibodies of the application is shown, with the HVR sequences according to Chothia highlighted by shading.

[0064] Figure 1D An alignment of the amino acid sequences of the VH and VL of the antibodies of the application is shown, with the HVR sequences according to Contact highlighted by shading.

[0065] Figure 2 Binding of the antibodies of the application to cells expressing CLDN18.2 is shown.

[0066] Figure 3 Binding of the antibodies of the present application to cells expressing CLDN18.1 is shown.

[0067] Figure 4 ADCC mediated by the antibodies of the present application on cells expressing CLDN18.2 is shown.

[0068] Figure 5 Binding curves of the antibodies of the present application to huCLDN18.2 are shown.

[0069] Figure 6 Binding curves of the antibodies of the present application to cells expressing huCLDN18.2 are shown.

[0070] Figure 7 Results of ADCC assays of the antibodies of the present application are shown.

[0071] Figure 8 Results of cell binding assays of the antibodies of the present application are shown.

[0072] Figure 9 Results of CDC assays of the antibodies of the present application are shown.

[0073] Figure 10 Results of ADCC assays of the antibodies of the present application are shown.

[0074] Figure 11 CDC effect mediated by the antibodies of the present application on CT26 expressing CLDN18.2 determined by LDH assay is shown.

[0075] Figure 12 CDC effect mediated by the antibodies of the present application on KATO III expressing CLDN18.2 determined by LDH assay is shown.

[0076] Figure 13 ADCC effect mediated by the antibodies of the present application on KATO III expressing CLDN18.2 determined by LDH assay is shown.

[0077] Figure 14 ADCC effect mediated by the antibodies of the present application on NCI-N87 expressing CLDN18.2 determined by LDH assay is shown. DETAILED DESCRIPTION

[0078] I. DEFINITIONS

[0079] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0080] “Antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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.

[0081] 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, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0082] The“class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The light chain can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0083] “Effector function” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0084] As used herein, the term“engineer,”“engineered,”“engineering” is taken to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, modification of the glycosylation pattern, or modification of the side chain groups of individual amino acids, and combinations of these approaches.

[0085] The term "Fc region" herein is used to define a 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 variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present. In one embodiment, an anti-CLDN18.2 antibody as described herein is of the IgG1 isotype and comprises a 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 indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0086] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is generally comprised of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in a VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0087] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region.

[0088] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include both primary transformed cells and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny can not be identical to that of the parent cell, but can contain mutations due to reasonably foreseeable mutations that occur during clonal propagation. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0089] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0090] The term "hypervariable region" or "HVR", as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contacts ("antigen contacts"). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs herein include:

[0091] (a) the hypervariable loops that are 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));

[0092] (b) the CDRs that are present at 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 Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0093] (c) the antigen contacts that are present at 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 identified according to the Chothia et al. definition (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)) using the abmino program in the ablnego package (Martin and Thornton, Acta Crystallogr. D 58: 192- 200 (2002), which is hereby incorporated by reference). https: / / www.novopro.cn / tools / cdr.html

[0096] Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in a variable domain are numbered herein according to Rabat et al., supra.

[0097] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and transgenic animals containing all or part of the human immunoglobulin loci, which methods and other exemplary methods are described herein.

[0098] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved with binding the antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures and each domain comprises 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 can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., 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. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0100] II. Exemplary Antibodies

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

[0102] The present application provides an isolated monoclonal antibody (particularly Fc engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises a VH comprising a HVR-H1 as set forth in SEQ ID NO: 17, a HVR-H2 as set forth in SEQ ID NO: 18, and a HVR-H3 as set forth in SEQ ID NO: 19, and a VL comprising a HVR-L1 as set forth in SEQ ID NO: 20, a HVR-L2 as set forth in SEQ ID NO: 21, and a HVR-L3 as set forth in SEQ ID NO: 22. In one embodiment, the antibody comprises a VH as set forth in SEQ ID NO: 3 and a VL as set forth in SEQ ID NO: 4. Optionally, the first two N-terminal amino acid residues of the VH are absent.

