EA050412B1 - Monoclonal antibodies to CLDN18.2 and their Fc-engineered versions - Google Patents

Abstract

The invention provides a panel of monoclonal antibodies or antigen-binding fragments thereof that specifically bind to CLDN18.2 (claudin 18.2), comprising HVRIII, HVR-H2 and HVR-H3 contained in the VH presented in SEQ ID NO: 5, and HVRL1, HVR-L2 and HVR-L3 contained in the VL presented in SEQ ID NO: 6. The antibodies according to the present invention do not bind specifically to CLDN18.1, and optionally have an engineered Fc region. The present invention also provides an isolated nucleic acid, a vector and a host cell based on the monoclonal antibodies of the invention, as well as a method for producing such an antibody and a composition for the treatment of cancer expressing CLDN18.2, containing said monoclonal antibody.

Description

Cross-reference to related applications

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

Field of technology

A panel of monoclonal antibodies that specifically bind to CLDN18.2 (claudin 18.2) and do not specifically bind to CLDN18.1, and optionally have an engineered Fc region, is provided.

State of the art

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

RT-PCR (reverse transcription polymerase chain reaction) did not detect CLDN18.2 expression in normal tissues, with the exception of the stomach. Immunohistochemistry with CLDN18.2-specific antibodies revealed the stomach as the only positive tissue.

CLDN18.2 is a highly selective gastric lineage antigen expressed exclusively on short-lived differentiated gastric epithelial cells. CLDN18.2 persists during malignant transformation and is thus frequently displayed on the surface of human gastric cancer cells. Furthermore, this pan-tumor antigen is ectopically upregulated at significant levels in esophageal, pancreatic, and lung adenocarcinomas. CLDN18.2 is also localized in lymph node metastases from gastric adenocarcinomas and in distant metastases, particularly in the ovary (so-called Krukenberg tumors).

Brief description of the invention

The invention provides antibodies to CLDN18.2.

The invention provides an isolated monoclonal antibody, in particular with an engineered Fc, that specifically binds to human CLDN18.2, wherein the antibody comprises:

(1) HVR-H1 (heavy chain hypervariable region 1), HVR-H2 and HVR-H3 contained in the VH (heavy chain variable region) shown in SEQ ID NO: 1, and HVR-L1 (light chain hypervariable region 1), HVR-L2 and HVR-L3 contained in the VL (heavy chain variable region) shown in SEQ ID NO: 2;

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

(3) HVR-H1, HVR-H2, and HVR-H3 contained in the VH shown in SEQ ID NO: 5 and HVR-L1, HVR-L2, and HVR-L3 contained in the VL shown in SEQ ID NO: 6; or (4) HVR-H1, HVR-H2, and HVR-H3 contained in the VH shown in SEQ ID NO: 7 and HVR-L1, HVR-L2, and HVR-L3 contained in the VL shown in SEQ ID NO: 8, such as shown in Fig. 1A, 1B, 1C, or 1D, and optionally comprises one or more mutations in the Fc region.

In one embodiment, the antibody comprises:

(1) HVR-H1 as shown in SEQ ID NO: 11, HVR-H2 as shown in SEQ ID NO: 12, HVRH3 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;

(2) HVR-H1 as shown in SEQ ID NO: 17, HVR-H2 as shown in SEQ ID NO: 18, HVRH3 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;

(3) HVR-H1 as shown in SEQ ID NO: 23, HVR-H2 as shown in SEQ ID NO: 24, HVRH3 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 (4) HVR-H1 as shown in SEQ ID NO: 29, HVR-H2 as shown in SEQ ID NO: 30, HVRH3 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.

In one embodiment, the antibody comprises:

(1) HVR-H1 as shown in SEQ ID NO: 41, HVR-H2 as shown in SEQ ID NO: 42, HVRH3 as shown in SEQ ID NO: 43, HVR-L1 as shown in SEQ ID NO:

NO: 44, HVR-L2 as shown in SEQ ID NO: 45, and HVR-L3 as shown in SEQ ID NO: 46;

(2) HVR-H1 as shown in SEQ ID NO: 47, HVR-H2 as shown in SEQ ID NO: 48, HVRH3 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 (3) HVR-H1 as shown in SEQ ID NO: 53, HVR-H2 as shown in SEQ ID NO: 54, HVRH3 as shown in SEQ ID NO: 55, HVR-L1 as shown in SEQ ID NO: 56, HVR-L2 as shown in

- 1 050412 presented in SEQ ID NO: 57, and HVR-L3 presented in SEQ ID NO: 58.

The present invention further provides an isolated monoclonal antibody, in particular an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises:

(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;

(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;

(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 (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 optionally comprising one or more mutations in the Fc region.

The present invention further provides an isolated monoclonal antibody, in particular an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises:

(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;

(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 (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 optionally comprising one or more mutations in the Fc region.

The present invention further provides an isolated monoclonal antibody, in particular an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises:

(1) VH as shown in SEQ ID NO (2) VH as shown in SEQ ID NO (3) VH as shown in SEQ ID NO

1, and VL, presented in SEQ ID NO: 2;

3, and VL, presented in SEQ ID NO: 4;

5, and the VL presented in SEQ ID NO: 6; or (4) the VH presented in SEQ ID NO: 7 and the VL presented in SEQ ID NO: 8, and optionally comprises one or more mutations in the Fc region, optionally wherein the first two N-terminal amino acid residues of VH are absent.

In one embodiment, the one or more mutations in the Fc region are one or more mutations that modify, e.g., increase or decrease, binding to an Fc receptor and/or effector function, e.g., ADCC (antibody-dependent cellular cytotoxicity) and/or CDC (complement-dependent cytotoxicity). 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.

The present invention further provides an isolated monoclonal antibody, in particular an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody:

i) competes for binding to human CLDN18.2 with an anti-CLDN18.2 antibody comprising: (1) the VH shown in SEQ ID NO: 1 and the VL shown in SEQ ID NO: 2; (2) the VH shown in SEQ ID NO: 3 and the VL shown in SEQ ID NO: 4; (3) the VH shown in SEQ ID NO: 5 and the VL shown in SEQ ID NO: 6; or (4) the VH shown in SEQ ID NO: 7 and the VL shown in SEQ ID NO: 8, and/or ii) binds to the same epitope on human CLDN18.2 as an anti-CLDN18.2 antibody comprising (1) the VH shown in SEQ ID NO: 1 and the VL shown in SEQ ID NO: 2; (2) the VH shown in

- 2 050412 in SEQ ID NO: 3, and the VL presented in SEQ ID NO: 4; (3) the VH presented in SEQ ID NO: 5, and the VL presented in SEQ ID NO: 6; or (4) the VH presented in SEQ ID NO: 7, and the VL presented in SEQ ID NO: 8; and/or

Hi) mediates ADCC of PBMCs (peripheral blood mononuclear cells) on cells (e.g., 293T cells or CHO cells or CT26 cells or KATOIII cells (human gastric cancer cells) or NCI-N87 cells) that express human CLDN18.2, e.g., with an EC ₅₀ (half-maximal effective concentration) 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, were determined by LDH (lactate dehydrokinase) or FACS (fluorescence-activated cell sorting); and/or iv) does not mediate ADCC of PBMC on cells (e.g., 293T cells or CHO cells (Chinese hamster ovary cells) or CT26 cells or KATOIII cells or NCI-N87 cells) that express human CLDN18.1; and/or

v) mediates CDC on cells (e.g., 293T cells or CHO cells or CT26 cells or KATOIII cells or NCI-N87 cells) that express human CLDN18.2, e.g., with an EC ₅₀ 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, such as determined by LDH or FACS; and/or vi) does not mediate CDC on cells (e.g., 293T cells or CHO cells or CT26 cells or KATOIII cells or NCI-N87 cells) that express human CLDN18.1; and/or vii) binds to cells (e.g., 293T cells or CHO cells) that express human CLDN18.2 on the cell surface, e.g., with a Kd (dissociation constant) value at 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 viii) does not bind to cells (e.g., 293T cells or CHO cells) that express human CLDN18.1 on the cell surface; and/or ix) specifically bind 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

x) do not specifically bind to human CLDN18.1.

