HK40044791B

HK40044791B - Anti-cldn18.2 antibody and uses thereof

Info

Publication number: HK40044791B
Application number: HK62021032760.2A
Authority: HK
Inventors: 杜靓; 张红艳; 凌文; 袁纪军
Original assignee: 上海吉倍生物技术有限公司
Filing date: 2018-10-22
Publication date: 2023-06-09

Description

anti-CLDN 18.2 antibodies and uses thereof

Technical Field

The present invention relates to the field of disease treatment and immunology, in particular, the present invention relates to an antibody or antigen-binding fragment thereof against CLDN18.2, a nucleic acid molecule encoding the same, an immunoconjugate, a bispecific molecule, a chimeric antigen receptor and a pharmaceutical composition comprising the same, and their use for preventing and/or treating a tumor.

Background

Gastric cancer is one of the most common malignant tumors worldwide, with a low 5-year survival rate, at the level of 20% -40% in most countries, and about 70 million people die of the disease each year. The survival rate of the gastric cancer in China is 35.9%, and statistics show that the number of new cases of the gastric cancer in China in 2015 reaches 679.1/10 ten thousand, and the number of cases of death reaches 498.0/10 ten thousand, so that the gastric cancer becomes the second largest tumor with high morbidity and mortality next to the lung cancer. The malignancy degree of the gastric cancer is high, and because the screening of the gastric cancer is not widely carried out, the patients are usually diagnosed at the late stage, the chance of radical operation is missed, and the patients can only be treated mainly by chemotherapy.

With the development of tumor molecular biology, the curative effects of targeted drugs and immunotherapy in hematological tumors, breast cancer and colorectal cancer are proved, but the application in the treatment of gastric cancer is relatively slow. The targets for the currently internationally approved targeted therapy of gastric cancer comprise human epidermal growth factor receptor 2 (HER 2) and Vascular Endothelial Growth Factor (VEGF), and the approved immunotherapy target of gastric cancer is programmed lethal protein 1 (PD-1). In 9 months 2017, based on the large phase three clinical trial ATTRACTION-2, the Japan Kyoho approved the use of the PD-1 antibody Opdivo for the treatment of chemotherapy-resistant advanced gastric cancer patients: compared with placebo, opdivo can reduce the mortality rate by 37%, with an effective rate of 11.2%. Based on the secondary clinical trial of Keynote 059, the FDA in the united states accelerated the approval of the PD-1 antibody Keytruda for the treatment of chemotherapy-resistant PD-L1 positive advanced gastric cancer patients: the effective rate is 15.5%. In the aspect of targeted therapy, vascular Endothelial Growth Factor (VEGF) not only promotes tumor angiogenesis, but also can be combined with receptors on the surface of tumor cells to activate downstream signaling pathways, and directly participate in processes such as formation, generation and migration of tumor stem cells. VEGFR2 inhibitors apatinib and ramucirumab have clinically proven that VEFR targeted therapy is safe and effective at the second and above second lines of advanced gastric cancer. In about 6% -35% of new annual cases of gastric cancer, the phenomenon of HER2 gene amplification or protein overexpression occurs, so that HER2 is one of important targets in anticancer therapy. The clinical trial of TOGA phase III firstly proves the advantages of trastuzumab in first-line treatment of HER2 (+) advanced gastric cancer, the OS of trastuzumab-chemotherapy group is obviously prolonged (13.8 months vs.11 months) compared with that of single chemotherapy group, and the secondary endpoints including PFS, ORR (objective remission rate), TTP (time to progression of disease) and the like are also obviously improved. However, the proportion of HER2 positive of patients in China is only about 10 percent, so that the search of a new gastric cancer treatment target is imperative.

Claudin (Claudin) is a transmembrane protein complex in the tight junction between epithelium and endothelium, is located on the apical side of the adjacent cell gap, has tissue-organ specificity in distribution, and mainly has the functions of intercellular adhesion, cell polarity maintenance, cell bypass permeability regulation, and participation in cell proliferation and differentiation regulation. Claudin18 (Claudin 18) molecules are tetraspanins with four transmembrane hydrophobic regions and two extracellular loops, present as two different splice variants CLDN18.1 and CLDN18.2. CLDN18.1 and CLDN18.2 differ in sequence at the intracellular nitrogen end and the first extracellular loop outside the cell, while the primary sequence of the proteins in the other parts are identical. Tissue distribution of CLDN18 showed that CLDN18.1 is selectively expressed only in lung cells, whereas CLDN18.2 is expressed only on stomach cells, and expression of CLDN18.2 in normal stomach is restricted to differentiated, short-lived gastric epithelial cells. CLDN18.2 is frequently retained during malignant transformation of gastric cells and is thus frequently displayed on the surface of human gastric Cancer cells, 60-80% of gastrointestinal adenomas show CLDN18.2 positivity (Clinical Cancer Research 2008,14 (23): 7624-34). Furthermore, CLDN18.2 is highly expressed in pancreatic duct Cancer and metastatic pancreatic Cancer, has a positive rate of up to 60-70%, and can be used as a pancreatic duct/pancreatic Cancer diagnostic marker and therapeutic target (Journal of Clinical Pathology 2012,65, 431-436, world Journal of Gastroenterology 2014,20 (31): 10813-10824, international Journal of Cancer 2014, 134. There is no systematic study on ectopic activation of CLDN18.2 on other tumors, and currently available literature shows that this protein is expressed in esophageal, bronchial, non-small cell lung (NSCLC), breast, ENT, ovarian, colon, liver and metastatic cancers, especially gastric Cancer metastasis such as Krukenberg tumor, peritoneal metastasis and lymph node metastasis (Clinical Cancer Research 2008,14 (23): 7624-34 international Journal of Cancer 2014, 134-739 Cancer Letters 2017, 403-73. CLDN18.2 is a tumor target with preventive and therapeutic value, its differential expression between cancer and normal cells, its membrane localization, its absence in most toxicity-related normal tissues, its expression in the stomach is limited to differentiated gastric cells that can be supplemented by target-negative stem cells (or locational unreachable stem cells) of the stomach, making CLDN18.2 an attractive target for cancer immunotherapy. Therefore, it is urgently and necessarily required to develop an anti-CLDN 18.2 antibody having higher specificity, lower toxic and side effects, and superior clinical efficacy, which will provide more drug options for cancer patients.

Disclosure of Invention

The antibodies of the invention are capable of specifically recognizing/binding to human CLDN18.2 and are capable of inducing killing of cells (e.g., tumor cells) expressing CLDN18.2 by ADCC and/or CDC and have superior functional properties compared to known anti-CLDN 18.2 antibodies. Therefore, the antibody of the invention has the potential for preventing and/or treating tumors and has great clinical value.

Antibodies of the invention

Accordingly, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, said antibody or antigen-binding fragment thereof comprising:

(a) A heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs):

(i) VH CDR1, consisting of the following sequence: SEQ ID NO:75, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to SEQ ID NO:75,

(ii) VH CDR2, consisting of the following sequence: SEQ ID NO:76, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) as compared with SEQ ID NO:76, and

(iii) A VH CDR3, consisting of the sequence: 77, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to SEQ ID NO: 77;

and/or

(b) A light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs):

(iv) VLCDR1, consisting of the following sequence: 78, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to SEQ ID NO:78,

(v) VLCDR2, consisting of the following sequence: 79, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to SEQ ID NO:79, and

(vi) VLCDR3, consisting of the following sequence: SEQ ID NO:80, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to SEQ ID NO: 80.

In certain preferred embodiments, the substitution recited in any one of (i) - (vi) is a conservative substitution;

in certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown as SEQ ID NO. 75, VH CDR2 shown as SEQ ID NO. 76, and VH CDR3 shown as SEQ ID NO. 77; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:78 or 96, VL CDR2 as shown in SEQ ID NO:79, and VL CDR3 as shown in SEQ ID NO: 80.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

(a) The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region set forth in SEQ ID NO: 73; and, the light chain variable region comprises the 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 74; or

(b) The heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region set forth in SEQ ID NO: 91; and, the light chain variable region comprises the 3 CDRs contained in the light chain variable region represented by SEQ ID NO: 92.

In certain preferred embodiments, the 3 CDRs contained in the heavy chain variable region, and/or the 3 CDRs contained in the light chain variable region, are defined by the Kabat, chothia, or IMGT numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 44F7 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof has one or more of the following biological functions:

(a) Binds to human CLDN18.2 with an EC50 of 0.1 μ g/ml or less (e.g., 0.05 μ g/ml or less);

(b) Binds mouse CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(c) Does not bind to human CLDN18.1;

(d) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as CLDN 18.2-expressing tumor cells) by antibody-dependent cell-mediated cytotoxicity (ADCC);

(e) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as CLDN 18.2-expressing tumor cells) by complement-dependent cytotoxicity (CDC);

(f) Prevent and/or treat a tumor (e.g., a tumor expressing CLDN 18.2) in a subject.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) VH CDR1 shown as SEQ ID NO. 75, VH CDR2 shown as SEQ ID NO. 76, and VH CDR3 shown as SEQ ID NO. 77; VL CDR1 as shown in SEQ ID NO. 78 or 96, VL CDR2 as shown in SEQ ID NO. 79, VL CDR3 as shown in SEQ ID NO. 80; or

(2) (a) 3 CDRs contained in the heavy chain variable region set forth in SEQ ID NO: 73; and 3 CDRs contained in the light chain variable region represented by SEQ ID NO: 74; or (b) 3 CDRs contained in the heavy chain variable region set forth in SEQ ID NO: 91; and 3 CDRs contained in the light chain variable region represented by SEQ ID NO 92.

In one exemplary embodiment, the antibody or antigen-binding fragment thereof comprises:

(a) A heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) 73 in SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 73; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 73;

and/or the presence of a gas in the gas,

(b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) 74, SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 74; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 74.

In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO. 73 and VL having the sequence shown as SEQ ID NO. 74.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is humanized.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) Framework Region (FR) derived from a human immunoglobulin, and/or the VL of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) Framework Region (FR) derived from a human immunoglobulin. In such embodiments, the heavy chain variable region FR and/or the light chain variable region FR of the antibody or antigen-binding fragment thereof may comprise one or more non-human (e.g., murine) amino acid residues, e.g., the heavy chain framework region FR and/or the light chain framework region FR may comprise one or more amino acid back mutations in which there is a corresponding murine amino acid residue.