[0103] The present invention provides an isolated monoclonal antibody (particularly Fc engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises a VH comprising a HVR-H1 as set forth in SEQ ID NO: 23, a HVR-H2 as set forth in SEQ ID NO: 24, and a HVR-H3 as set forth in SEQ ID NO: 25, and a VL comprising a HVR-L1 as set forth in SEQ ID NO: 26, a HVR-L2 as set forth in SEQ ID NO: 27, and a HVR-L3 as set forth in SEQ ID NO: 28. The present invention provides an isolated monoclonal antibody (particularly Fc engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises a VH comprising a HVR-H1 as set forth in SEQ ID NO: 41, a HVR-H2 as set forth in SEQ ID NO: 42, and a HVR-H3 as set forth in SEQ ID NO: 43, and a VL comprising a HVR-L1 as set forth in SEQ ID NO: 44, a HVR-L2 as set forth in SEQ ID NO: 45, and a HVR-L3 as set forth in SEQ ID NO: 46. The present invention provides an isolated monoclonal antibody (particularly Fc engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises a VH comprising a HVR-H1 as set forth in SEQ ID NO: 47, a HVR-H2 as set forth in SEQ ID NO: 48, and a HVR-H3 as set forth in SEQ ID NO: 49, and a VL comprising a HVR-L1 as set forth in SEQ ID NO: 50, a HVR-L2 as set forth in SEQ ID NO: 51, and a HVR-L3 as set forth in SEQ ID NO: 52. The present invention provides an isolated monoclonal antibody (particularly Fc engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises a VH comprising a HVR-H1 as set forth in SEQ ID NO: 53, a HVR-H2 as set forth in SEQ ID NO: 54, and a HVR-H3 as set forth in SEQ ID NO: 55, and a VL comprising a HVR-L1 as set forth in SEQ ID NO: 56, a HVR-L2 as set forth in SEQ ID NO: 57, and a HVR-L3 as set forth in SEQ ID NO: 58. In one embodiment, the antibody comprises a VH as set forth in SEQ ID NO: 5 and a VL as set forth in SEQ ID NO: 6. Optionally, the first two N-terminal amino acid residues of the VH are absent.

[0104] The present application provides an isolated monoclonal antibody (in particular Fc engineered) that specifically binds to human CLDN18.2, wherein the antibody comprises a VH comprising a HVR-H1 as set forth in SEQ ID NO: 29, a HVR-H2 as set forth in SEQ ID NO: 30, and a HVR-H3 as set forth in SEQ ID NO: 31, and a VL comprising a HVR-L1 as set forth in SEQ ID NO: 32, a HVR-L2 as set forth in SEQ ID NO: 33, and a HVR-L3 as set forth in SEQ ID NO: 34. In one embodiment, the antibody comprises a VH as set forth in SEQ ID NO: 7 and a VL as set forth 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) binding to an 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 group consisting of 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 present application is a murine antibody, a chimeric antibody, or a humanized antibody.

[0107] In one embodiment, the anti-CLDN18.2 monoclonal antibody according to the present application is an antigen-binding antibody fragment, optionally selected from the group consisting of a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFv fragment, and a diabody.

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

[0109] In one embodiment, the monoclonal antibody (mAb) or Fab fragment of the application has a cross-over format (x-mAb or x-Fab), wherein the variable domains of the light and heavy chains or the (first) constant domains are exchanged.

[0110] III. Recombinant methods and compositions

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

[0112] In the case of a native antibody or a native antibody fragment, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. The nucleic acid(s) encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., one or more light chains and / or heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0113] In one embodiment, an isolated nucleic acid encoding an antibody as used in a method as reported herein is provided.

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

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

[0116] In one such embodiment, the host cell comprises (e.g., has been transformed with):

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

[0118] (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody.

[0119] In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell or host cell culture medium.

[0120] For recombinant production of an antibody, a nucleic acid encoding an antibody, e.g., as described above, 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 are specific to genes encoding the heavy and light chains of the antibody), or produced by recombinant methods or by chemical synthesis.

[0121] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. See also Charlton, K. A., in: Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0122] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibodies-encoding vectors, including fungal and yeast strains whose glycosylation pathway has been "humanized" to produce antibodies with partial or fully human glycosylation pattern. See Gerngross, T. U., 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 are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculoviral strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0124] Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES TM technology) for production of antibodies in transgenic plants.

[0125] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. 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, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., 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.

[0126] IV. Assays

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

[0128] Binding assays and other assays

[0129] In one aspect, the antigen binding activity of the antibodies of the application is tested, e.g., by known methods such as ELISA, Western blotting, and the like.