In one embodiment, the anti-CLDN18.2 monoclonal antibody of the invention is a murine, chimeric or humanized antibody.

In one embodiment, the monoclonal antibody to CLDN18.2 of the invention is an antigen-binding fragment of an antibody, optionally selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an scFv fragment, and a diabody.

In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention is a full-length antibody. In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention comprises a human IgG heavy chain constant region, in particular IgG1, optionally as shown in SEQ ID NO: 9. In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention comprises a mutated human IgG heavy chain constant region, in particular IgG1, optionally as shown in SEQ ID NO: 40. In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention comprises a human kappa light chain constant region, optionally as shown in SEQ ID NO: 10. In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention is a chimeric antibody, such as a mouse/human chimeric antibody. In one embodiment, the anti-CLDN18.2 monoclonal antibody of the invention has an engineered Fc.

In one embodiment, the monoclonal antibody (mAb) or Fab fragment of the present invention has a crossover format (x-mAb or x-Fab), wherein either the variable domains or the (first) constant domains of the light and heavy chains are exchanged.

The present invention provides an isolated nucleic acid encoding a monoclonal antibody of the invention. The present invention provides a vector, such as a cloning vector or an expression vector, comprising the nucleic acid of the present invention. The present invention provides a host cell comprising the nucleic acid of the present invention or the vector of the present invention. The present invention provides a method for producing the monoclonal antibody of the invention, comprising culturing the host cell to produce the antibody. In one embodiment, the method further comprises isolating the antibody from the host cell or cell culture.

The present invention provides a composition comprising a monoclonal antibody of the present invention. The present invention provides a pharmaceutical composition comprising a monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.

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The present invention provides a monoclonal antibody of the invention for use as a medicament. The present invention provides a monoclonal antibody of the invention for use in the treatment of cancer. The present invention provides the use of a monoclonal antibody of the invention in the manufacture of a medicament. In one embodiment, the medicament is for the treatment of cancer. The present invention provides a method of treating an individual suffering from cancer, comprising administering to the individual an effective amount of a monoclonal antibody of the invention.

Brief description of graphic materials

Fig. 1A shows an alignment of the amino acid sequences of the VH and VL antibodies of the present invention, where the Kabat HVR sequences are shaded.

Fig. 1B shows an alignment of the amino acid sequences of the VH and VL antibodies of the present invention, where the HVR sequences from IMGT (ImmunoGeneTics database) are shaded.

Fig. 1C shows an alignment of the amino acid sequences of the VH and VL antibodies of the present invention, with the Chothia HVR sequences shaded.

Fig. 1D shows an alignment of the amino acid sequences of the VH and VL antibodies of the present invention, where the HVR sequences along the Contact are shaded.

Fig. 2 shows the binding of antibodies of the present invention to cells expressing CLDN18.2.

Fig. 3 shows the binding of antibodies of the present invention to cells expressing CLDN18.1.

Fig. 4 shows ADCC on cells expressing CLDN18.2 mediated by antibodies of the present invention.

Fig. 5 shows a binding curve of antibodies of the present invention to huCLDN18.2.

Fig. 6 shows a binding curve of antibodies of the present invention to cells expressing huCLDN18.2.

Fig. 7 shows the results of the ADCC assay of the antibodies of the present invention.

Fig. 8 shows the results of a cellular binding assay of antibodies of the present invention.

Fig. 9 shows the results of the CDC analysis of the antibodies of the present invention.

Fig. 10 shows the results of the ADCC assay of the antibodies of the present invention.

Fig. 11 shows the effect of CDC on CT26 cells expressing CLDN18.2 mediated by antibodies of the present invention, determined by LDH assay.

Fig. 12 shows the effect of CDC on KATOIII expressing CLDN18.2 mediated by antibodies of the present invention, determined by the LDH assay.

Fig. 13 shows the effect of ADCC on KATOIII expressing CLDN18.2 mediated by antibodies of the present invention, determined by the LDH assay.

Fig. 14 shows the effect of ADCC on NCI-N87 expressing CLDN18.2 mediated by antibodies of the present invention, determined by the LDH assay.

Detailed description of the invention

I. Definition

The term "antibody" is used herein in its broadest sense and includes, but is not limited to, various antibody structures, including monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired antigen-binding activity.

An antibody fragment refers to a molecule that is not an intact antibody but contains a fragment of an intact antibody that binds an antigen to which an intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and polyspecific antibodies formed from antibody fragments.

The term chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a specific source or animal species, while the remainder of the heavy and/or light chain is derived from a different source or animal species.

An antibody class refers to the type of constant domain, or constant region, contained in its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, _IgG2 , _IgG3 , _IgG4 , IgA1, and _IgA2 . The constant domains of the heavy chain that correspond to the different immunoglobulin classes are called δ, ε, γ, and μ, respectively. The light chain can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

Effector functions refer to the biological activity attributed to the Fc region of an antibody and are dependent on the antibody isotype. Examples of antibody effector functions include: binding

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C1q and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor) and B cell activation.

As used herein, the terms "design," "designed," and "engineering" include any manipulation of the peptide backbone or post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Design includes modifications of the amino acid sequence, glycosylation pattern, or side chain moiety of individual amino acids, as well as combinations of these approaches.

The term "Fc region" is used herein to refer to the C-terminal region of an immunoglobulin heavy chain that comprises at least a fragment of a constant region. This term includes a native sequence Fc region and Fc region variants. In one embodiment, the Fc region of a human IgG heavy chain is located from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In one embodiment, an anti-CLDN18.2 antibody as described herein has an IgG1 isotype and comprises the heavy chain constant region of SEQ ID NO: 9 or SEQ ID NO: 40. In one embodiment, the antibody further comprises a C-terminal lysine (Lys447). Unless otherwise noted, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also called the EU index, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The framework, or FR, refers to the residues of the variable domain excluding the hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences in VH (or VL) are typically arranged in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms full-length antibody, intact antibody, and whole antibody are used interchangeably herein and refer to an antibody that has a structure substantially similar to that of a native antibody or that contains heavy chains containing an Fc region.

The terms host cell, host cell line, and host cell culture are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cells and the progeny derived from them, regardless of passage number. Progeny may not be completely identical in nucleic acid content to the parent cell but may contain mutations. This definition includes mutant progeny that possess the same function or biological activity observed during screening or selected from the initially transformed cells.

A humanized antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., the CDR (complementarity determining region)) correspond to similar regions from a non-human antibody, and all or substantially all of the FR regions correspond to the sequence of a human antibody. In some cases, a humanized antibody may comprise at least a fragment of the constant region of an antibody derived from a human antibody. A humanized form of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term hypervariable region or HVR refers to each region of the variable domain of an antibody that possesses hypervariable sequences (complementarity-determining regions or CDRs) and/or forms structurally defined loops (hypervariable loops) and/or contains residues that contact an antigen (antigenic contacts). Antibodies typically contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring 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));

(c) antigen contacts occurring with 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));

(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).

HVR residues can be identified on websites such as https://www.novopro.cn/tools/cdr.html.

Unless otherwise noted, HVR residues and other residues in the variable domain (e.g., FR

- 5 050412 residues) in this document are numbered according to Kabat et al., supra.