(a) A heavy chain framework region of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) in comparison to the sequence from which it is derived; and/or

(b) A light chain framework region of a human immunoglobulin or a variant thereof, which variant has conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a framework region of a human immunoglobulin, such as a framework region comprised in an amino acid sequence encoded by a human germline antibody gene. In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region comprised in the amino acid sequence encoded by the human heavy chain germline gene and/or a light chain framework region comprised in the amino acid sequence encoded by the human light chain germline gene.

In such embodiments, the framework regions (heavy chain framework regions and/or light chain framework regions) of the antibody or antigen-binding fragment thereof may comprise one or more amino acid residues of non-human (e.g., murine) origin. In certain preferred embodiments, the framework regions (heavy chain framework regions and/or light chain framework regions) comprise one or more amino acid residues that are back-mutated to the corresponding murine residue or conservative amino acid substitutions of the corresponding murine residue (such mutations are referred to as back mutations).

Thus, in certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a framework region of a human immunoglobulin (e.g., a framework region comprised in an amino acid sequence encoded by a human germline antibody gene), which optionally comprises one or more back mutations from human residues to murine residues.

(i) 91 in SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 91; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 91;

and/or the presence of a gas in the gas,

(iv) 92, SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 92; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO 92.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO.91 and VL having the sequence shown as SEQ ID NO. 92.

(i) 99 in SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 99; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO 99;

and/or the presence of a gas in the atmosphere,

(iv) 100 in SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 100; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID No. 100.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having a sequence shown as SEQ ID NO. 99 and VL having a sequence shown as SEQ ID NO. 100.

(i) 101 is shown in SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 101; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO 101;

and/or the presence of a gas in the gas,

(iv) 102 in SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 102; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 102.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH with a sequence shown as SEQ ID NO. 101 and VL with a sequence shown as SEQ ID NO. 102.

In another aspect, the present invention provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising:

(i) VH CDR1, consisting of the following sequence: 3, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(ii) VH CDR2, consisting of the following sequence: 4, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(iii) VH CDR3, consisting of the following sequence: 5, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto;

and/or

(iv) VL CDR1, consisting of the following sequence: 6, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(v) VL CDR2, consisting of the following sequence: 7, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(vi) VL CDR3, consisting of the following sequence: 8, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto.

In certain preferred embodiments, the substitution recited in any one of (i) - (vi) is a conservative substitution.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown as SEQ ID NO. 3, VH CDR2 shown as SEQ ID NO.4, and VH CDR3 shown as SEQ ID NO. 5; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 6, VL CDR2 as shown in SEQ ID NO. 7, and VL CDR3 as shown in SEQ ID NO. 8.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region set forth in SEQ ID No. 1; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 2.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 1D10 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(a) Binds to human CLDN18.2 with an EC50 of 0.1 μ g/ml or less (e.g., 0.05 μ g/ml, 0.02 μ g/ml or less);

(b) Binds mouse CLDN18.2 with an EC50 of 1 μ g/ml or less;

(c) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as CLDN 18.2-expressing tumor cells) by antibody-dependent cell-mediated cytotoxicity (ADCC);

(d) Inducing killing of cells expressing human CLDN18.2 (e.g., tumor cells, such as CLDN 18.2-expressing tumor cells) by complement-dependent cytotoxicity (CDC);

(e) Mediates internalization of CLDN18.2 into cells (e.g., tumor cells), e.g., at a level of internalization of at least 10% (e.g., at least 15%, at least 20% or more) as measured by FACS or flow cytometry; the cell expresses CLDN18.2 on its surface;

(1) VH CDR1 shown as SEQ ID NO. 3, VH CDR2 shown as SEQ ID NO.4, and VH CDR3 shown as SEQ ID NO. 5; VL CDR1 as shown in SEQ ID NO. 6, VL CDR2 as shown in SEQ ID NO. 7, and VL CDR3 as shown in SEQ ID NO. 8; or

(2) 1, 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO. 2.

(i) 1, SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence shown in SEQ ID NO. 1; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in SEQ ID NO. 1;

and/or the presence of a gas in the gas,

(iv) The sequence shown as SEQ ID NO. 2;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 2; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 2.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having a sequence shown as SEQ ID NO. 1 and VL having a sequence shown as SEQ ID NO. 2.

(i) VH CDR1, consisting of the following sequence: 11, or a sequence having substitution, deletion or addition of one or several amino acids (e.g.substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(ii) VH CDR2, consisting of the following sequence: 12, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(iii) A VH CDR3, consisting of the sequence: 13, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto;

and/or

(iv) VL CDR1, consisting of the following sequences: 14, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids),

(v) VL CDR2, consisting of the following sequence: 15, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(vi) VL CDR3, consisting of the following sequence: 16, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown in SEQ ID NO. 11, VH CDR2 shown in SEQ ID NO. 12, and VH CDR3 shown in SEQ ID NO. 13; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 14, VL CDR2 as shown in SEQ ID NO. 15, and VL CDR3 as shown in SEQ ID NO. 16; .

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region represented by SEQ ID No. 9; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region set forth in SEQ ID NO 10.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 2F12 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(b) Binds mouse CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(c) Does not bind to human CLDN18.1;

(1) VH CDR1 shown in SEQ ID NO. 11, VH CDR2 shown in SEQ ID NO. 12 and VH CDR3 shown in SEQ ID NO. 13; VL CDR1 as shown in SEQ ID NO. 14, VL CDR2 as shown in SEQ ID NO. 15, and VL CDR3 as shown in SEQ ID NO. 16; or

(2) The heavy chain variable region shown in SEQ ID NO 9 comprises 3 CDRs; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO 10.

(i) A sequence shown as SEQ ID NO. 9;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 9; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 9;

and/or the presence of a gas in the gas,

(iv) 10, SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 10; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 10.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having a sequence shown as SEQ ID NO.9 and VL having a sequence shown as SEQ ID NO. 10.

(i) VH CDR1, consisting of the following sequence: 19 or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g. substitution, deletion or addition of 1, 2 or 3 amino acids),

(ii) VH CDR2, consisting of the following sequence: 20, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(iii) VH CDR3, consisting of the following sequence: 21, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto;

and/or

(iv) VL CDR1, consisting of the following sequence: 22, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(v) VL CDR2, consisting of the following sequence: 23, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and

(vi) VL CDR3, consisting of the following sequence: 24, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown in SEQ ID NO. 19, VH CDR2 shown in SEQ ID NO. 20, and VH CDR3 shown in SEQ ID NO. 21; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 22, VL CDR2 as shown in SEQ ID NO. 23, and VL CDR3 as shown in SEQ ID NO. 24.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region set forth in SEQ ID No. 17; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 18.

In certain preferred embodiments, the 3 CDRs contained in the heavy chain variable region, and/or the 3 CDRs contained in the light chain variable region, are defined by the Kabat, chothia or IMGT numbering system.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 3F2 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(b) Binds mouse CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(c) Does not bind to human CLDN18.1;

(1) VH CDR1 shown in SEQ ID NO. 19, VH CDR2 shown in SEQ ID NO. 20, and VH CDR3 shown in SEQ ID NO. 21; VL CDR1 as shown in SEQ ID NO. 22, VL CDR2 as shown in SEQ ID NO. 23, and VL CDR3 as shown in SEQ ID NO. 24; or

(2) 17 in the heavy chain variable region containing 3 CDR; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO 18.

(i) 17, SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 17; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO 17;

and/or the presence of a gas in the gas,

(iv) 18 in SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 18; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 18.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: a VH having a sequence shown as SEQ ID NO. 17 and a VL having a sequence shown as SEQ ID NO. 18.

(i) VH CDR1, consisting of the following sequence: 27, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(ii) VH CDR2, consisting of the following sequence: 28, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and

(iii) A VH CDR3, consisting of the sequence: 29, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g. substitution, deletion or addition of 1, 2 or 3 amino acids);

and/or

(iv) VL CDR1, consisting of the following sequence: 30, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(v) VL CDR2, consisting of the following sequence: 31, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(vi) VL CDR3, consisting of the following sequence: 32, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown in SEQ ID NO. 27, VH CDR2 shown in SEQ ID NO. 28, and VH CDR3 shown in SEQ ID NO. 29; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 30, VL CDR2 as shown in SEQ ID NO. 31, and VL CDR3 as shown in SEQ ID NO. 32.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region represented by SEQ ID No. 25; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 26.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 5F9 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(a) Binds to human CLDN18.2 with an EC50 of 0.5 μ g/ml or less;

(b) Binds mouse CLDN18.2 with an EC50 of 0.2 μ g/ml or less;

(1) VH CDR1 shown in SEQ ID NO. 27, VH CDR2 shown in SEQ ID NO. 28, and VH CDR3 shown in SEQ ID NO. 29; VL CDR1 as shown in SEQ ID NO. 30, VL CDR2 as shown in SEQ ID NO. 31, and VL CDR3 as shown in SEQ ID NO. 32; or

(2) The heavy chain variable region shown in SEQ ID NO. 25 comprises 3 CDRs; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO: 26.

(i) The sequence shown as SEQ ID NO. 25;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 25; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 25;

and/or the presence of a gas in the gas,

(iv) 26 is shown as SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 26; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 26.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO. 25 and VL having the sequence shown as SEQ ID NO. 26.

(i) VH CDR1, consisting of the following sequence: 35, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(ii) VH CDR2, consisting of the following sequence: 36, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and

(iii) A VH CDR3, consisting of the sequence: 37, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto;

and/or

(iv) VL CDR1, consisting of the following sequence: 38, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(v) VL CDR2, consisting of the following sequence: 39, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(vi) VL CDR3, consisting of the following sequence: 40, or a sequence having substitution, deletion or addition of one or several amino acids (e.g. substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown in SEQ ID NO. 35, VH CDR2 shown in SEQ ID NO. 36, and VH CDR3 shown in SEQ ID NO. 37; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 38, VL CDR2 as shown in SEQ ID NO. 39, and VL CDR3 as shown in SEQ ID NO. 40.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region represented by SEQ ID No. 33; and, the light chain variable region comprises the 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 34.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 9F3 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(a) Binds to human CLDN18.2 with an EC50 of 0.2 μ g/ml or less;

(b) Binds mouse CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(c) Does not bind to human CLDN18.1;

(1) VH CDR1 shown in SEQ ID NO. 35, VH CDR2 shown in SEQ ID NO. 36, and VH CDR3 shown in SEQ ID NO. 37; VL CDR1 as shown in SEQ ID NO. 38, VL CDR2 as shown in SEQ ID NO. 39, VL CDR3 as shown in SEQ ID NO. 40; or

(2) 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 33; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO: 34.