[0130] In another aspect, a competition assay can be used to identify antibodies that compete with aCLDN18.2 for binding to CLDN18.2. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) as aCLDN18.2. Detailed exemplary methods for mapping the epitope bound by an antibody are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0131] In an exemplary competition assay, immobilized CLDN18.2 is incubated in a solution containing a first labeled antibody that binds to CLDN18.2 and a second unlabeled antibody, and the ability of the second unlabeled antibody to compete with the first antibody for binding to CLDN18.2 is tested. The second antibody can be present in a 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 that allow 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 is significantly reduced in the test sample relative to the control sample, then the second antibody competes with the first antibody for binding to CLDN18.2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0132] Activity assays

[0133] In one aspect, assays are provided for identifying anti-CLDN18.2 antibodies that have biological activity. Biological activity can include, for example, the effect of an anti-CLDN18.2 antibody on ADCC of PBMCs against target cells expressing CLDN18.2. Antibodies that have such biological activity in vivo and / or in vitro are also provided.

[0134] Examples

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

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

[0137] This example demonstrates cloning of the H chain and L chain genes from mouse hybridoma cells to obtain variable region sequences specific for CLDN18.2 to produce chimeric antibodies.

[0138] RNA was isolated and purified from hybridoma cells using the Quick-RNA TM RNA was isolated and purified from hybridoma cells using the Quick-RNA RACE 5’ / 3’ kit (Takara Bio USA, Inc., Cat# 634858) along with IgGl 3’ constant primer (SEQ ID NO: 35), IgG2a 3’ constant primer (SEQ ID NO: 36), and Kappa 3’ constant primer (SEQ ID NO: 37). Gel extraction was performed on RACE DNA products with NuceloSpin gel and PCR Clean-Up kit (Takara, Cat# 740986.20). In-fusion reaction mixtures of linearized pRACE vectors and gel purified PACE products were transformed into Stellar competent cells (Clontech, Cat# 636766). Plasmid DNA was isolated from transformants using QIAprep Spin Miniprep kit (Qiagen, Cat# 27104) and Sanger sequenced (GENEWIZ) using M13 sequencing primers. Final gene sequences for four pairs of heavy and light chains were obtained (data not shown), which were identified as CLDN18.2 mAbs (mAb1, mAb2, mAb3, and mAb4) below.

[0139] Example 2: Construction of chimeric antibodies by replacing mouse constant regions with constant regions from humans

[0140] This example demonstrates construction of chimeric antibodies by replacing the constant regions of the four molecular cloned mouse mAbs with constant regions from human IgGl heavy chain and kappa light chain.

[0141] Plasmids pFUSE-CHIg-hG1 (InvivoGen, Cat# pfuse-hchg1) containing the constant region of human IgG1 heavy chain (SEQ ID NO: 9) and pFUSE2-CLIg-hk (InvivoGen, Cat# pfuse2-hclk) containing the constant region of human kappa light chain (SEQ ID NO: 10) were digested with Hind III and Nhe I (for IgG1) or BsiW I (for kappa) (all from NEB lab). 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 using HiFi HotStart (Kapa (Roche), KK2602) from the plasmids obtained in Example 1 along with specific primers (data not shown) and purified by gel purification using NucleoSpin Gel and PCR Clean-up kit (Takara, Cat# 740986.20). Linearized vectors and inserts were assembled using Gibson HiFi One Step kit (SGI (VWR), Cat# GA1100-50). The assembly reaction mixture was transformed into Stellar competent cells (Clontech, Cat# 636766). Plasmid DNA was isolated from transformants using QIAprep Spin Miniprep kit (Qiagen, Cat# 27104) and subjected to Sanger sequencing (GENEWIZ). Four chimeric antibodies were constructed, each comprising the variable region from one of the four mouse mAbs produced in Example 1. The new 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 specificity of the four chimeric monoclonal antibodies via 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] A density of 2x10 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. 6PBMC (50 μL) at 1 cell / ml, and the plate was incubated at 37°C in the dark for 2 hours. Cells were washed twice with PBS, and 100 μL eBioscience TM Fixable viability dye eFluor TM 660 (Thermo, Cat. No. 65-0864) was added to each well. The plate was incubated on ice in the dark for 30 minutes. After washing with PBS, cells were resuspended by adding 75 μL / well PBS and 25 μL / well 4% paraformaldehyde, and subjected to FACS using a BD LSR II flow cytometer (HTS). Data analysis was performed using the percentage of killing (CFSE / FVD-AF660 double-positive population divided by the CFSE-positive population), and Prism GraphPad was used for plotting. The ADCC activity obtained by the FACS-based method showed that the four chimeric antibodies could mediate ADCC activity against CLDN18.2-expressing cells (see Figure 4 ).