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, with the exception of possible variant antibodies, such as those containing natural mutations or arising during production of the monoclonal antibody, such variants being present in small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody from a monoclonal antibody preparation is directed against a single determinant of an antigen. Thus, the adjective "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies; it should not be interpreted as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be produced by a variety of methods, including, but not limited to, the hybridoma method, recombinant DNA techniques, phage display techniques, and methods using transgenic animals containing all or part of the human immunoglobulin loci, such methods and other typical methods for producing monoclonal antibodies are described herein.

The term variable region or variable domain refers to the domain of the heavy or light chain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al. Kuby Immunology, ^6th ed., W. H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by using the VH or VL domain of an antibody that binds to 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).

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes both a vector as a self-replicating nucleotide structure and a vector incorporated into the genome of the host cell into which it is introduced. Some vectors are capable of directing the expression of nucleic acids to which they are functionally linked. Such vectors are referred to herein as expression vectors.

II. Exemplary antibodies

The present invention provides an isolated monoclonal antibody, particularly an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises 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. In one embodiment, said antibody comprises the VH as shown in SEQ ID NO: 1 and the VL as shown in SEQ ID NO: 2. Optionally, the first two N-terminal amino acid residues of the VH are absent.

The present invention provides an isolated monoclonal antibody, particularly an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises 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. In one embodiment, said antibody comprises the VH as shown in SEQ ID NO: 3 and the VL as shown in SEQ ID NO: 4. Optionally, the first two N-terminal amino acid residues of the VH are absent.

The present invention provides an isolated monoclonal antibody, particularly with an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises 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. The present invention provides an isolated monoclonal antibody, particularly with an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises a VH comprising HVRH1 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. The present invention provides an isolated monoclonal antibody, particularly an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises 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

- 6 050412

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. The present invention provides an isolated monoclonal antibody, particularly an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises 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. In one embodiment, said antibody comprises a VH as shown in SEQ ID NO: 5, and VL as shown in SEQ ID NO: 6. Optionally, the first two N-terminal amino acid residues of VH are missing.

The present invention provides an isolated monoclonal antibody, particularly an Fc engineered antibody, that specifically binds to human CLDN18.2, wherein the antibody comprises 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. In one embodiment, said 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.

In one embodiment, the one or more mutations in the Fc region are one or more mutations that modify, for example, increase or decrease binding to an Fc receptor and/or effector function, for example, ADCC (antibody-dependent cellular cytotoxicity) and/or CDC (complement-dependent cytotoxicity). 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.

In one embodiment, the anti-CLDN18.2 monoclonal antibody of the invention is a murine, chimeric or humanized antibody.

In one embodiment, the monoclonal antibody to CLDN18.2 of the invention is an antigen-binding fragment of an antibody, optionally selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an scFv fragment, and a diabody.

In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention is a full-length antibody. In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention comprises a human IgG heavy chain constant region, in particular IgG1, optionally as shown in SEQ ID NO: 9. In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention comprises a mutated human IgG heavy chain constant region, in particular IgG1, optionally as shown in SEQ ID NO: 40. In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention comprises a human kappa light chain constant region, optionally as shown in SEQ ID NO: 10. In one embodiment, an anti-CLDN18.2 monoclonal antibody of the invention is a chimeric antibody, such as a mouse/human chimeric antibody. In one embodiment, the anti-CLDN18.2 monoclonal antibody of the invention has an engineered Fc.

In one embodiment, the monoclonal antibody (mAb) or Fab fragment of the present invention has a crossover format (x-mAb or x-Fab), wherein either the variable domains or the (first) constant domains of the light and heavy chains are exchanged.

III. Recombinant methods and compositions

Antibodies can be produced using recombinant techniques and compositions, such as those described in U.S. Patent No. 4,816,567. These methods provide one or more isolated nucleic acids encoding the antibody.

For 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. These nucleic acids encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of the antibody (e.g., the light and/or heavy chains of the antibody). These nucleic acids may be present on the same expression vector or on different expression vectors.

In one embodiment, isolated nucleic acids encoding an antibody used in the methods described herein are provided.

In a further embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acid(s) are provided.

In a further embodiment, a host cell comprising such nucleic acid(s) is provided.

In one such embodiment, the host cell comprises (e.g., is transformed with):

(1) a vector containing nucleic acids encoding an amino acid sequence containing a VL antibody and an amino acid sequence containing a VH antibody, or

- 7 050412 (2) a first vector containing a nucleic acid encoding an amino acid sequence containing a VL antibody, and a second vector containing a nucleic acid encoding an amino acid sequence containing a VH antibody.

In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a lymphoid cell (e.g., a YO, NSO, Sp2/0 cell). In one embodiment, a method for producing an antibody is provided, comprising culturing a host cell containing nucleic acids encoding the antibody, as described above, under conditions suitable for expression of the antibody, and optionally isolating the antibody from the host cell or the cultural environment of the host cell.

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

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, 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, which describes 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.

In addition to prokaryotes, suitable hosts for cloning or expression of antibody-encoding vectors include eukaryotic microorganisms such as filamentous fungi or yeast, including fungal and yeast strains with humanized glycosylation pathways that result in the production of antibodies with partially or completely human glycosylation patterns. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409–1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210–215.

Host cells suitable for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

In addition, plant cell cultures can be used as hosts. See, for example, US 5959177, US 6040498, US 6420548, US 7125978, and US 6417429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Additionally, vertebrate cells can be used as hosts. For example, mammalian cell lines adapted to growth in suspension can be used. Other examples of suitable mammalian host cell lines are the SV40-transformed monkey kidney line CV1 (C0S-7); the human embryonic kidney line (293 or 293 cells, as described, for example, 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, for example, in Mather, J.P., Biol. Reprod. 23 (1980) 243-251); 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 cells (MMT 060562); TRI cells, as described, for example, by Mather et al., Annals N.Y. Acad. Sci. 383 (1982) 44–68; MRC 5 cells; and FS4 cells. Other suitable 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. A review of some lines mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

IV. Analyses

The antibodies provided herein can be identified, screened, or examined for their physical/chemical properties and/or biological activity using various assays known in the art.

Binding and other assays

In one aspect, the antigen-binding activity of the antibody according to the invention was examined, for example, using known methods such as ELISA (enzyme-linked immunosorbent assay), Western blotting, etc.

In another aspect, competitive assays can be used to identify an antibody,

- 8 050412 that competes with aCLDN18.2 for binding to CLDN18.2. In some embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) that is bound by CLDN18.2. Detailed examples of methods for mapping the epitope to which an antibody binds are provided in Morris (1996) Epitope Mapping Protocols, in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized CLDN18.2 was incubated in a solution containing a primary labeled antibody that binds to CLDN18.2 and a second unlabeled antibody that was tested for its ability to compete with the primary antibody for binding to CLDN18.2. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CLDN18.2 was incubated in a solution containing the primary labeled antibody but not the second unlabeled antibody. After incubation under conditions favorable for binding of the primary antibody to CLDN18.2, excess unbound antibody was removed, and the amount of label bound to immobilized CLDN18.2 was measured. If the amount of label bound to immobilized CLDN18.2 is significantly reduced in the test sample compared to the control sample, this indicates that the second antibody is competing with the first antibody for binding to CLDN18.2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Activity analysis

In one aspect, assays are provided for identifying anti-CLDN18.2 antibodies exhibiting biological activity. Biological activity can include, for example, the effect of anti-CLDN18.2 antibodies on ADCC by PBMC on target cells expressing CLDN18.2. Antibodies exhibiting such biological activity in vivo and/or in vitro are also provided.