(i) 33, SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 33; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 33;

and/or the presence of a gas in the gas,

(iv) 34, SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 34; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 34.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having a sequence shown as SEQ ID NO. 33 and VL having a sequence shown as SEQ ID NO. 34.

(i) VH CDR1, consisting of the following sequence: 43 or a sequence having substitution, deletion or addition of one or several amino acids (e.g.substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto,

(ii) VH CDR2, consisting of the following sequence: 44, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(iii) A VH CDR3, consisting of the sequence: 45, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

and/or

(iv) VL CDR1, consisting of the following sequence: 46, or a sequence having substitution, deletion or addition of one or several amino acids (e.g.substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(v) VL CDR2, consisting of the following sequence: 47, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(vi) VL CDR3, consisting of the following sequence: 48, or a sequence having substitution, deletion or addition of one or several amino acids (e.g. substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown as SEQ ID NO. 43, VH CDR2 shown as SEQ ID NO. 44, and VH CDR3 shown as SEQ ID NO. 45; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 46, VL CDR2 as shown in SEQ ID NO. 47, and VL CDR3 as shown in SEQ ID NO. 48.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region represented by SEQ ID No. 41; and, the light chain variable region comprises the 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 42.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 10B11 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(b) Binds mouse CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(c) Does not bind to human CLDN18.1;

(1) VH CDR1 shown as SEQ ID NO. 43, VH CDR2 shown as SEQ ID NO. 44 and VH CDR3 shown as SEQ ID NO. 45; VL CDR1 as shown in SEQ ID NO. 46, VL CDR2 as shown in SEQ ID NO. 47, and VL CDR3 as shown in SEQ ID NO. 48; or

(2) (ii) 3 CDRs contained in the heavy chain variable region shown in SEQ ID No. 41; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO: 42.

(i) 41 in SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 41; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 41;

and/or the presence of a gas in the atmosphere,

(iv) 42, SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 42; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 42.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having a sequence shown as SEQ ID NO. 41 and VL having a sequence shown as SEQ ID NO. 42.

(i) VH CDR1, consisting of the following sequence: 51, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(ii) VH CDR2, consisting of the following sequence: 52, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(iii) A VH CDR3, consisting of the sequence: 53, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto;

and/or

(iv) VL CDR1, consisting of the following sequence: 54, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids),

(v) VL CDR2, consisting of the following sequences: 55, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and

(vi) VL CDR3, consisting of the following sequence: 56, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown as SEQ ID NO. 51, VH CDR2 shown as SEQ ID NO. 52 and VH CDR3 shown as SEQ ID NO. 53; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 54, VL CDR2 as shown in SEQ ID NO. 55, and VL CDR3 as shown in SEQ ID NO. 56.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region represented by SEQ ID No. 49; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 50.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 27B5 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(a) Binds to human CLDN18.2 with an EC50 of 0.2 μ g/ml or less;

(b) Binds to mouse CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(c) Does not bind to human CLDN18.1;

(1) VH CDR1 shown as SEQ ID NO. 51, VH CDR2 shown as SEQ ID NO. 52, and VH CDR3 shown as SEQ ID NO. 53; VL CDR1 as shown in SEQ ID NO. 54, VL CDR2 as shown in SEQ ID NO. 55, and VL CDR3 as shown in SEQ ID NO. 56; or

(2) 3 CDRs contained in the heavy chain variable region set forth in SEQ ID NO. 49; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO: 50.

(i) 49 of SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 49; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 49;

and/or the presence of a gas in the gas,

(iv) 50 in SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 50; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 50.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having a sequence shown as SEQ ID NO. 49 and VL having a sequence shown as SEQ ID NO. 50.

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) Framework Region (FR) derived from a human immunoglobulin, and/or the VL of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) Framework Region (FR) derived from a human immunoglobulin. In such embodiments, the heavy chain variable region FRs and/or light chain variable region FRs of the antibody or antigen-binding fragment thereof may comprise one or more non-human (e.g., murine) amino acid residues, e.g., the heavy chain framework region FRs and/or light chain framework region FRs may comprise one or more amino acid back mutations in which there is a corresponding murine amino acid residue.

(i) VH CDR1, consisting of the following sequence: 59, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(ii) VH CDR2, consisting of the following sequence: 60, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(iii) A VH CDR3, consisting of the sequence: 61, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto;

and/or

(iv) VL CDR1, consisting of the following sequence: 62 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(v) VL CDR2, consisting of the following sequence: 63, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(vi) VL CDR3, consisting of the following sequence: 64, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown as SEQ ID NO. 59, VH CDR2 shown as SEQ ID NO. 60, and VH CDR3 shown as SEQ ID NO. 61; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:62, VL CDR2 as shown in SEQ ID NO:63, and VL CDR3 as shown in SEQ ID NO: 64.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region represented by SEQ ID No. 57; and, the light chain variable region comprises the 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 58.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 37B1 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(b) Binds mouse CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(c) Does not bind to human CLDN18.1;

(1) VH CDR1 shown as SEQ ID NO. 59, VH CDR2 shown as SEQ ID NO. 60, and VH CDR3 shown as SEQ ID NO. 61; VL CDR1 as shown in SEQ ID NO:62, VL CDR2 as shown in SEQ ID NO:63, and VL CDR3 as shown in SEQ ID NO: 64; or

(2) 57, 3 CDRs contained in the heavy chain variable region set forth in SEQ ID NO; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO: 58.

(i) 57, SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 57; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 57;

and/or the presence of a gas in the gas,

(iv) 58 in SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 58; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 58.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO. 57 and VL having the sequence shown as SEQ ID NO. 58.

(i) VH CDR1, consisting of the following sequence: 67, or a sequence having one or more amino acid substitutions, deletions or additions thereto (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto,

(ii) VH CDR2, consisting of the following sequence: 68, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and

(iii) A VH CDR3, consisting of the sequence: 69, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto;

and/or

(iv) VL CDR1, consisting of the following sequence: 70, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(v) VL CDR2, consisting of the following sequence: 71, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(vi) VL CDR3, consisting of the following sequence: 72, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 shown in SEQ ID NO. 67, VH CDR2 shown in SEQ ID NO. 68, and VH CDR3 shown in SEQ ID NO. 69; and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO. 70, VL CDR2 as shown in SEQ ID NO. 71, and VL CDR3 as shown in SEQ ID NO. 72.

The present invention also provides an antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 CDRs contained in the heavy chain variable region represented by SEQ ID No. 65; and, the light chain variable region comprises 3 CDRs contained in the light chain variable region set forth in SEQ ID NO: 66.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof is 44A8 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

(a) Binds human CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(b) Binds mouse CLDN18.2 with an EC50 of 0.1 μ g/ml or less;

(c) Does not bind to human CLDN18.1;

(f) Mediates internalization of CLDN18.2 into cells (e.g., tumor cells), e.g., at an internalization level of at least 10% (e.g., at least 15%, at least 20% or more) as measured by FACS or flow cytometry; the cell expresses CLDN18.2 on its surface;

(g) Prevent and/or treat a tumor (e.g., a tumor expressing CLDN 18.2) in a subject.

(1) VH CDR1 shown in SEQ ID NO. 67, VH CDR2 shown in SEQ ID NO. 68, and VH CDR3 shown in SEQ ID NO. 69; VL CDR1 as shown in SEQ ID NO. 70, VL CDR2 as shown in SEQ ID NO. 71, and VL CDR3 as shown in SEQ ID NO. 72; or

(2) The heavy chain variable region shown in SEQ ID NO 65 contains 3 CDRs; and 3 CDRs contained in the light chain variable region shown in SEQ ID NO: 66.

(i) 65 of SEQ ID NO;

(ii) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 65; or

(iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 65;

and/or the presence of a gas in the gas,

(iv) 66, SEQ ID NO;

(v) A sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 66; or

(vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 66.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO. 65 and VL having the sequence shown as SEQ ID NO. 66.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention may further comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived. In certain preferred embodiments, the variant has conservative substitutions of one or more amino acids compared to the sequence from which it is derived.

In certain preferred embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived; and/or the presence of a gas in the atmosphere,

the light chain of the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived.

In certain preferred embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, igG2, igG3, or IgG4 heavy chain constant region. In certain preferred embodiments, the heavy chain constant region is a murine IgG1, igG2, igG3, or IgG4 heavy chain constant region. In certain preferred embodiments, the heavy chain constant region is a human IgG1, igG2, igG3, or IgG4 heavy chain constant region. In certain embodiments, preferably the heavy chain constant region is a human IgG1 or IgG4 heavy chain constant region.

In certain preferred embodiments, the light chain constant region is a kappa light chain constant region. In certain preferred embodiments, the light chain constant region is a murine kappa light chain constant region. In certain preferred embodiments, the light chain constant region is a human kappa light chain constant region.

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise the heavy chain constant region (CH) shown in SEQ ID NO: 81; and/or, a light chain constant region (CL) as set forth in SEQ ID NO: 82.

In certain preferred embodiments, the antibodies of the invention are murine, chimeric, humanized, bispecific or multispecific antibodies. In certain preferred embodiments, the antigen binding fragment of the invention is selected from the group consisting of Fab, fab ', (Fab') ₂ Fv, disulfide-linked Fv, scFv, diabody (diabody), and single domain antibody (sdAb).

In the present invention, an antibody or antigen-binding fragment thereof of the present invention may include variants that differ from the antibody or antigen-binding fragment thereof from which it is derived only by conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids) amino acid residues, or that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody or antigen-binding fragment thereof from which it is derived, and that substantially retain the above-described biological functions of the antibody or antigen-binding fragment thereof from which it is derived.

Preparation of antibodies

The antibodies of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cells are then cultured under specific conditions and the antibodies of the invention are expressed.

Antigen-binding fragments of the invention can be obtained by hydrolysis of the whole antibody molecule (see Morimoto et al, J.Biochem.Biophys.methods 24, 107-117 (1992) and Brennan et al, science 229 (1985). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, curr. Opin. Immunol.11:548-557 (1999); little et al, immunol.today, 21. For example, fab' fragments can be obtained directly from the host cell; fab 'fragments can be chemically coupled to form F (ab') ₂ Fragment (Carter et al, bio/Technology, 10. In addition, fv, fab or F (ab') ₂ The fragments may also be isolated directly from the culture medium of the recombinant host cell. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the invention, or a heavy chain variable region and/or a light chain variable region thereof. In certain preferred embodiments, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and/or a light chain variable region thereof.