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

[0149] In addition, four Fc-engineered chimeric antibodies were constructed. Compared with the first generation of original chimeric antibodies YL-G1-19-01, YL-G1-19-02, YL-G1-19-03 and YL-G1-19-04, the second generation of Fc-engineered chimeric antibodies YL-G2-A, YL-G2-B, YL-G2-C and YL-G2-D (compared with YL-G1-19-04, the first two N-terminal amino acid residues of the VH domain are absent in YL-G2-D) comprise five substitutions in the Fc region, i.e. L235V, F243L, R292P, Y300L and P396L (according to EU numbering). The mutant constant region of human IgG1 heavy chain (including CH1, hinge, CH2 and CH3) is shown in SEQ ID NO: 40.

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

[0151] 5.1: Antigen-antibody binding interaction.

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

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

[0154] To determine affinity using the KinExA method, serial dilutions of binding partner B (called the titrant) are performed in the context of a constant concentration of binding partner A (called the constant binding partner, CBP). This means that the CBP will remain at a constant concentration, while the concentration of the titrant will vary. Once these solutions have reached equilibrium, the KinExA 4000 instrument can directly measure the amount of unbound or free binding partner of the CBP remaining in solution. Using Sapidyne’s software, the percentage of free CBP can be plotted against the total titrant concentration to generate a binding curve and determine the affinity.

[0155] Kd was first determined with the antibody and recombinant human CLDN18.2 purchased from Sino Biological (P / N: 20047-H02H). For the equilibrium experiments, the titrant (huCLDN18.2) was serially diluted five-fold in the context of the CBP. Two equilibrium experiments were performed: one with a high concentration of CBP (20 nM binding sites) at 10 nM and the titrant serially diluted five-fold at 150 nM, and one with a low concentration of CBP (200 pM binding sites) at 100 pM and the titrant serially diluted five-fold at 150 nM. The data were collected on the KinExA 4000 and analyzed using Sapidyne Instruments n-Curve analysis software version 4.4.26. The binding curves are shown in Figure 5 .

[0156] The binding affinity of the antibody to the surface protein of whole cells (i.e., CHO cells overexpressing huCLDN18.2) was also measured using the KinExA 4000 instrument. The antibody concentration was kept constant (CBP), and the concentration of whole cells with surface protein was varied (titrant). The concentration of whole cells with surface protein was diluted three-fold. The titrated cells were incubated with the constant binding partner (CBP). Once equilibrium was reached, the samples were centrifuged, the supernatant was recovered, and the free CBP was detected with a fluorescently labeled anti-CBP molecule. The binding curves are shown in Figure 6 .

[0157] To establish a more accurate Kd, two equilibrium curves were prepared and analyzed. One curve used a low concentration of CBP (200 pM binding sites) at 100 pM and 10 6 cells / mL with three-fold dilutions, and one curve used a high concentration of CBP (20 nM binding sites) at 10 nM and 10 6cells / mL. KinExA 4000 measured the amount of unbound CBP in solution. Analysis was performed using Sapidyne Instruments n-Curve analysis software version 4.4.26. Summary of equilibrium dissociation constants Kd is shown in Table 1.

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

[0159]

[0160] 5.2: Cell binding assay.

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

[0162] Binding of the antibodies was evaluated using CHO cells expressing huCLDN18.2. For each sample, cells were seeded at 5 x 10 5 cells / 100 μΐ into wells of a 96-well plate. Then, 100 μΐ of each serially diluted antibody was added to the cells, starting at 40 μg / ml and diluting 5-fold. Thus, the final concentration of each antibody started at 20 μg / ml and was serially diluted 5-fold. After 1 hour, the cells were washed twice, and 100 μΐ of GAH-FITC (1 :200 dilution) was added to each well. After 30 minutes, the cells were washed twice and resuspended in 120 μΐ of FACS buffer. The cells were analyzed via flow cytometry.