Examples

After initial screening, four positive hybridoma cell lines from immunized mice were identified with specific binding to CLDN18.2-expressing cells but not to CLDN18.1-expressing cells.

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

This example illustrates the cloning of the H and L chain genes (H - heavy, L - light) of an antibody from mouse hybridoma cells to obtain variable region sequences specific for CLDN18.2 to generate chimeric antibodies.

Following standard procedures in the art, RNA was isolated and purified from hybridoma cells using the Quick-RNA™ Microprep Kit (ZYMO Research, Cat. No. R1050). First-strand cDNA was synthesized and RACE was performed using the SMARTer® RACE 573' Kit (Takara Bio USA, Inc. Cat. No. 634858) with IgG1 3' Constant Primer (SEQ ID NO: 35), IgG2a 3' Constant Primer (SEQ ID NO: 36), and Kappa 3' Constant Primer (SEQ ID NO: 37). RACE DNA products were gel extracted using the NuceloSpin Gel and PCR Clean-Up kit (Takara, Cat. No. 740986.20). The fusion reaction mixture of the linearized pRACE vector and the gel-purified PACE product was transformed into competent Stellar cells (Clontech, cat. #636766). Plasmid DNA was isolated from transformants using the QIAprep Spin Miniprep Kit (Qiagen, cat. #27104) and subjected to Sanger sequencing (GENEWIZ) using M13 sequencing primers. The final gene sequences of four pairs of heavy and light chains (data not shown) were obtained and are defined below as mAb CLDN18.2 (mAb1, mAb2, mAb3, and mAb4).

Example 2. Construction of chimeric antibodies to replace the mouse constant region with the human constant region

This example illustrates the construction of chimeric antibodies by replacing the constant regions of 4 molecularly cloned mouse mAbs with constant regions from human IgG1 heavy chain and kappa light chain.

Plasmid pFUSE-CHIg-hG1 (InvivoGen, Cat# pfuse-hchg1), which contains the human IgG1 heavy chain constant region (SEQ ID NO: 9), and plasmid pFUSE2-CLIg-hk (InvivoGen, Cat# pfuse2-hclk), which contains the human kappa light chain constant region (SEQ ID NO: 10), were digested with HindIII and either NheI for IgG1 or BsiWI for kappa (all from NEB lab). Linearized plasmids were purified by gel purification using NucleoSpin gel and PCR purification kit (Takara, Cat# 740986.20). The coding sequences for the variable regions of the heavy and light chains of mouse mAb1, mAb2, mAb3, and mAb4 were amplified by PCR 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 Purification Kit (Takara, Cat. No. 740986.20). The linearized vector and insert fragment were assembled using the Gibson Assembly® HiFi 1 Step Kit (SGI (VWR), Cat. No. GA1100-50). The assembly reaction mixture was transformed into Stellar competent cells (Clontech, Cat. No. 636766). Plasmid DNA

- 9 050412 was isolated from transformants using the QIAprep Spin Miniprep Kit (Qiagen, Cat. No. 27104) and subjected to Sanger sequencing (GENEWIZ). Four chimeric antibodies were constructed, each incorporating the variable region of one of the four mouse mAbs obtained in Example 1. The new chimeric antibodies are designated YL-G1-19-01, YL-G1-19-02, YL-G1-19-03, and YL-G1-19-04.

Example 3. Confirmation of the specificity of these four chimeric monoclonal antibodies by surface staining

This example illustrates the test of 4 chimeric monoclonal antibodies with their binding specificity to CLDN18.2 compared to a reference mAb (IMAB362, Ganymed).

CLDN18.2-expressing 293T cells and CLDN18.1-expressing 293T cells at a density of 2x106 cells/mL in FACS buffer (50 μL) were mixed with a dilution series (60.00, 20.00, 6.67, 2.22, 0.74, 0.25, 0.08 μg/mL in FACS buffer) of chimeric antibodies, reference antibody, or negative control IgG (50 μL) in a 96-well V-bottom plate and incubated for 30 min on ice. After washing with 200 µl/well of FACS buffer, cells were resuspended in 30 µl/well of Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG secondary antibody, Fcy fragment specific (Jackson, cat. #109-605-098) and incubated for 20 min on ice. After three washes with 200 µl/well of FACS buffer, cells were resuspended in 150 µl/well of FACS buffer and subjected to FACS using a BD LSR II flow cytometer (HTS). Data were analyzed using the geometric mean and plotted using Prism GraphPad. Flow cytometry analysis showed that the four chimeric antibodies had higher specific binding to cells expressing CLDN18.2 compared to the reference mAb (see Fig. 2). Binding to cells expressing CLDN18.1 was not shown (see Fig. 3), demonstrating the high specificity of these four chimeric antibodies.

Example 4. Identification of the functional activity of these four chimeric antibodies using ADCC assay

This example illustrates the ADCC-mediated killing activity test of these 4 chimeric antibodies compared to a reference mAb (IMAB362, Ganymed).

CLDN18.2-expressing 293T cells and CLDN18.1-expressing 293T cells were labeled with CFSE using eBioscience™ CFSE (Thermo, Cat. No. 65-0850-84). CFSE-labeled cells at a density of 4x105 cells/mL in Lympholyte® cell separation medium (Cedarlane, Cat. No. CL5110) (50 µL) were mixed with serial dilutions (20.00, 6.67, 2.22, 0.74, 0.25, 0.08, 0.03 µg/mL in medium) of chimeric antibodies, reference antibody, or negative control IgG (100 µL) in a 96-well V-bottom plate and incubated for 15 min at room temperature in the dark. PBMCs at a density of 5x106 cells/mL (50 µL) were then added and the plate was incubated for 2 h at 37°C in the dark. Cells were washed twice with PBS, and 100 µL of eBioscience™ Fixable Viability Dye eFluor™ 660 working solution (Thermo, cat. #650864) were added per well. The plate was incubated for 30 min on ice in the dark. 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). Percent kill (CFSE/FVD-AF660 double-positive population versus CFSE-positive population) was used for data analysis, and graphs were generated using Prism GraphPad. ADCC activity by FACS-based method showed that the 4 chimeric antibodies could mediate ADCC activity against CLDN18.2-expressing cells (see Fig. 4).

Example 5. Characterization of chimeric antibodies constructed on the basis of Fc

In addition, four chimeric antibodies with modified Fc were constructed. Compared with the ^1st generation parental chimeric antibodies YL-G1-19-01, YL-G1-19-02, YL-G1-19-03 and YL-G119-04, the ^2nd generation chimeric antibodies YL-G2-A, YL-G2-B, YL-G2-C and YL-G2-D, with engineered Fc (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, namely L235V, F243L, R292P, Y300L and P396L, according to EU numbering. The mutant constant region (including CH1, hinge, CH2, and CH3) of the human IgG1 heavy chain is shown in SEQ ID NO: 40.

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

5.1: Antigen-antibody binding interaction.

This example illustrates the characteristics of the antigen-antibody binding interaction of YLG2-B, YL-G2-C, and YL-G2-D.

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

To determine affinity using the KinExA method, serial dilutions of binding partner B (known as the titrant) were performed in a background of binding partner A (known as the constant binding partner, CBP). This means that CBP will remain in constant concentration.

- 10 050412 concentrations, while the titrant will differ in concentration. Once these solutions have reached equilibrium, the KinExA 4000 instrument can directly measure the amount of unbound or free-form CBP remaining in the solutions. Using Sapidyne software, the percentage of free CBP can be plotted against the total titrant concentration to generate a binding curve and determine affinity.