In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention. In certain preferred embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages and the like. In certain preferred embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (e.g., a mammal, e.g., a human).

In another aspect, the invention provides a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention. Such host cells include, but are not limited to, prokaryotic cells such as e.coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). In certain preferred embodiments, the host cell of the invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DG 44).

In another aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

Derivatized antibodies

The antibodies or antigen-binding fragments thereof of the invention can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to CLDN18.2 (particularly human CLDN 18.2). Thus, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or otherwise) to one or more other molecular moieties, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and/or a protein or polypeptide (e.g., avidin or a polyhistidine tag) capable of mediating binding of the antibody or antigen-binding fragment to another molecule. In addition, the antibodies or antigen-binding fragments thereof of the present invention may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, for example to increase serum half-life.

Thus, in certain preferred embodiments, the antibodies of the invention or antigen-binding fragments thereof are labeled. In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention carry a detectable labelFor example, an enzyme, a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material), or biotin. The detectable label of the present invention may be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., ³ H、 ¹²⁵ I、 ³⁵ S、 ¹⁴ c or ³² P), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., cy7, alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridine ester compounds), magnetic beads (e.g.,) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above. Patents teaching the use of such labels include, but are not limited to, U.S. Pat. nos. 3,817,837;3,850,752;3,939,350;3,996,345;4,277,437;4,275,149; and 4,366,241 (all incorporated herein by reference). Detectable labels as described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation calculators, and fluorescent labels can be detected using photodetectors to detect the emitted light. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label. In certain embodiments, such labels can be suitable for use in immunological assays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, and the like). In certain embodiments, the detectability as described above may be by linkers of varying lengthsThe label to be tested is attached to the antibody or antigen binding fragment thereof of the present invention to reduce potential steric hindrance.

Bispecific or multispecific molecules

The antibodies or antigen binding fragments thereof of the invention can be used to form bi-or multispecific molecules. The antibodies or antigen-binding fragments thereof of the present invention can be part of a bispecific or multispecific molecule comprising a second functional moiety (e.g., a second antibody) having a binding specificity different from that of the antibody or antigen-binding fragment thereof of the present invention, thereby being capable of binding to at least two different binding sites and/or target molecules. For example, an antibody or antigen-binding fragment thereof of the invention can be linked to a second antibody or antigen-binding fragment thereof that is capable of specifically binding any protein that can be used as a potential target for combination therapy. To produce such bispecific or multispecific molecules, an antibody or antigen-binding fragment thereof of the invention can be linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).

Thus, in another aspect, the invention provides a bispecific or multispecific molecule comprising an antibody or antigen-binding fragment thereof of the invention.

In certain preferred embodiments, the bispecific or multispecific molecule specifically binds to CLDN18.2 and additionally specifically binds to one or more other targets.

In certain preferred embodiments, the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity for a second target (e.g., a second antibody).

Immunoconjugates

The antibodies or antigen-binding fragments thereof of the invention can be linked to a therapeutic agent to form an immunoconjugate. Due to the ability of immunoconjugates to selectively deliver one or more therapeutic agents to a target tissue (e.g., a tumor-associated antigen such as a CLDN 18.2-expressing tumor), immunoconjugates can increase the therapeutic efficacy of an antibody or antigen-binding fragment thereof of the invention in treating a disease (e.g., cancer).

Thus, in another aspect, the invention provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof of the invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

In certain preferred embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

In certain preferred embodiments, the therapeutic agent is a cytotoxic agent. In the present invention, the cytotoxic agent includes any agent that is detrimental to cells (e.g., kills cells).

In certain preferred embodiments, the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof.

Examples of alkylating agents that can be used in the immunoconjugates of the invention include, but are not limited to, nitrogen mustards (e.g., mechlorethamine, chlorambucil, melphalan, cyclophosphamide, etc.), vinyl imines (e.g., thiotepa, etc.), sulfates and polyols (e.g., busulfan, dibromomannitol), nitrosoureas (e.g., carmustine, lomustine, etc.), platinum antineoplastic agents (e.g., cisplatin, oxaliplatin, carboplatin, etc.), and the like.

Examples of mitotic inhibitors that can be used in the immunoconjugates of the invention include, but are not limited to, maytansinoids (e.g., maytansine, maytansinol, the C-3 ester of maytansinol, etc.), taxanes (e.g., docetaxel, paclitaxel, or nanoparticulate paclitaxel, etc.), vinca alkaloids (e.g., vindesine sulfate, vincristine, vinblastine, or vinorelbine, etc.)

Examples of antitumor antibiotics that can be used in the immunoconjugates of the invention include, but are not limited to, actinomycin, anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, etc.), calicheamicin, duocarmycin, and the like.

Examples of antimetabolites that may be used in the immunoconjugates of the invention include, but are not limited to, folate antagonists (e.g., methotrexate, etc.), pyrimidine antagonists (e.g., 5-fluorouracil, floxuridine, cytarabine, capecitabine, gemcitabine, etc.), purine antagonists (e.g., 6-mercaptopurine, 6-thioguanine, etc.), adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nelarabine, pentostatin, etc.).

Examples of topoisomerase inhibitors that can be used in the immunoconjugates of the invention include, but are not limited to (camptothecin and derivatives thereof (e.g., irinotecan, topotecan, etc.), amsacrine, daunorubicin, doxorubicin, epipodophyllotoxins, ellipticine, epirubicin, etoposide, propinimine, teniposide, and the like.

Examples of tyrosine kinase inhibitors that may be used in the immunoconjugates of the invention include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sunitinib, vandetanib, and the like.

Examples of radionuclide agents that can be used in the immunoconjugates of the invention include, but are not limited to, those described in I ¹³¹ 、In ¹¹¹ 、Y ⁹⁰ 、Lu ¹⁷⁷ And the like.

In certain exemplary embodiments, the therapeutic agent is selected from the group consisting of platinum-based antineoplastic agents, anthracycline antibiotics, taxanes, nucleoside analogs, camptothecin-based compounds, analogs or homologs thereof, and any combination thereof.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is conjugated to the therapeutic agent, optionally through a linker.

In the present invention, cytotoxic agents may be conjugated to the antibodies of the invention or antigen binding fragments thereof using linker technology available in the art. Examples of types of linkers that have been used to couple cytotoxic agents to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Linkers can be selected, for example, that are susceptible to cleavage within the lysosomal compartment by low pH or by proteases (e.g., proteases preferentially expressed in tumor tissue, such as cathepsins, such as cathepsin B, C, D).

Further discussion of the type of cytotoxic agent, linker, and method of coupling a therapeutic agent to an antibody can also be found in Saito, g. et al (2003) adv.drug deliv.rev.55:199-215; trail, p.a. et al (2003) Cancer immunol.immunoher.52: 328-337; payne, G. (2003) cancer Cell 3:207-212; allen, t.m. (2002) nat. Rev. Cancer 2:750-763; pasan, i.and Kreitman, r.j. (2002) curr.opin.investig.drugs 3:1089-1091; se nter, p.d.and Springer, c.j. (2001) adv.drug deliv.rev.53:247-264.

Chimeric antigen receptors

The antibodies or antigen-binding fragments thereof of the invention can be used to construct Chimeric Antigen Receptors (CARs) comprising an extracellular antigen-binding domain (e.g., scFv) that specifically binds to CLDN18.2 linked to a transmembrane domain linked to one or more intracellular T cell signaling domains. The intracellular T cell signaling domain can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or a combination thereof. By T cell receptor signaling domain is meant the portion of the CAR that comprises the intracellular domain of the T cell receptor (e.g., the intracellular portion of the CD3 zeta protein). A costimulatory signaling domain refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule, a cell surface molecule other than the antigen receptor or its ligand required for a high response of lymphocytes to an antigen.

Features of the CARs of the invention include their ability to direct T-cell specificity and reactivity to cells expressing CLDN18.2 (e.g., tumor cells) in a non-MHC restricted manner. non-MHC-restricted CLDN18.2 recognition ability confers on T cells expressing the CARs of the invention the ability to recognize antigen independently of antigen processing.

Accordingly, in another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain of an antibody or antigen binding fragment thereof of the invention.

In certain preferred embodiments, the antigen binding domain comprises the heavy chain variable region and the light chain variable region of an antibody or antigen binding fragment thereof of the invention.

In certain preferred embodiments, the antigen binding domain is an scFv.

In certain preferred embodiments, the chimeric antigen receptor comprises an antigen-binding fragment (e.g., scFv) of an antibody of the invention.

In certain preferred embodiments, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

In certain preferred embodiments, a spacer domain comprising a polypeptide sequence may be present between the antigen binding domain and the transmembrane domain of the CAR. The spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In some embodiments, the spacer domain may comprise an immunoglobulin domain, such as a human immunoglobulin sequence. In certain exemplary embodiments, the immunoglobulin domain comprises immunoglobulin CH2 and CH3 domain sequences. In such embodiments, without being bound by a particular theory, it is believed that the CH2 and CH3 domains extend the antigen binding domain of the CAR away from the membrane of the CAR-expressing cell and can more accurately mimic the size and domain structure of a native TCR.

In certain preferred embodiments, the transmembrane domain may be derived from natural or synthetic sources. In such embodiments, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains useful in the CARs of the invention may comprise at least a transmembrane region of the alpha, beta or zeta chain of a T cell receptor, which may be selected from CD28, CD3 epsilon, CD45, CD4, CD5, CDs, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine.

In certain exemplary embodiments, the transmembrane domain comprises a transmembrane domain of a T cell receptor, such as a CD8 transmembrane domain.

In certain exemplary embodiments, the transmembrane domain comprises a transmembrane domain of a T cell costimulatory molecule (e.g., CD137 or CD 28).

In certain preferred embodiments, examples of intracellular T cell domains useful in the CARs include the cytoplasmic sequences of the T Cell Receptor (TCR) and costimulatory molecules that synergistically initiate signal transduction upon antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequence with the same functional capability.