[0163] Using unstained cells as a reference to set the overall target cell gate and establish the FITC negative population, we were able to establish a FITC positive cell gate for each cell line. In addition, the mean fluorescence intensity (MFI) of the entire cell population was calculated to confirm the FITC positive results in duplicate. The ratio of the number of gated positive cells to the total number of live cells was taken as the percentage of positive cells. Results are shown in Table 2. Figure 7

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

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

[0166] Target cells (i.e., CHO cells expressing huCLDN18.2) were washed once with DPBS. Then, the cells were seeded at 2 x 10 4 ​Cells were seeded at 100 μL / well into RPMI in U-bottom plates. Antibodies were serially diluted at 1 :2 and incubated with cells at 50 μl / well for 15 min at room temperature. Then 20% pooled serum was added to all wells at 50 μl / well, including spontaneous release and maximum release wells. Plates were incubated at 37°C for 3.5 h in an incubator. Forty-five minutes before the end of the incubation period, plates were centrifuged at 1200 rpm for 5 min and 20 μL lysis buffer (CyQUANT TM LDH Cytotoxicity Assay Kit, Cat. No. C20300 and C20301) was added to the maximum release control wells containing target cells only. Fifty μL supernatant was transferred to a black-walled 96-well plate together with 50 μL / well of reaction buffer (CyQUANT TM LDH Cytotoxicity Assay Kit, Cat. No. C20300 and C20301) was added to the maximum release control wells containing target cells only. Fifty μL supernatant was transferred to a black-walled 96-well plate together with 50 μL / well of reaction buffer (CyQUANT TM LDH Cytotoxicity Assay Kit, Cat. No. C20300 and C20301) was added to the maximum release control wells containing target cells only. Fifty μL supernatant was transferred to a black-walled 96-well plate together with 50 μL / well of reaction buffer (CyQUANT Figure 8 LDH Cytotoxicity Assay Kit, Cat. No. C20300 and C20301) was added to the maximum release control wells containing target cells only. Fifty μL supernatant was transferred to a black-walled 96-well plate together with 50 μL / well of reaction buffer (CyQUANT

[0167] 5.4: Internalization assay.

[0168] This example illustrates an internalization assay for YL-G2-B, YL-G2-C and YL-G2-D.

[0169] CHO cells expressing huCLDN18.2 were prepared at 5 x 10 5 / mL in DMEM medium. 100 μL of cells were seeded per well of a U-bottom 96-well plate. Test antibodies and control antibodies were diluted to 40 μg / ml and then each antibody was serially diluted 1 :4 in culture medium. Each diluted antibody was added to the cells at 50 μl / well. The plates were incubated at 37°C for 30 min. Then, 40 μg / ml PEP-ZAP (a small Fc-binding peptide fused to a cytotoxic peptide, developed by AB Studio Inc.; see WO 2020 / 018732 Al) was added to each well at 50 μl / well to give a final concentration of 10 μg / ml PEP-ZAP. The plates were incubated at 37°C for 72 h. Finally, the cells were spun down and 100 μl of supernatant was taken for measurement of LDH. The results are shown in Figure 9 Table 5.4.

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

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

[0172] ADCC function of antibodies 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 seeded at 2x10 4 cells / 50 μl into the wells of a 96-well plate in DMEM-F12 + 10% FBS medium. Then, 100 μl of each 1:10 serial dilution of antibody was added to the cells. After 20 minutes, NK cells were added to the plate at 2x10 5 cells / 50 μl, resulting in a target:effector ratio of 1:10. After this addition, the resulting final concentrations of each antibody started at 50 μg / ml, followed by 5, 0.5, and 0.05 μg / ml. The plate was placed in a 37°C, CO2 incubator for 24 hours. Then, the cells were stained with 7AAD, washed twice, and resuspended in approximately 200 μl FACS buffer. Cells were analyzed via flow cytometry.