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

The KinExA 4000 instrument was also used to measure the binding affinity of antibodies to the surface protein of intact cells, i.e., CHO cells overexpressing huCLDN18.2. The antibody concentration was maintained constant (CBP), while the concentration of whole cells with surface protein was varied (titrant). The concentration of whole cells with surface proteins was diluted threefold. The titrated cells were incubated with a constant binding partner (CBP). After reaching equilibrium, the samples were centrifuged, the supernatants were recovered, and free CBP was detected using a fluorescently labeled anti-CBP molecule. The binding curves are shown in Fig. 6.

To establish a more accurate Kd (equilibrium dissociation constant), two equilibrium curves were prepared and analyzed. One curve with a low CBP concentration at 100 pM (200 pM binding sites) and ¹⁰⁶ cells/mL with three-fold dilutions, and one curve with a high CBP concentration at 10 nM (20 nM binding sites) and ¹⁰⁶ cells/mL with three-fold dilutions. The amount of unbound CBP in solution was measured using the KinExA 4000. The analysis was performed using Sapidyne Instruments n-curve analysis software version 4.4.26. A summary of the equilibrium dissociation constant Kd is given in Table 1.

Table 1. Kd values of YL-G2-B, YL-G2-C and YL-G2-D

sh Binding to recombinant huCLDN18.2-Fc (nM) Binding to CHO cells overexpressing huCLDN18.2 (pM) YL-G2-B 1.7 9.4 YL-G2-C 3.8 38.6 YL-G2-D 6.4 13.1

5.2: Cell Binding Assay

This example illustrates the binding analysis of YL-G2-B, YL-G2-C, and YL-G2-D cells.

To assess antibody binding, CHO cells expressing huCLDN18.2 were used. Cells were seeded at 5x105 cells/100 µl per well of a 96-well plate for each sample. Then, 100 µl of each serially diluted antibody was added to the cells, each dilution starting at 40 µg/ml and diluted 5-fold. Thus, the final concentration of each antibody started at 20 µg/ml followed by 5-fold serial dilutions. After 1 h, the cells were washed twice, and 100 µl of GAH-FITC (GAH - goat anti-human, FITC-fluorescein isothiocyanate) (1:200 dilution) were added to each well. After 30 min, the cells were washed twice and resuspended in 120 µl of FACS buffer. Cells were analyzed by flow cytometry.

Unstained cells were used as a reference for setting overall gating of target cells and for identifying FITC-negative populations, allowing us to establish a FITC-positive cell gate for each cell line. Additionally, the mean fluorescence intensity (MFI) of the entire cell population was calculated for secondary validation of FITC-positive results. The ratio of the number of gated positive cells to the total number of viable cells is taken as the percentage of positive cells. The results are shown in Fig. 7.

5.3: Complement-Dependent Cytotoxicity (CDC) Assay

This example illustrates the complement-dependent cytotoxicity (CDC) assay of YL-G2-B, YLG2-C, and YL-G2-D.

Target cells (i.e., CHO cells expressing huCLDN18.2) were washed once with DPBS (Dulbecco's phosphate-buffered saline). Cells were then seeded in a U-bottom plate at 2 x 104 cells, 100 μl/well in RPMI. Antibodies were serially diluted 1:2 and coincubated with cells at 50 μl/well for 15 min at room temperature. Then, 20% pooled serum was added to all wells, including the spontaneous release and maximum release wells, at 50 μl/well. The plate was incubated in an incubator at 37°C for 3.5 h. Forty-five min before the end of the incubation period, the plate was centrifuged at 1200 rpm.

- 11 050412 for 5 min and 20 µl of lysis buffer (CyQUANT™ LDH Cytotoxicity Assay Kit, Cat. No. C20300 and C20301) was added to the maximum release control well containing target cells only. Fifty µl of supernatant was transferred to a black-walled 96-well plate along with 50 µl/well of reaction buffer (CyQUANT™ LDH Cytotoxicity Assay Kit, Cat. No. C20300 and C20301), which was added to all wells as well as the maximum release and spontaneous release wells. The plate was incubated in the dark for 30 min. At the end of the incubation, 50 µl of stop solution (CyQUANT™ LDH Cytotoxicity Assay Kit, Cat. No. C20300 and C20301) was added to all wells and mixed gently by tapping. OD (optical density) was measured at 490 nm and 680 nm. Activity was represented as percent cytotoxicity: % cytotoxicity = (experimental value - spontaneous target cell release, without volume correction)/(maximum target cell release - spontaneous target cell release, volume correction) x 100. The results are shown in Fig. 8.

5.4: Internalization Analysis

This example illustrates the analysis of internalization of YL-G2-B, YL-G2-C and YL-G2-D.

CHO cells expressing huCLDN18.2 were obtained at 5 x 105/ml in DMEM (Dulbecco's Modified Eagle's Minimum Essential Medium). One hundred µl of cells were seeded into each well of a 96-well U-bottom plate. Test antibodies and control antibodies were diluted to 40 µg/ml, then each antibody was serially diluted in culture medium at a ratio of 1:4. Each diluted antibody was added to the cells at 50 µl/well. The plate was incubated at 37°C for 30 min. Then, 40 μg/mL PEP-ZAP (a small Fc-binding peptide fused to a cell-toxic peptide developed by AB Studio Inc.; see WO 2020/018732 A1) was added to each well at 50 μL/well to obtain a final PEP-ZAP concentration of 10 μg/mL. The plate was incubated at 37°C for 72 h. Finally, the cells were centrifuged, and 100 μL of the supernatant was collected for LDH measurement. The results are shown in Fig. 9.

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

This example illustrates the antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

To evaluate the ADCC function of antibodies, target (CHO cells expressing huCLDN18.2) and effector cells (NK 8837-F cells, ATCC PTA-8837) were used. Target cells were seeded at ^2x104 cells per 50 μl per well of a 96-well plate for each sample in DMEMF12 + 10% FBS (fetal bovine serum). Then, 100 μl of each serially diluted antibody 1:10 were added to the cells. After 20 min, NK cells were added to the plate at ^2x105 cells per 50 μl, resulting in a target:effector ratio of 1:10. After this addition, the final concentration of each antibody started at 50 μg/ml and then 5, 0.5, and 0.05 μg/ml. The plate was placed in _{a CO2} incubator at 37°C for 24 h. The cells were then stained with 7AAD, washed twice, and resuspended in approximately 200 µl of FACS buffer. The cells were analyzed by flow cytometry.

Unstained cells were used as a reference to set up global gating of target cells and to generate 7AAX-negative populations, which allowed differentiation between 7AAD (dead cells) and live cells.

CT26 cells expressing huCLDN18.2 can also be used as target cells, and ADCC can also be determined by LDH.

A comparison of ADCC activity between YL-G1-02 and YL-G2-B (which have the same amino acid sequence except for the VLPLL substitutions in the Fc region) is shown in Fig. 10. A summary of EC ₅₀ information is shown in Table 2.

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

ID LDH FACS EC50 (nM) Max (%) EC50 (nM) Max (%) YL-G1-02 0.30 29.8 -0.2244 32.0 YL-G2-B 0.012 55.1 0.0073 44.1 Isotype NA (not applicable) 1.5 NA 8.3

Example 6. Functional characteristics of antibodies

6.1: Cells

CT26 CLDN18.2 cells (mouse colon cancer cells, Kyinno biotechnology, Cat. No. KC-1195) maintained in DMEM (Gibco, Cat. No. 31053-036) containing 10% FBS (ExCell Bio, Cat. No. FND500), KATOIII CLDN18.2 cells (human gastric cancer cells, Kyinno biotechnology, Cat. No. KC1453) maintained in RPMI1640 (RPMI - Roswell Park Cancer Institute) medium (Gibco, Cat. No. 22400-089) containing 10% FBS and NCI-N87 CLDN18.2 cells (human gastric cancer cells, Kyinno biotechnology, Cat. No. KC-1222) maintained in RPMI1640 containing 10% FBS represent all tumor cells overexpressing human CLDN18.2 and are used

- 12 050412 was used to determine the CDC activity and ADCC activity of the subject's antibodies.