In certain preferred embodiments, the intracellular region of the CAR may comprise a primary cytoplasmic signal sequence that functions in a stimulatory manner, which may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs comprising a primary cytoplasmic signal sequence that can be included in the CAR include those from CD3 ζ, fcR γ, fcR β, CD3 γ, CD3 δ, CD3 epsilon, CDs, CD22, CD79a, CD79b, and CD66d proteins.

In certain preferred embodiments, the intracellular region of the CAR may comprise an ITAM comprising the primary cytoplasmic signaling domain (e.g., CD3 ζ) by itself or in combination with any other desired cytoplasmic domain that may be used in the environment of the CAR. For example, the cytoplasmic domain of the CAR comprises a CD3 zeta chain portion and an intracellular costimulatory signaling domain. The costimulatory signaling domain refers to the portion of the CAR that comprises the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand required for the efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD 137), OX40 (CD 134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

In certain preferred embodiments, the CAR can comprise a CD3 zeta signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain, or any combination thereof. The order of the one or more T cell signaling domains on the CAR can be varied as desired by one of skill in the art.

Methods of making chimeric antigen receptors, T cells comprising such receptors, and their uses (e.g., for treating cancer) are known in the art, a detailed description of which can be found, for example, in Brentjens et al, 2010, molecular therapy,18, 4,666-668; morgan et al, 2010, molecular therapy, published online February 23,2010, pages 1-9; till et al, 2008, blood, 112; park et al, trends biotechnol, 29, 550-557,2011; grupp et al, NEnglJMed.,368 1509-1518,2013; han et al, j.hematol oncol, 6, 47,2013; PCT patent publications WO2012/079000, WO2013/126726; and u.s. Patent publication 2012/0213783, which is incorporated by reference in its entirety). For example, a nucleic acid molecule encoding a chimeric antigen-binding receptor of the invention can be included in an expression vector (e.g., a lentiviral vector) for expression in a host cell, such as a T cell, to produce the CAR. In certain exemplary embodiments, methods of using the chimeric antigen receptor comprise isolating T cells from a subject, transforming T cells with an expression vector (e.g., a lentiviral vector) encoding the chimeric antigen receptor, and administering the engineered T cells expressing the chimeric antigen receptor to the subject for treatment, e.g., for treatment of a tumor in the subject.

Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of the invention. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor of the invention.

In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain preferred embodiments, the vector of the invention is, for example, a plasmid.

In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

Therapeutic formulationsMethods and pharmaceutical compositions

The antibody or antigen-binding fragment thereof of the present invention can kill cells by inducing ADCC and/or CDC by binding to CLDN18.2, thereby being useful for the prevention and/or treatment of tumors.

Thus, in another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention, and a pharmaceutically acceptable carrier and/or excipient.

In certain preferred embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

In certain preferred embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, anti-metabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers (e.g., gemcitabine, 5-fluorouracil, taxanes, cisplatin, etc.), anti-angiogenic agents, cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), molecularly targeted drugs (e.g., CD20 antibodies such as rituximab, her2 antibodies such as trastuzumab, VEGF antibodies such as bevacizumab, EGFR antibodies such as cetuximab, etc.), immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, etc.), oncolytic viruses, and the like.

In certain preferred embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention is provided as a separate component or as a component of the same composition with the additional pharmaceutically active agent. Thus, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In certain exemplary embodiments, the pharmaceutical composition comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

In another aspect, the present invention provides a method of reducing the expression level of CLDN18.2 on the surface of a cell, comprising contacting the cell with an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the invention such that the expression level of CLDN18.2 on the surface of the cell is reduced; wherein the cell expresses CLDN18.2 on its surface.

In certain preferred embodiments, the cell is a tumor cell expressing CLDN18.2.

In certain preferred embodiments, the method is for reducing expression levels of CLDN18.2 on the cell surface in vitro for non-diagnostic purposes.

In another aspect, there is provided a use of an antibody or antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, or a pharmaceutical composition of the invention in the manufacture of a medicament for reducing the expression level of CLDN18.2 on the surface of a cell.

In another aspect, there is provided an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the invention for use in reducing the expression level of CLDN18.2 on the surface of a cell.

In another aspect, the invention provides a method of inhibiting the growth of and/or killing a tumor cell expressing CLDN18.2 comprising contacting the tumor cell with an effective amount of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor of the invention (e.g., a chimeric antigen receptor T cell (CAR-T)).

The method may be used for therapeutic purposes, or for non-therapeutic purposes. In certain preferred embodiments, the method for inhibiting growth and/or killing of tumor cells expressing CLDN18.2 in vitro may be used for non-therapeutic purposes.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, a pharmaceutical composition, a chimeric antigen receptor, or a host cell expressing the chimeric antigen receptor (e.g., a chimeric antigen receptor T cell (CAR-T)) of the invention in the manufacture of a medicament for inhibiting the growth and/or killing a tumor cell expressing CLDN18.2.

In another aspect, there is provided an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing said chimeric antigen receptor (e.g., chimeric antigen receptor T cell (CAR-T)) of the invention for use in inhibiting growth and/or killing a tumor cell expressing CLDN18.2.

In another aspect, the invention provides a method for preventing and/or treating a tumor in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell (e.g., chimeric antigen receptor T cell (CAR-T)) of the invention.

In certain preferred embodiments, the tumor involves tumor cells expressing CLDN18.2. In certain preferred embodiments, said CLDN18.2 is expressed on the surface of said tumor cells.

In certain preferred embodiments, the tumor expresses CLDN18.2.

In certain preferred embodiments, the tumor is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, otorhinolaryngological (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, cancer of the gallbladder, and metastatic cancers thereof (e.g., gastric cancer metastasis, such as Krukenberg tumor, peritoneal metastasis, or lymph node metastasis).

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors or host cells expressing the chimeric antigen receptors (e.g., chimeric antigen receptor T cells (CAR-T)) of the invention are used in combination with an additional agent having anti-tumor activity. Such additional agents having anti-tumor activity can be administered prior to, concurrently with, or subsequent to administration of the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell (e.g., CAR-T) expressing the chimeric antigen receptor.

In certain preferred embodiments, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors or host cells (e.g., CAR-T) expressing the chimeric antigen receptors of the present invention are administered in combination with additional therapy. Such additional therapy may be any therapy known for tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy, or palliative therapy. Such additional therapies may be administered prior to, concurrently with, or subsequent to administration of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor (e.g., CAR-T) of the invention.

The antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell (e.g., T cell) expressing the chimeric antigen receptor of the present invention may be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections, and concentrated solutions for injections), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the antibody of the present invention is incorporated in a suitable solvent in the necessary dosage and, optionally, with other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier, e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffered solution (e.g., phosphate buffered solution), ringer's solution, and any combination thereof, prior to use.

In addition, the antibodies or antigen binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors or host cells (e.g., T cells) expressing the chimeric antigen receptors of the invention can be present in a pharmaceutical composition in unit dosage form for ease of administration.

The antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions, chimeric antigen receptors, or host cells (e.g., T cells) expressing the chimeric antigen receptors of the invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell (e.g. T cell) expressing the chimeric antigen receptor of the present invention is administered by intravenous injection or bolus injection.

The pharmaceutical compositions of the invention can include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, pharmaceutical composition, chimeric antigen receptor, or host cell (e.g., T cell) expressing the chimeric antigen receptor of the invention. A "prophylactically effective amount" is an amount sufficient to prevent, or delay the onset of disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. A therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention may vary according to the following factors: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g. age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, etc.

In the present invention, the dosage regimen may be adjusted to obtain the optimal desired response (e.g., a therapeutic or prophylactic response). For example, the dosage may be given in a single dose, may be given multiple times over a period of time, or may be reduced or increased proportionally with the exigencies of the therapeutic situation.

In the present invention, the subject may be a mammal, such as a human.

Detection method and kit

The antibodies or antigen-binding fragments thereof of the invention are capable of specifically binding to CLDN18.2 and are thus useful for detecting the presence or level of CLDN18.2 in a sample.

Thus, in another aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof of the invention. In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention are detectably labeled. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody of the invention or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable label.

In the present invention, the detectable label may be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels be capable of being adapted for immunological detection (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., ³ H、 ¹²⁵ I、 ³⁵ S、 ¹⁴ c or ³² P), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., cy7, alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridine ester compounds), magnetic beads (e.g.,) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above. Patents that teach the use of such markers include, but are not limited to, U.S. Pat. nos. 3,817,837;3,850,752;3,939,350;3,996,345;4,277,437;4,275,149; and 4,366,241 (incorporated herein by reference in its entirety). The markers encompassed by the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation calculators, and fluorescent labels can be detected using photodetectors to detect the emitted light. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label. In certain embodiments, linkers as described above may be attached through linkers of different lengthsThe label to be detected is attached to the antibody or antigen-binding fragment thereof of the present invention to reduce potential steric hindrance.

In another aspect, the present invention provides a method of detecting the presence or amount of CLDN18.2 in a sample comprising the steps of:

(1) Contacting the sample with an antibody or antigen-binding fragment thereof of the invention;

(2) Detecting the formation of a complex between the antibody or antigen binding fragment thereof and CLDN18.2 or detecting the amount of the complex.

The formation of the complex indicates the presence of CLDN18.2 or cells expressing CLDN18.2.

In certain preferred embodiments, the sample is a cell sample, i.e., a sample comprising cells (e.g., tumor cells). In such embodiments, preferably, the complex is formed between the antibody, antigen-binding fragment or conjugate and CLDN18.2 expressed by cells in the sample.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention is further provided with a detectable label. In another preferred embodiment, in step (2), the antibody or antigen-binding fragment thereof of the invention is detected using a reagent bearing a detectable label.

The method may be used for diagnostic purposes, or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient). In certain preferred embodiments, said CLDN18.2 is human CLDN18.2.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the invention in the preparation of a kit for detecting the presence or amount of CLDN18.2 in a sample. In certain preferred embodiments, said CLDN18.2 is human CLDN18.2.

In another aspect, the present invention provides a method for detecting whether a tumor can be treated by an anti-tumor therapy targeting CLDN18.2, comprising the steps of:

(1) Contacting a sample containing said tumor cells with an antibody or antigen-binding fragment thereof of the invention;

(2) Detecting the formation of a complex between the antibody or antigen binding fragment thereof and CLDN18.2.

In certain preferred embodiments, the complex is formed between the antibody or antigen-binding fragment thereof and CLDN18.2 expressed by tumor cells in the sample.