[0173] The use of unstained cells as a reference to set the overall target cell gate and establish the 7AAD negative population allowed for the differentiation between 7AAD (dead cells) and 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 has the same amino acid sequence except for a VLPLL substitution in the Fc region) is shown in Figure 10 Table 2. A summary of EC50s 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, catalog number KC-1195) maintained in DMEM medium (Gibco, catalog number 31053-036) containing 10% FBS (ExCell Bio, catalog number FND500), KATO III CLDN18.2 cells (human gastric cancer cells, Kyinno biotechnology, catalog number KC-1453) maintained in RPMI1640 medium (Gibco, catalog number 22400-089) containing 10% FBS, and NCI-N87 CLDN18.2 cells (human gastric cancer cells, Kyinno biotechnology, catalog number KC-1222) maintained in RPMI1640 medium containing 10% FBS are all tumor cells overexpressing human CLDN18.2, and they were used to determine the CDC activity and ADCC activity of the subject antibodies.

[0181] 6.2: CDC assay

[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] Data were analyzed by four-parameter non-linear regression using GraphPad Prism 7 software and EC 50 values were calculated and obtained.

[0185] 6.3: ADCC assay

[0186] The ADCC activity of the subject antibodies was evaluated by measuring the change in LDH level released into the culture medium after cell lysis. NCI-N87 CLDN18.2 cells or KATOIII CLDN18.2 cells were suspended in phenol red-free RPMI 1640 medium at a density of 6E+05 cells / mL. The subject antibodies were diluted 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 with 1% FBS-containing phenol red-free RPMI 1640 medium. Fresh human PBMC cells (Saily, from volunteer #XC11057W) were suspended in 1% FBS-containing phenol red-free RPMI 1640 medium at a density of 1.2E+07 cells / mL. To each well of a round-bottom 96-well microplate, 50 μL of antibody dilution, 50 μL of human PBMC cell suspension and 50 μL of tumor cell suspension were added. Human IgG1 isotype was included as a negative control. Reference mAb (IMAB362, Ganymed) was included as a positive control. The microplate was incubated in an incubator set at 37°C and 5% CO2 for 4-6 hours. After incubation, the release of LDH into the supernatant of the cell culture was detected according to the instruction provided with the LDH cytotoxicity assay kit as described above. The ADCC activity of the subject antibodies was calculated by 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] Data were analyzed by four-parameter non-linear regression using GraphPad Prism 7 software and EC 50 values were calculated and obtained.

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

[0190] As Figure 11 and shown in Table 3, both batches of YL-G2-B exhibited comparable CDC effect on CT26 CLDN18.2 cells Figure 11 YL-G1-19-02, YL-G2-B, YL-G1-19-03, YL-G2-C, YL-G1-19-04 and YL-G2-D all exhibited 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 KATO III CLDN18.2 cells by LDH assay

[0195] As Figure 12 and shown in Table 4, both batches of YL-G2-B exhibited comparable ADCC effect on KATO III CLDN18.2 cells Figure 12 , 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 exhibited stronger ADCC effect on KATO III CLDN18.2 cells than the positive control, the positive control Figure 12 .

[0196] Table 4: ADCC effect on KATO III CLDN18.2 cells

[0197]

[0198] 6.6: ADCC effect on KATO III CLDN18.2 cells by LDH assay

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

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

[0201]

[0202]

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

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

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

[0206]

[0207] 6.8: SPR

[0208] Binding affinity of the subject antibodies was determined via SPR using Human Antibody Capture Kit Type 2 (Cytiva, Cat# 29234600) according to USP 43 IMMUNOLOGICAL TEST METHODS— SURFACE PLASMON RESONANCE <1105> and CP, 2020 edition, Part IV, General Rules, 3429 IMMUNOCHEMISTRY, NON-LABELING IMMUNOCHEMICAL METHODS (IV) SURFACE PLASMON RESONANCE. Briefly, anti-human IgG (Fc) antibody was diluted to 25 pg / mL with immobilization buffer and injected at a flow rate of 10 pL / min for 6 min onto a Series S Sensor CM5 chip (Cytiva, Cat# BR100530) to achieve a coupled secondary antibody of about 7000-14000 response units (RU). The subject antibodies were then diluted to 5 pg / mL with running buffer and injected at a flow rate of 10 pL / min to achieve a coupled primary antibody of about 200 RU. For kinetics measurements, two serial dilutions (0.195-50 nM) of His-tagged human Claudin 18.2 were injected at a flow rate of 30 pL / min and binding was monitored on a Biacore 8K (Cytiva) for 120 s for association and 300 s for dissociation. Association rates (ka) and dissociation rates (kd) were calculated by simultaneous fitting of association and dissociation sensorgrams using a simple one-to-one binding model. The equilibrium dissociation constant (Kd) was calculated as the ratio kd / ka. Results are shown in Table 7 below. D