6.2: CDC Analysis

The activity of the subject's CDC antibodies was assessed by measuring the change in the level of LDH released into the culture medium after cell lysis. CT26 CLDN18.2 cells or KATOIII CLDN18.2 cells were suspended in RPMI 1640 medium without phenol red (Gibco, cat. no. 11835-030) containing 1% FBS at a density of 4E+05 cells/mL or 6E+05 or 1E+06 cells/mL, respectively. The antibodies in question were diluted in RPMI 1640 medium without phenol red containing 1% FBS to 200, 50, 12.5, 3.13, 0.78, 0.195, 0.0488, 0.0122, 0.00305, 0.000763, and 0.000191 nM. Normal human serum complement (Quidel, cat. no. A113) was diluted in RPMI 1640 medium without phenol red containing 1% FBS at 1:50. To each round-bottomed well of a 96-well microplate (Corning, cat. #3799), 50 μl of antibody diluent, 50 μl of normal human serum complement diluent, and 50 μl of tumor cell suspension were added. Human IgG1 isotype antibody was included as a negative control. The reference antibody (IMAB362, Ganymed) was included as a positive control. The microplate was incubated for 3-4 hours in an incubator set at 37°C and 5% CO ₂ . After incubation, LDH release into the cell culture supernatant was detected according to the instructions provided with the LDH cytotoxicity assay kit (Roche, cat. #11644793001). Briefly, the microplate was centrifuged (Eppendorf model 5810R) at 1500 rpm for 5 minutes, and 70 μl of supernatant from each well was removed and transferred to a well on a new microplate. Fifty μl of LDH detection substrate was then 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 detected using a SpectraMax M5e (Molecular Devices LLC) minus the optical density at 690 nm (OD _{492 nm} – OD _{690 nm} ). The CDC activity of the subject's antibodies was calculated from the percentage of specific cell lysis using the following formula:

Specific cell lysis (%) = (OD antibody + complement + tumor cell OD complement + tumor cell) * 100/(OD tumor cell + Triton - OD tumor cell).

Data were analyzed using four-parameter nonlinear regression using GraphPad Prism 7 software and EC50 value was calculated and obtained.

6.3: ADCC Analysis

The ADCC activity of the subject's antibodies was assessed by measuring changes in the level of LDH released into the culture medium after cell lysis. NCI-N87 CLDN18.2 cells or KATOIII CLDN18.2 cells were suspended in RPMI 1640 medium without phenol red at a density of 6E+05 cells/mL. The antibodies in question were diluted in RPMI 1640 medium without phenol red containing 1% FBS to 20, 4, 0.8, 0.16, 0.032, 0.0064, 1.28E-03, 2.56E-04, 5.12E-05, 1.02E-05, 2.05E-06, 4.10E-07, 8.19E-08, 1.64E-08, 3.28E-09, 6.55E-10, 1.31E-10, 2.62E-11 and 5.24E-12 nM. Fresh human PBMCs (Saily, from volunteer # XC11057W) were suspended in RPMI 1640 medium without phenol red containing 1% FBS at a density of 1.2E+07 cells/mL. A 96-well microplate, 50 μL of antibody diluent, 50 μL of human PBMC cell suspension, and 50 μL of tumor cell suspension were added to each round-bottom well of the plate. Human IgG1 isotype was included as a negative control. The reference mAb (IMAB362, Ganymed) was included as a positive control. The microplate was incubated for 4-6 hours in an incubator set at 37°C and 5% CO2. Following incubation, LDH release into the cell culture supernatant was detected according to the instructions provided with the LDH cytotoxicity assay kit, as described above. The ADCC activity of the subject's antibodies was calculated from the percentage of specific cell lysis using the formula below:

Specific cell lysis (%) = (OD antibody + PBMC + tumor cell ^- OD PBMC + tumor cell) * 100 / (OD tumor cell + Triton OD tumor cell)

Data were analyzed using four-parameter nonlinear regression using GraphPad Prism 7 software and EC50 value was calculated and obtained.

6.4: Effect of CDC on CT26 CLDN18.2 cells by LDH assay

As shown in Fig. 11 and Table 3, the two lots of YL-G2-B showed comparable CDC effect on CT26 CLDN18.2 cells (Fig. 11, panel A). YL-G1-19-02, YL-G2-B, YL-G1-19-03, YL-G2C, YL-G1-19-04, and YL-G2-D showed stronger CDC effect on CT26 CLDN18.2 cells than the positive control (Fig. 11).

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Table 3. Effect of CDC on CT26 CLDN18.2 cells

Antibody Concentration (mg/ml) Purity (%) CT26 CLDN18.2 EC ₅ o (nM) Max. (% cytotoxicity) Positive control, series A 20.63 96.80% -11.54 72.52 YL-G1-19-02 0.74 97.30% 0.26 73.44 YL-G2-B lot 1 18.96 97.90% 0.14 75.99 YL-G2-B lot 2 50 98.70% 0.12 74.74 hIgGl_H3OTnn, series A 13.6 99.40% NA 10.80 Positive control, series B 20.63 96.80% -10.70 54.74 YL-G1-19-03 10.73 96.00% 0.24 62.21 YL-G2-C 5.55 93.22% 0.10 65.24 hIgGl_H3OTnn, series B 13.6 99.40% NA -0.23 Positive control, series C 20.63 96.80% -11.86 51.65 YL-G1-19-04 9.03 98.00% 0.19 59.21 YL-G2-D 4.37 98.01% 0.12 71.85 hIgGl_H3OTnn, series C 13.6 99.40% NA -0.38

6.5: Effect of CDC on KATOIII CLDN18.2 cells by LDH assay

As shown in Fig. 12 and Table 4, the two lots of YL-G2-B showed comparable CDC effect on CT26 CLDN18.2 cells (Fig. 12, panel A). YL-G1-19-02, YL-G2-B, YL-G1-19-03, YL-G2C, YL-G1-19-04, and YL-G2-D showed stronger CDC effect on KATOIII CLDN18.2 cells than the positive control (Fig. 12).

Table 4. Effect of CDC on KATOIII CLDN18.2 cells

Antibody Concentration (mg/ml) Purity (%) KATOIII CLDN18.2 EC ₅ o (nM) Max. (% cytotoxicity) Positive control, series A 20.63 96.80% -38.08 29.71 YL-G1-19-02 0.74 97.30% 1.95 35.43 YL-G2-B lot 1 18.96 97.90% 1.24 39.65 YL-G2-B lot 2 50 98.70% 1.33 39.42 hlgGl Isotype, series A 13.6 99.40% NA -0.85 Positive control, series B 20.63 96.80% -22.73 45.96 YL-G1-19-03 10.73 96.00% -4.98 67.00 YL-G2-C 5.55 93.22% -6.04 74.56 hlgGl Isotype, series IN 13.6 99.40% NA -2.46 Positive control, series C 20.63 96.80% -27.48 34.89 YL-G1-19-04 9.03 98.00% 4.76 48.32 YL-G2-D 4.37 98.01% 2.40 49.19 hlgGl Isotype, series C 13.6 99.40% NA -1.53

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6.6: Effect of ADCC on KATOIII CLDN18.2 cells by LDH assay

As shown in Fig. 13 and Table 5, the two lots of YL-G2-B showed comparable ADCC effect on KATOIII CLDN18.2 cells (Fig. 12, panel A). YL-G2-B (EC50 = 0.028 nM or 0.018 nM for different lots), YL-G2-C (EC50 = 0.019 nM), and YL-G2-D (EC50 = 0.021 nM) showed a stronger ADCC effect (lower EC50), and YL-G1-19-02 ( _EC50 = 0.16 nM), YL-G1-19-03 ( _EC50 = 0.21 nM), and YL-G1-19-04 ( _EC50 = 0.14 nM) showed a comparable ADCC effect on KATOIII CLDN18.2 cells compared to the positive control (ECso = 0.12 nM, 0.22 nM, or 0.27 nM for three cycles) (Fig. 13).