In certain preferred embodiments, the sample is from a subject having, suspected of having, or at risk of having a tumor. In certain preferred embodiments, the sample is from a tissue or organ wherein the cells do not substantially express CLDN18.2 when the tissue or organ is not afflicted with cancer. In certain preferred embodiments, the tissue is selected from stomach tissue, lung tissue, esophageal tissue, pancreatic tissue, or breast tissue, and the tissue has optionally been diagnosed as affected by cancer, for example by visual inspection or culture testing of cells of the tissue or organ. In certain preferred embodiments, the tissue is other than stomach tissue. In certain preferred embodiments, the tissue is lung tissue, esophageal tissue, pancreatic tissue, or breast tissue. In such embodiments, when there is an increase in the amount of CLDN18.2 or cells expressing CLDN18.2, and/or cells expressing CLDN18.2 as compared to a reference level (e.g., as compared to a patient without a tumor disease), the subject is indicated as being suitable for anti-tumor therapy targeting CLDN18.2.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention further carries a detectable label. In another preferred embodiment, in step (2), the antibody or antigen-binding fragment thereof of the present invention is detected using a reagent bearing a detectable label.

In certain preferred embodiments, said CLDN18.2 is human CLDN18.2.

In certain preferred embodiments, the tumor is selected from the group consisting of esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, otorhinolaryngological (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastatic cancers thereof (e.g., gastric cancer metastasis, such as Krukenberg tumor, peritoneal metastasis, or lymph node metastasis.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the invention in the preparation of a kit for detecting whether a tumor is treatable by an anti-tumor therapy targeting CLDN18.2.

In certain preferred embodiments, the antibody or antigen-binding fragment thereof carries a detectable label.

In certain preferred embodiments, said CLDN18.2 is human CLDN18.2.

In certain preferred embodiments, the tumor is selected from esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, otorhinolaryngological (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastatic cancers thereof (e.g., gastric cancer metastasis such as Krukenberg tumor, peritoneal metastasis, or lymph node metastasis.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunological laboratories and the like used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "CLDN18 (Claudin 18 )" has the meaning commonly understood by those skilled in the art, belongs to the Claudin family, is a transmembrane protein within the epithelial-endothelial tight junction, and exists in the form of two splice variants CLDN18.1 and CLDN18.2. The sequences of CLDN18.1 and CLDN18.2 are well known in the art and can be found in NCBI database accession numbers NP _057453.1 and NP _001002026.1, respectively.

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, igD, igG, igA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each V _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antigen-binding sites, respectively. The assignment of amino acids to regions or domains may follow Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia&Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342, 878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs, designated CDR1, CDR2 and CDR3, in the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed. Public Health service, national Institutes of Health, bethesda, md., 1991), chothia numbering system (Chothia & Lesk (1987) J.mol.biol.196:901-917, chothia et al (1989) Nature 342. For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, e.g., lefranc et al, dev. Complex. Immunol.27:55-77,2003).

In the present invention, the CDRs contained in the antibodies of the present invention or antigen binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat, chothia, or IMGT numbering system. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat numbering system.

As used herein, the terms "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be of different isotypes, e.g., igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment comprising a full-length antibodyA polypeptide that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, also referred to as an "antigen-binding portion". See generally, fundamental Immunology, ch.7 (Paul, W., ed., 2nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes ₂ Fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabodies (diabodies), single domain antibodies (single domain antibodies), chimeric antibodies, linear antibodies (linear antibodies), nanobodies (technical from Domantis), probodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding capability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al, 2005; nat Biotechnol, 23.

As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein "full-length heavy chain" refers to a polypeptide chain consisting of, in the N-terminal to C-terminal direction, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, when the full length antibody is of IgE isotype, optionally further comprising a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. Two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric antibodies or humanized antibodies are also contemplated. The full-length antibody of the present invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, that specifically recognize/bind to the same antigen.

As herein describedAs used, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al, nature 341 544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reductively linked F (ab') ₂ The fragment obtained after disulfide bonding of the two heavy chain fragments of the fragment consists of one complete fragment of Fd of the light and heavy chains (consisting of the VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that form an entire antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity on the antibody. However, even one variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the entire binding site.

As used herein, the term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain and the second and third constant regions of a second heavy chain of an antibody. The Fc fragment of an antibody has a number of different functions, but is not involved in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein The VL and VH are connected by a linker (see, e.g., bird et al, science 242 423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA 85. Such scFv molecules can have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a peptide having an amino acid sequence (GGGGS) ₄ To be connected withFirst, but variants thereof can also be used (Holliger et al (1993), proc.natl.acad.sci.usa 90. Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J. Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Rovers et al (2001), cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but that a linker is used that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and create two antigen binding sites (see, e.g., holliger p. Et al, proc.natl.acad.sci.usa.90: 6444-6448 (1993), and Poljak r.j. Et al, strucure 2 1121-1123 (1994)).

As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen to which the full-length antibody binds. Single domain antibodies are also known as nanobodies (nanobodies).

As used herein, the term "probody" has the meaning commonly understood by those skilled in the art, and refers to a masked antibody that remains inert in healthy tissue, but is specifically activated in the disease environment (e.g., via protease cleavage by proteases that are either abundant or characteristic in the disease environment). For detailed teachings thereof see, e.g., desnoyers et al, sci. Trans. Med., 5. Similar masking techniques can be used for any of the antibodies or antigen-binding portions thereof described herein.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or competes with the full length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (e.g., antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of antibodies are specifically screened for specificity in the same manner as for intact antibodies.

Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

As used herein, the terms "monoclonal antibody," "mAb," and "mAb" have the same meaning and are used interchangeably and refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. The monoclonal antibody has high specificity to a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" is used merely to indicate that the antibody is characterized as being obtained from a population of highly homologous antibodies, and is not to be construed as requiring production of the antibody by any particular method.

Monoclonal antibodies of the invention can be prepared by a variety of techniques, such as hybridoma techniques (see, e.g., kohler et al, nature,256, 495, 1975), recombinant DNA techniques (see, e.g., U.S. Pat. No.4,816,567), or phage antibody library techniques (see, e.g., clackson et al, nature352:624-628,1991, or Marks et al, J.mol.biol.222:581-597, 1991).

Antibodies can be purified by well-known techniques, such as affinity chromatography using protein a or protein G. Subsequently or alternatively, the specific antigen (the target molecule recognized by the antibody) or an epitope thereof may be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography. Purification of immunoglobulins can be found, for example, in D.Wilkinson (The Scientist, published by The Scientist, inc., philadelphia Pa., vol.14, no.8 (Apr.17, 2000), pp.25-28).

As used herein, the term "Chimeric antibody (Chimeric antibody)" refers to an antibody, a portion of which light chain or/and heavy chain is derived from one antibody (which may be derived from a certain species or belonging to a certain antibody class or subclass) and another portion of which light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belonging to the same or different antibody class or subclass), but which nevertheless retains binding activity to an antigen of interest (u.s.p 4,816,567to Cabilly et al; morrison et al, proc.natl.acad.sci.usa, 81. For example, the term "chimeric antibody" can include an antibody (e.g., a human murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody) and the heavy and light chain variable regions of the antibody are from a second antibody (e.g., a human antibody).

As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence modified to increase homology to the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having a desired property (e.g., antigen specificity, affinity, reactivity, etc.).

In the present application, the desired properties of the antibodies of the invention include: (1) Specifically recognizes/binds to CLDN18.2 (in particular human CLDN 18.2); (2) mediates internalization of CLDN18.2; (3) Inducing killing of cells expressing human CLDN18.2 by antibody-dependent cell-mediated cytotoxicity (ADCC); (4) Inducing killing of cells expressing human CLDN18.2 by Complement Dependent Cytotoxicity (CDC); (5) ability to prevent and/or treat tumors. The antibodies of the invention have one or more of the desired properties described above.

The chimeric antibody or humanized antibody of the present invention can be prepared based on the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chains can be obtained from a murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

To make chimeric antibodies, murine immunoglobulin variable regions can be joined to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Pat. No.4,816,567 to Cabilly et al). For example, DNA encoding a VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene. The sequence of the Human heavy chain constant region gene is known in the art (see, e.g., kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, igG2, igG3, igG4, igA, igE, igM or IgD constant region, but is typically preferably an IgG1 or IgG4 constant region. For example, the DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region CL to obtain a full-length light chain gene (as well as the Fab light chain gene). The sequence of the Human light chain constant region gene is known in the art (see, e.g., kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is typically preferably a kappa constant region.

To make humanized antibodies, murine CDR regions can be inserted into human framework sequences using Methods known in the art (see, e.g., U.S. Pat. No.5,225,539 to Winter; U.S. Pat. Nos.5,530,101 to Queen et al; 5,585,089;5,693,762; and 6,180,370; and Lo, benny, K.C., edition, in Antibody Engineering: methods and Protocols, volume 248, humana Press, N.J., 2004). Alternatively, transgenic animals can also be used which are capable of not producing endogenous immunoglobulins after immunization and which are capable of producing a complete human antibody repertoire. For example, antibodies have been reported in chimeric and germline mutant miceHomozygous deletion of the heavy chain joining region (JH) gene can completely suppress endogenous antibody production, and then transfer of a human germline immunoglobulin gene array into the germline mutant mouse will result in the mouse producing human antibodies upon encountering antigen stimulation (see, e.g., jakobovits et al, 1993, proc.natl.acad.sci.usa 90. Non-limiting examples of such transgenic animals include, huMAb mice (Metarex, inc.), which contain a human immunoglobulin gene minilocus (minioci) encoding unrearranged human heavy (μ and γ) and kappa light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and kappa chain loci (see, e.g., lonberg et al (1994) Nature368 (6474): 856-859); or "KM mice carrying human heavy chain transgenes and human light chain transchromosomes ^TM "(see patent application WO 02/43478). Other methods of antibody humanization engineering also include phage display techniques (Hoogenboom et al, 1991, J.mol.biol.227, marks et al, J.mol.biol.1991, 222, 581-597, vaughan et al, 1996, nature Biotech 14.