[0209] Table 7

[0210] Subject Antibody Capture Level (RU) k a (1 / Ms)]]> k d (1 / s) K D (M)]]> Rmax (RU) Chi 2 (RU 2 )]]> YL-G1-19-02 162.4 6.73E+05 1.41F-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 LISTING

[0212]

[0213]

[0214] ​

Claims

1. A monoclonal antibody that specifically binds to CLDN18.2, the monoclonal antibody comprising: (1) HVR-H1 as set forth in SEQ ID NO: 23, HVR-H2 as set forth in SEQ ID NO: 24, HVR-H3 as set forth in SEQ ID NO: 25, HVR-L1 as set forth in SEQ ID NO: 26, HVR-L2 as set forth in SEQ ID NO: 27, and HVR-L3 as set forth in SEQ ID NO: 28, according to IMGT numbering; (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, according to Kabat numbering; (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, according to Chothia numbering; or (4) 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, according to Contact numbering.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises: a VH as set forth in SEQ ID NO: 5 and a VL as set forth in SEQ ID NO:

6.

3. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody is a murine antibody.

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

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

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

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

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

9. The monoclonal antibody of claim 7, wherein the monoclonal antibody comprises a human IgGl heavy chain constant region as set forth in SEQ ID NO: 9 and / or a human kappa light chain constant region as set forth in SEQ ID NO:

10.

10. The monoclonal antibody of 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 an antigen-binding antibody fragment selected from the group consisting of a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFv fragment, and a diabody.

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

13. The monoclonal antibody of 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 set forth in SEQ ID NO: 23, HVR-H2 as set forth in SEQ ID NO: 24, HVR-H3 as set forth in SEQ ID NO: 25, HVR-L1 as set forth in SEQ ID NO: 26, HVR-L2 as set forth in SEQ ID NO: 27, and HVR-L3 as set forth in SEQ ID NO: 28, according to IMGT numbering; (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, according to Kabat numbering; (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, according to Chothia numbering; or (4) 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, according to Contact numbering, and one or more mutations in the Fc region.

15. The monoclonal antibody of claim 14, wherein the monoclonal antibody comprises: (1) HVR-H1 as set forth in SEQ ID NO: 23, HVR-H2 as set forth in SEQ ID NO: 24, HVR-H3 as set forth in SEQ ID NO: 25, HVR-L1 as set forth in SEQ ID NO: 26, HVR-L2 as set forth in SEQ ID NO: 27, and HVR-L3 as set forth in SEQ ID NO: 28, according to IMGT numbering; (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, according to Kabat numbering; (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, according to Chothia numbering; or (4) 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, according to Contact numbering, and one or more mutations in the Fc region. a VH as set forth in SEQ ID NO: 5 and a VL as set forth in SEQ ID NO:

6.

16. The monoclonal antibody of claim 14, wherein the monoclonal antibody comprises: a VH as set forth in SEQ ID NO: 5 and a VL as set forth in SEQ ID NO: 6, wherein the first two N-terminal amino acid residues of the VH are absent.

17. The monoclonal antibody of 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 of 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 an Fc receptor.

20. The monoclonal antibody of 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 of 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 of 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 of 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 group consisting of: L235V, F243L, R292P, Y300L, and P396L.

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

25. The monoclonal antibody of claim 24, wherein the monoclonal antibody comprises a human IgGl heavy chain constant region.

26. The monoclonal antibody of claim 24, wherein the monoclonal antibody comprises a human IgGl heavy chain constant region as set forth in SEQ ID NO: 40, and / or a human kappa light chain constant region as set forth in SEQ ID NO:

10.

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

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

29. An isolated nucleic acid encoding the monoclonal antibody of any one of claims 1 to 28.

30. A vector comprising the nucleic acid of claim 29.

31. A host cell comprising the nucleic acid of claim 29 or the vector of claim 30, which host cell is not a plant or animal variety.

32. A method of producing a monoclonal antibody according to any one of claims 1 to 28, by culturing a host cell according to claim 31 so that the antibody is produced, 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.

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