Table 5. Effect of ADCC on KATOIII CLDN18.2 cells

Antibody Concentration (mg/ml) Purity (%) KATOIII CLDN18.2 EC50 (nM) Max. (% cytotoxicity) Positive control, series A 20.63 96.80% 0.12 27.89 YL-G1-19-02 0.74 97.30% 0.16 19.90 YL-G2-B lot 1 18.96 97.90% 0.028 19.90 YL-G2-B lot 2 50 98.70% 0.018 15.78 hIgGl_H3OTHn, series A 13.6 99.40% NA oh, s Positive control, series B 20.63 96.80% 0.22 24.46 YL-G1-19-03 10.73 96.00% 0.21 16.72 YL-G2-C 5.55 93.22% 0.019 17.57 hlgGl HsoTHn, B series 13.6 99.40% NA -5.51 Positive control, series C 20.63 96.80% 0.27 24.54 YL-G1-19-04 9.03 98.00% 0.14 15.44 YL-G2-D 4.37 98.01% 0.021 18.42 hIgGl_H3OTHn, series C 13.6 99.40% NA -7.64

6.7: Effect of ADCC on NCI-N87 CLDN18.2 cells using LDH assay

As shown in Fig. 14 and Table 6, the two lots of YL-G2-B showed comparable ADCC effect on NCI-N87 CLDN18.2 cells (Fig. 14, panel A). YL-G2-B (EC50 = 0.0067 nM or 0.012 nM for different lots), YL-G2-C ( _EC50 = 0.0078 nM), and YL-G2-D (EC50 = 0.0089 nM) showed stronger ADCC effect (lower EC50), and YL-G1-19-02 ( _EC50 = 0.072 nM), YL-G1-19-03 ( _EC50 = 0.13 nM), and YL-G1-19-04 ( _EC50 = 0.067 nM) showed comparable ADCC effect on KATOIII CLDN18.2 cells compared to the positive control (EC50 = 0.057 nM, 0.082 nM, or 0.10 nM for the three cycles) (Fig. 14).

Table 6. Effect of ADCC on NCI-N87 CLDN18.2 cells

Antibody Concentration (mg/ml) Purity (%) NCI-N87 CLDN18.2 EC ₅ o (nM) Max. (% cytotoxicity) Positive control, series A 20.63 96.80% 0.057 26.05 YL-G1-19-02 0.74 97.30% 0.072 16.80 YL-G2-B lot 1 18.96 97.90% 0.0067 19.04 YL-G2-B lot 2 50 98.70% 0.012 21.05 hIgGl_H3OTHn, series A 13.6 99.40% NA -6.73

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Positive control, series B 20.63 96.80% 0.082 22.06 YL-G1-19-03 10.73 96.00% 0.13 15.21 YL-G2-C 5.55 93.22% 0.0078 14.93 hlgGl HsoTHn, B series 13.6 99.40% NA -11.72 Positive control, series C 20.63 96.80% 0.10 21.11 YL-G1-19-04 9.03 98.00% 0.067 12.59 YL-G2-D 4.37 98.01% 0.0089 18.60 hlgGl HsoTHn, C series 13.6 99.40% NA -9.22

6.8: SPR (Surface Plasmon Resonance)

The binding affinity of the antibodies in question was determined by SPR in accordance with 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, using the Human Antibody Capture Kit, type 2 (Cytiva, cat. no. 29234600). Briefly, anti-human IgG (Fc) antibody was diluted to 25 μg/mL in capture buffer and injected onto a Sensor CM5 S-Series chip (Cytiva, cat. #BR100530) at a flow rate of 10 μL/min for 6 min to yield approximately 7,000-14,000 response units (RU) of bound secondary antibody. The antibody was then diluted to 5 μg/mL in running buffer and injected at a flow rate of 10 μL/min to yield approximately 200 RU of bound primary antibody. For kinetic measurements, two-fold serial dilutions (0.195–50 nM) of His-tagged human claudin-18.2 were injected at a flow rate of 30 μl/min and binding was monitored for 120 s for association and 300 s for dissociation on a Biacore 8K (Cytiva). Association rates (ka) and dissociation rates (kd) were calculated using a simple one-to-one binding model by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) was calculated as the ratio kd/ _ka . The results are shown in Table 7 below.

Table 7

The antibody in question Capture Level (RU) to _a (1/Ms) k _d (1/s) K _D (M) Yamax (RU) 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,PE-09 177.7 3.85 YL-G2-D 169.4 7.07E+05 7.22E-04 1.02E-09 187.2 3.67

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Sequence table

SEQ Ш NO Description Subsequence 1 YL-G1-19-01 VH QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLI EWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGK ATLTADKSSNTAYMQLSSLTSEDSAVYFCARVYYG NSFGYWGQGTLVTVSA 2 YL-G1-19-01 VL DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQ KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFT GSGSGTDFTLTISSVQAEDLAVYYCQNEYFYPFTF GSGTKLEIK 3 YL-G1-19-02 VH EVQLQQSGPELEKPGASVKISCKASGYSFTGYKM NWVKQSNGKSLEWIGNIDPYYGGTTYNQKFKGK ATLTVDKSSSTAYMQLKSLTSEDSAVYYCARYGK GNTMDYWGQGTSVTVSS 4 YL-G1-19-02 VL DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQ KNYLTWYQQRPGQPPKLLIYWASTRESGVPVRFT GSGSGTDFTLTISSVQAEDLAVYYCQNAYIYPLTF GTGTKLELK 5 YL-G1-19-03 VH QIQLVQSGPELRKPGETVKISCKASGFPFTTDGMS WVKQAPGKGLKWMGWINTYSGVPTYADDFKGR VAF SLETS ASTAYLQIKNLKNEDTAT YFC ARFRRG NALDNWGQGTS VT VS S 6 YL-G1-19-03 VL DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQ KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFT

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GSGSGTDFTLTISSVQAEDLAIYYCQNNYFYPLTF GAGTRLELK 7 YL-G1-19-04 VH EVQLQQSGPELEKPGASVKISCKASGYSFTGYKM NWVKQSNGESLEWIGNIDPYYGDTTYTQKFKGK ATFTVDTSSSTAYMQLKSLTSEDSAVYFCARYNRG NTMDYWGQGTSVTVSS 8 YL-G1-19-04 VL DIVMTQSPSSLTVTAGEKVTLSCKSSQSLLNSGNQ KNYLTWYQQRPGQPPKLLIYWASTRESGVPVRFT GSGSGADFTLTIS S VQAEDLAVYFCQNAYF YPLTF GTGTKLELR 9 Human IgGl CH ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK 10 Human Kappa CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 11 YL-G1-19-01 HVR-H1 GYAFTNYL 12 YL-G1-19-01 HVR-H2 INPGSGGT 13 YL-G1-19-01 HVR-H3 ARVYYGNSFGY 14 YL-G1-19-01 HVR-L1 QSLLNSGNQKNY 15 YL-G1-19-01 HVR-L2 WAS