As used herein, the term "germline antibody gene (germline antibody gene)" or "germline antibody gene segment (germline antibody gene segment)" refers to immunoglobulin-encoding sequences present in the genome of an organism that have not undergone a maturation process that can lead to genetic rearrangements and mutations that express specific immunoglobulins. In the present invention, the expression "heavy chain germline gene" means the germline antibody gene or gene segment encoding the immunoglobulin heavy chain, which includes the V gene (variable), the D gene (diversity), the J gene (conjugation), and the C gene (constant); similarly, the expression "light chain germline gene" refers to germline antibody genes or gene segments encoding immunoglobulin light chains, which include the V gene (variable), the J gene (junction), and the C gene (constant). In the present invention, the amino acid sequence encoded by the germline antibody gene or germline antibody gene segment is also referred to as "germline sequence". Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from specialized databases (e.g., IMGT, unsmig, NCBI, or VBASE 2).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction may be the equilibrium dissociation constant (K) of the interaction _D ) And (4) showing. In the present invention, the term "K _D "refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody has less than about 10 ^-9 M, e.g. less than about 10 ^-9 M、10 ^-10 M、10 ^-11 M or 10 ^-12 M or less affinity (K) _D ) Binding the antigen. Specific binding properties between two molecules can be determined using methods well known in the art, for example in a BIACORE instrument using Surface Plasmon Resonance (SPR).

As used herein, the term "cytotoxic agent" includes any agent that is harmful to (e.g., kills) a cell, such as a chemotherapeutic drug, a bacterial toxin, a plant toxin, or a radioisotope, and the like.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or artificial chromosome (PAC) of P1 origin; bacteriophage such as lambda bacteriophage or M13 bacteriophage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK293 cells, or human cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of e.meyers and w.miller (comput.appl biosci.,4, 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. In addition, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, as well as the GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and the length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al, proc. Natl Acad. Set USA 94 412-417 (1997), which are incorporated herein by reference.

The twenty conventional amino acids referred to herein are written following conventional usage. See, for example, immunology-A Synthesis (2 nd edition, E.S. Golub and D.R.Gren, eds., sinauer Associates, sunderland, mass. (1991)) which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "Chimeric Antigen Receptor (CAR)" refers to an engineered T cell receptor having an extracellular antibody-derived targeting domain (e.g., scFv) that is joined to one or more intracellular signaling domains of the T cell receptor. In the present invention, the term "chimeric antigen receptor T cell" is a T cell that expresses a CAR and has an antigen specificity determined by the targeting domain of the CAR. Methods of making CARs (e.g., for cancer treatment) are known in the art, see, e.g., park et al, trends biotechnol, 29; grupp et al, NEnglJMed.,368 1509-1518,2013; han et al, j.hematolncol, 6, 47,2013; PCT patent publications WO2012/079000, WO2013/059593; and U.S. patent publication 2012/0213783, which is incorporated by reference in its entirety herein.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., remington's Pharmaceutical sciences. Edited by geno AR, 9th ed. Pennsylvania: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to stabilize the desired activity of the active ingredient in a medicament, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as milk albumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

As used herein, the term "prevention" refers to a method performed in order to prevent or delay the onset of a disease or disorder or symptom (e.g., tumor) in a subject. As used herein, the term "treatment" refers to a method performed in order to obtain a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., no longer worsening) of the state of the disease, delay or slowing of disease progression, amelioration or palliation of the state of the disease, and remission (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to prolonging survival as compared to expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, e.g., a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., human) has a tumor (e.g., a tumor expressing CLDN 18.2), or is at risk for having a disease as described above.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a prophylactically effective amount (e.g., tumor) refers to an amount sufficient to prevent, or delay the onset of a disease (e.g., tumor); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

As used herein, the term "immune effector cell" includes cells having hematopoietic origin and functioning in an immune response, such as lymphocytes, e.g., B cells and T cells; a natural killer cell; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes. In certain preferred embodiments, the immune effector cell is a T cell.

As used herein, the term "metastasis" refers to the spread of cancer cells from their original site to other parts of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and vessels and subsequent infiltration of target organs following transport from the blood. Finally, the growth of new tumors (i.e., secondary or metastatic tumors) at the target site is dependent on angiogenesis. Tumor metastasis often occurs even after resection of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the invention relates to "distant metastasis", which relates to metastasis distant from the primary tumor and regional lymph node system. Cells of secondary or metastatic tumors are similar to those in the original tumor. This means that, for example, if ovarian cancer metastasizes to the liver, the secondary tumor is composed of abnormal ovarian cells (rather than abnormal liver cells). Then the tumor in the liver is called metastatic ovarian cancer (rather than liver cancer).

Advantageous effects of the invention

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the antibodies of the invention are capable of specifically recognizing/binding to CLDN18.2 and are capable of inducing killing of cells (e.g., tumor cells) expressing CLDN18.2 by ADCC and/or CDC. Thus, the antibodies of the invention have the potential for use in the prevention and/or treatment of tumors, in particular tumors expressing CLDN18.2. The humanized antibodies of the invention not only retain the function and properties of the parent antibody, but also have a high degree of humanization, and thus can be safely administered to human subjects without eliciting an immunogenic response. It is particularly surprising that the antibodies of the invention have significantly improved affinity activity and tumor killing activity compared to known anti-CLDN 18.2 antibodies. Thus, the antibodies of the invention (particularly humanized antibodies) are of significant clinical value.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIGS. 1A-1D show the results of measurements of the binding activity of anti-CLDN 18.2 murine antibodies to different cell surfaces CLDN18.2 or CLDN18.1, respectively. FIG. 1A; FIG. 1B: HEK 293T-mCLDNN 18.2; FIG. 1C: HEK 293T-hLDN 18.1; FIG. 1D: HEK293T.

Fig. 2 shows the results of the determination of the binding activity of the anti-CLDN 18.2 chimeric antibody to cell surface CLDN18.2.

FIGS. 3A-3C show the results of the determination of ADCC activity of anti-CLDN 18.2 chimeric antibodies against HEK 293T-hCDLN18.2, KATO-III and NUGC4, respectively. FIG. 3A: HEK 293T-hLDN 18.2; FIG. 3B; fig. 3 c.

FIGS. 4A-4B show the results of measurements of CDC activity of anti-CLDN 18.2 chimeric antibodies against HEK 293T-hCLDNN 18.2 and KATO-III, respectively. FIG. 4A: HEK 293T-hLDN 18.2; FIG. 4B.

Fig. 5 shows the results of measurement of the binding activity of the anti-CLDN 18.2 humanized antibody to cell surface CLDN18.2.

FIG. 6 shows the results of an assay of ADCC activity of anti-CLDN 18.2 humanized antibody against KATO-III.

FIG. 7 shows the results of determination of CDC activity of anti-CLDN 18.2 humanized antibody against KATO-III.

Fig. 8A-8B show the effect of anti-CLDN 18.2 antibodies on tumor volume (a) and survival (B), respectively, in a mouse tumor model.

Sequence information

Information on the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

Detailed Description

The invention will now be described with reference to the following examples which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. Molecular biology laboratory Manual, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Example 1: production of anti-CLDN 18.2 murine antibodies

To obtain anti-human CLDN18.2 antibodies, mice (strain code 216, beijing wilfordii laboratory animal technology limited) were vaccinated using a different immunization strategy (table 2) to elicit the production of murine-derived mabs. Wherein the antigen comprises: expression plasmid expressing nucleic acid sequence encoding human CLDN18.2 extracellular 1 region (CLDN 18.2-ECL1 DNA; SEQ ID NO:83, vector pcDNA3.1), expression plasmid expressing nucleic acid sequence encoding human CLDN18.2 extracellular 1 region-complement C3 (CLDN 18.2-ECL1-C3d DNA; SEQ ID NO:84, vector pcDNA3.1), expression plasmid expressing nucleic acid sequence encoding human CLDN18.2 full length (hCLDN 18.2 DNA; SEQ ID NO:85, vector pcDNA3.1), chinese hamster ovary (CHO-hCLDN 18.2) transfected to express human CLDN18.2, and kidney embryo cell (LDHEK 293-hCN18.2) transfected to express human CLDN18.2 high expression. Adjuvants include: in vivo-jetPEI (Polyplus Transfection Inc., cat # 201-50G), ODN 1826VacciGrade (InvivoGen, cat # vac-1826-1), complete Freund's adjuvant CFA (InvivoGen, cat # vac-CFA-60). Routes of administration include: muscle (im), intraperitoneal (ip) and subcutaneous (sc). Splenocytes from immunized mice were fused with mouse myeloma cells SP2/0 using the polyethylene glycol method 3 days after the booster immunization to obtain B-cell fusions that both express antibodies and proliferate indefinitely in vitro, and were cultured in HAT selective medium. The fused hybridoma cells were plated in 96-well cell culture plates and positive clones were selected by primary screening for 2-3 rounds of subcloning.

Table 2: immunization protocols for the production of monoclonal antibodies

Primary screening: the supernatant of the growing clones was tested for cell surface CLDN18.2 binding capacity in the primary screen by using cells expressing human CLDN18.2. Assessment of binding capacity was performed on a whole-field cell scanning analyzer by revealing the presence of reactive antibodies in the supernatant with a DyLight488 goat anti-mouse IgG (Abcam catalog No. ab 97015) secondary antibody (see example 3).

Secondary screening: supernatants of the above fusion clones binding to human CLDN18.2 were tested for binding ability to cell surface CLDN18.1 using cells expressed with human CLDN18. Assessment of binding capacity was performed on a whole-field cell scanning analyzer by revealing the presence of reactive antibodies in the supernatant with a DyLight488 goat anti-mouse IgG (Abcam catalog No. ab 97015) secondary antibody (see example 3 for detailed experimental procedures). Finally obtaining 10 positive hybridoma monoclonal cell strains, and then separating and purifying the culture supernatant to obtain the following antibodies: 1D10, 2F12, 3F2, 5F9, 9F3, 10B11, 27B5, 37B1, 44A8, 44F7.

Example 2: evaluation of antigen binding Activity of anti-CLDN 18.2 murine antibody

2.1 construction of cell lines expressing CLDN18.2

Human CLDN18.2 (SEQ ID NO: 86) or CLDN18.1 (SEQ ID NO: 88), or mouse CLDN18.2 (SEQ ID NO: 87), respectively, were overexpressed on HEK293T cells (ATCC), CHOS cells (Invitrogen), OCUM-1 gastric cancer tumor cells (Byokoku), by the method of lentivirus infection and resistance selection (MOI =3-10,5 μ g/ml polybrene). The lentivirus is provided by Shanghai Ji Kai Gen-chemical technology GmbH, the cells are infected for 72 hours, then added with corresponding resistance to be cultured for 2-4 weeks, amplified and frozen for subsequent experiments.