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16 YL-G1-19-01 HVR-L3 QNEYFYPFT 17 YL-G1-19-02 HVR-H1 GYSFTGYK 18 YL-G1-19-02 HVR-H2 IDPYYGGT 19 YL-G1-19-02 HVR-H3 ARYGKGNTMDY 20 YL-G1-19-02 HVR-L1 QSLLNSGNQKNY 21 YL-G1-19-02 HVR-L2 WAS 22 YL-G1-19-02 HVR-L3 QNAYIYPLT 23 YL-G1-19-03 HVR-H1 IMGT GFPFTTDG 24 YL-G1-19-03 HVR-H2 IMGT INTYSGVP 25 YL-G1-19-03 HVR-H3 IMGT ARFRRGNALDN 26 YL-G1-19-03 HVR-L1 IMGT QSLLNSGNQKNY 27 YL-G1-19-03 HVR-L2 IMGT WAS 28 YL-G1-19-03 HVR-L3 IMGT QNNYFYPLT 29 YL-G1-19-04 HVR-H1 GYSFTGYK 30 YL-G1-19-04 HVR-H2 IDPYYGDT 31 YL-G1-19-04 HVR-H3 ARYNRGNTMDY 32 YL-G1-19-04 HVR-L1 QSLLNSGNQKNY

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33 YL-G1-19-04 HVR-L2 WAS 34 YL-G1-19-04 HVR-L3 QNAYFYPLT 35 3'-constant primer IgGl GATTACGCCAAGCTTTCATTTACCAGGAGAGTG GGAGAGGCTC 36 3'-constant IgG2a primer GATTACGCCAAGCTTTCATTTACCCGGAGTCCGG GAGAAGCTC 37 3'-constant kappa primer GATTACGCCAAGCTTTCAACACTCATTCCTGTTG AAGCTCTTG 38 huCLDN18.2 (261 aa (amino acid)) MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDL YNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTL LGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIR IGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFA NMLVTNFWMSTANMYTGMGGMVQTVQTRYTFG AALFVGWVAGGLTLIGGVMMCIACRGLAPEETN YKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKK IYDGGARTEDEVQSYPSKHDYV 39 huCLDN18.1 (261 aa) MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQD LYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTI LGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIR IGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFA NMLVTNFWMSTANMYTGMGGMVQTVQTRYTFG AALFVGWVAGGLTLIGGVMMCIACRGLAPEETN YKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKK IYDGGARTEDEVQSYPSKHDYV 40 Mutant IgGl human CH ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

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KTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 41 YL-G1-19-03 HVR-H1 no Kabatu TDGMS 42 YL-G1-19-03 HVR-H2 no Kabatu WINTYSGVPTYADDFKG 43 YL-G1-19-03 HVR-H3 no Kabatu FRRGNALDN 44 YL-G1-19-03 HVR-L1 no Kabatu KSSQSLLNSGNQKNYLT 45 YL-G1-19-03 HVR-L2 no Kabatu WASTRES 46 YL-G1-19-03 HVR-L3 no Kabatu QNNYFYPLT 47 YL-G1-19-03 HVR-H1 no Chotia GFPFTTD 48 YL-G1-19-03 HVR-H2 no Chotia NTYSGV 49 YL-G1-19-03 HVR-H3 no Chotia FRRGNALDN 50 YL-G1-19-03 HVR-L1 no Chotia KSSQSLLNSGNQKNYLT

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Claims (26)

51 YL-G1-19-03 HVR-L2 by Chotia WASTRES 52 YL-G1-19-03 HVR-L3 by Chotia QNNYFYPLT 53 YL-G1-19-03 HVR-H1 by Contact TTDGMS 54 YL-G1-19-03 HVR-H2 by Contact WMGWINTYSGVPT 55 YL-G1-19-03 HVR-H3 by Contact ARFRRGNALD 56 YL-G1-19-03 HVR-L1 by Contact LNSGNQKNYLTWY 57 YL-G1-19-03 HVR-L2 by Contact LLIYWASTRE 58 YL-G1-19-03 HVR-L3 by Contact QNNYFYPL FORMULA OF THE INVENTION (1) HVR-H1 as shown in SEQ ID NO: 23, HVR-H2 as shown in SEQ ID NO: 24, HVRH3 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, according to IMGT numbering; 1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 (claudin 18.2), containing HVR-H1 (heavy chain hypervariable region 1), HVR-H2 and HVR-H3 contained in the VH (heavy chain variable region) presented in SEQ ID NO: 5, and HVR-LI (light chain hypervariable region 1), HVR-L2 and HVR-L3 contained in the VL (light chain variable region) presented in SEQ ID NO: 6. (2) HVR-H1 as shown in SEQ ID NO: 41, HVR-H2 as shown in SEQ ID NO: 42, HVRH3 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 Kabat numbering; 2. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, containing: 3. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising VH as presented in SEQ ID NO: 5 and VL as presented in SEQ ID NO: 6. (3) HVR-H1 as shown in SEQ ID NO: 47, HVR-H2 as shown in SEQ ID NO: 48, HVRH3 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 Chothia numbering; or (4) HVR-H1 as shown in SEQ ID NO: 53, HVR-H2 as shown in SEQ ID NO: 54, HVRH3 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 Contact numbering. 4. A monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising a VH as presented in SEQ ID NO: 5 and a VL as presented in SEQ ID NO: 6, wherein the first two N-terminal amino acid residues of VH are absent. 5. A monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a murine antibody, a chimeric antibody or a humanized antibody or an antigen-binding fragment thereof. 6. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antigen-binding fragment of the antibody is selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab') ₂ fragment, an scFv fragment, and a diabody. 7. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the 8. A monoclonal antibody or antigen-binding fragment thereof according to claim 7, comprising a constant region of a human IgG heavy chain and/or a constant region of a human kappa light chain. 9. A monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein the constant region of the human IgG heavy chain is the constant region of the human IgG1 heavy chain. 10. A monoclonal antibody or antigen-binding fragment thereof according to claim 9, wherein the constant region of the human IgG1 heavy chain is presented in SEQ ID NO: 9. 11. A monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein the constant region of the human kappa light chain is presented in SEQ ID NO: 10. 12. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 7 to 11, wherein the antibody comprises one or more mutations in the Fc region. 13. A monoclonal antibody or antigen-binding fragment thereof according to claim 12, wherein the one or more mutations in the Fc region are one or more mutations that modify Fc receptor binding and/or effector function. 14. A monoclonal antibody or antigen-binding fragment thereof according to claim 13, wherein one or more mutations in the Fc region are one or more mutations that increase or decrease Fc receptor binding and/or effector function. 15. A monoclonal antibody or antigen-binding fragment thereof according to claim 13 or 14, wherein the effector function is ADCC (antibody-dependent cellular cytotoxicity) and/or CDC (complement-dependent cytotoxicity). 16. A monoclonal antibody or antigen-binding fragment thereof according to claim 13, wherein 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. 17. A monoclonal antibody or antigen-binding fragment thereof according to claim 9, wherein the constant region of the human IgG1 heavy chain is presented in SEQ ID NO: 40. 18. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, which is isolated. 19. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, which is naked. 20. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, which is conjugated. 21. An isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 20. 22. A vector containing a nucleic acid according to claim 21. - 22 050412 titelo is a full-length antibody. 23. A host cell containing the nucleic acid of claim 21 or the vector of claim 22. 24. A method for producing a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 20, comprising culturing a host cell according to claim 23 to obtain the antibody or an antigen-binding fragment thereof. 25. The method according to claim 24, further comprising isolating the antibody or antigen-binding fragment thereof from the host cell or cell culture. 26. A composition for treating a cancer expressing CLDN18.2, comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 20. -