2.2 detection of binding of murine antibodies to cell surfaces CLDN18.2 and CLDN18.1 by cell scanning Analyzer

Using HEK293T-hcldn18.2 and OCUM-1-hcldn18.2 recombinant for expression of human CLDN18.2 (hcldn 18.2), or HEK293T-mcldn18.2 for expression of mouse CLDN18.2 (mcldn 18.2), and the corresponding negative control cell line HEK293T; alternatively, a titration binding curve was generated using HEK 293T-hCDTN 18.1, CHOS-hCDTN 18.1 recombinantly expressing human CLDN18.1 (hCDN18.1), and DyLight488 goat anti-mouse IgG (Abcam catalog number ab 97015) or DyLight488 goat anti-human IgG (Abcam catalog number ab 97003) as the second antibody using the following method.

10000 cells were plated in 100. Mu.L DMEM +10% FBS/well, flat bottom 96 well plates were used, cells were allowed to adhere or settle to the bottom of the wells overnight, and the supernatant was removed the next day. A3-fold gradient dilution was performed by diluting 1/3 volume (100. Mu.L) in 200. Mu.L DMEM. mu.L of diluted antibody (fusion clone supernatant or subclone supernatant was used for selection) was added to each well of the cell plate, and 100. Mu.L of DMEM was added to the corresponding negative control well, and incubated at room temperature for 1 hour. After removing the supernatant, 100. Mu.L of secondary antibody (5. Mu.g/mL, diluted in DMEM) was added to each well and incubated at room temperature for 0.5 hour. After staining was complete, the supernatant was removed, washed once with PBS and 100. Mu.L PBS was added to each well, and read on the machine.

Using a full-field cell scanning analyzer (Nexcelom, model)Image Cytometer) take measurement readings on the plate. When the method is used for determination, a second antibody is selected to simultaneously carry out high-speed scanning imaging on cells in a hole corresponding to a green fluorescence channel and a bright field channel, the imaging obtained by the green fluorescence channel counts the cells bound with the antibody according to the cell morphology and fluorescence intensity setting parameters, the imaging obtained by the bright field channel counts adherent cells according to the cell morphology setting parameters, then two groups of data are divided to obtain the percentage of the cells bound with the antibody and showing green fluorescence to the total number of the adherent cells (% fluorescent cells), the binding activity of the anti-CLDN 18.2 antibody and the cells expressing CLDN18.2 is determined according to the ratio, the lower the percentage represents the poorer the ability of the determined anti-CLDN 18.2 antibody to bind to CLDN18.2 on the surface of the cells, and the higher the percentage represents the better the ability of the determined anti-CLDN 18.2 antibody to bind to CLDN18.2 on the surface of the cells. Data analysis used GraphPad.

The results of measurement of binding activity of the anti-CLDN 18.2 antibody to HEK293T expressing human CLDN18.2, HEK293T expressing murine CLDN18.2, HEK293T expressing human CLDN18.1 and control HEK293T are shown in fig. 1A-1D, respectively, on the abscissa using the logarithm of the antibody concentration and on the ordinate using the percentage of cells showing green fluorescence bound to CLDN18.2 antibody to the total number of adherent cells. The EC50 for the anti-CLDN 18.2 antibody binding antigen activity was further fitted to a curve, the results of which are shown in table 3. Among these, the reference antibody is 175D10 (Ganymed Pharmaceuticals AG) disclosed in, for example, CN101312989B, CN103509114B.

Table 3: measurement of binding Activity of anti-CLDN 18.2 antibody to CLDN18.2

Note: n.b. indicates no binding over the range of concentrations determined.

The above results show that 1D10, 2F12, 3F2, 5F9, 9F3, 10B11, 27B5, 37B1, 44A8, 44F7 all bound to cells expressing human CLDN18.2 and were significantly better than the reference antibody 175D10, while these antibodies did not bind to negative control cells (HEK 293T) that do not express CLDN18.2.

The EC50 of the binding activity of the anti-CLDN 18.2 antibody to CLDN18.1 is shown in table 4. The results show that none of 2F12, 3F2, 9F3, 10B11, 27B5, 37B1, 44A8, 44F7 bound to CLDN18.1, showing good binding specificity for CLDN18.2.

Table 4: measurement of binding Activity of anti-CLDN 18.2 antibody to CLDN18.1

Note: n.b. indicates no binding over the range of concentrations determined, blank indicates that the antibody was not measuring this data.

Example 3: sequencing of anti-CLDN 18.2 murine antibody and preparation of chimeric antibody

3.1 determination of sequences of variable regions of anti-human CLDN18.2 murine antibodies

Collecting hybridoma cells by centrifugation at 5-10 × 10 ⁶ Adding 1ml TRIzol and 0.2ml chloroform into each cell, shaking vigorously for 15 s, standing at room temperature for 3 min, centrifuging to obtain water phase, adding 0.5ml isopropanol, standing at room temperature for 10 minCollecting the precipitate, washing with ethanol, and drying to obtain RNA. Adding template RNA and primers into an ice bath centrifuge tube, correctly matching the primers and the template, performing reverse transcription, and performing PCR amplification. After the amplification is finished, 4 microcentrifuge tubes are respectively added with 2.5 mul of dNTP/ddNTP mixture, and the mixture is subjected to warm bath at 37 ℃ for 5min for later use. 1pmol of PCR amplification product, 10pmol of sequencing primer and 2. Mu.l of 5 Xsequencing buffer were added to an empty microcentrifuge tube, double distilled water was added to a total volume of 10. Mu.l, heated at 96 ℃ for 8min, cooled in ice bath for 1min and centrifuged at 4 ℃ for 10s at 10000 g. Mu.l of the pre-cooled labeling mix (dCTP, dGTP, dTTP 0.75. Mu. Mol/L each), alpha-32P-dATP 5. Mu. Ci, 1. Mu.l of 0.1mol/L DDT, and 2U of sequencer enzyme were added, water was added to 15. Mu.l, mixed well and placed on ice for 2min. Mu.l of the labeled reaction mixture was added to 4 prepared microcentrifuge tubes and incubated at 37 ℃ for 5min. Mu.l of stop solution was added to each tube. Samples were heat denatured in a water bath at 80 ℃ for 5min, 2. Mu.l of each lane was applied to the sequencing gel, the fragments were electrophoretically separated, and sequence information was collected.

The VH and VL sequences of 10 murine antibodies are shown in Table 5 below. Further, the CDR Sequences of 10 murine mAbs were determined using the method described by Kabat et al (Kabat et al, sequences of Proteins of Immunological Interest, fifth edition, public Health Service, national institutes of Health, besseda, md. (1991), pp 647-669) (Table 6).

TABLE 5 murine antibody light and heavy chain variable region amino acid sequences

TABLE 6 CDR sequences of murine antibodies

3.2 preparation of human-murine chimeric antibody and evaluation of antigen-binding Activity

The gene sequences encoding the heavy and light chain variable regions of the above murine antibody (see SEQ ID N Os: 103-122) were ligated to sequences encoding the heavy chain constant region (SEQ ID NO: 81) and light chain constant region (SEQ ID NO: 82), respectively, of a human antibody, and recombinant expression was carried out in HEK293 cells (ATCC), thereby obtaining the corresponding chimeric antibodies 1D10-chIgG1, 2F12-chIgG1, 3F2-chIgG1, 5F9-chIgG1, 9F3-chIgG1, 10B11-chIgG1, 27B5-chIgG1, 37B1-c hIgG1, 44A8-chIgG1, 44F7-chIgG1. And binding activity of the chimeric antibodies at different concentrations to HEK293T cells expressing human CLDN18.2 (HEK 293T-h CLDN 18.2) was tested by the method described in example 2. The results are shown in fig. 2, using the logarithm of the antibody concentration on the abscissa and the percentage of cells showing green fluorescence (% fluorescent cells) to the total number of adherent cells bound to CLDN18.2 antibody on the ordinate. Further curve-fitting revealed that E C had the activity of chimeric antibody binding to antigen as shown in table 7. The results showed that the chimeric antibodies 3F2-chIgG1, 5F9-chIgG1, 9F3-chIg G1, 10B11-chIgG1, 27B5-chIgG1, 37B1-chIgG1, 44A8-chIgG1, and 44F7-chI gG1 all were able to recognize/bind to human CLDN18.2.

Table 7: measurement of binding Activity of chimeric antibody to CLDN18.2

Example 4: evaluation of Activity of anti-CLDN 18.2 chimeric antibody to induce ADCC

Target cells used HEK 293T-hLDN 18.2 or human gastric carcinoma tumor cell lines KATO-III and NUGC4 expressing hLDN 18.2 naturally, and effector cells used human Peripheral Blood Mononuclear Cells (PBMC) isolated by Ficoll. Target cells were harvested, washed 2 times with PBS, and the viable cell dye Calcein AM (50. Mu.g of Calcein AM dry powder dissolved by addition of 50. Mu.l DMSO, life Technologies, cat # C3100 MP) was diluted to 3. Mu.M at 5% CO% ₂ The target cells were stained at 37 ℃ for 30 minutes. Staining was complete, washed 2 times with PBS, 5000 target cells were plated in 100 μ L DMEM/well using flat bottom 96 well plates. A3-fold gradient dilution was performed by diluting 1/3 volume (100. Mu.L) in 200. Mu.L DMEM. 50 μ L of diluted antibody (50 μ L of LDMEM medium in the non-specific killing control well) was added to the corresponding well of the cell plate at 5% CO ₂ After incubation with target cells for 30 minutes at 37 deg.C, 50. Mu.L of isolated PBMC 50000 cells per well were added as effector cells. Centrifuge at 1000rpm for 3 minutes to allow the cells to settle to the bottom of the plate. The whole field cell scanning analyzer (Nexcelom, model) was used at different time points for 0 hour, 2 hours, 3 hours, 4 hours, and 6 hoursImage Cytometer) take measurement readings on the plate. During measurement, calcein AM is selected to correspond to a green fluorescence channel and a bright field channel and simultaneously carry out high-speed scanning imaging on cells in the hole, live cells in the hole are counted according to the imaging obtained by the green fluorescence channel and the fluorescence intensity setting parameters, total cells in the hole are counted according to the cell shape setting parameters obtained by the bright field channel, and then the two groups of data are divided to obtain the percentage of the live cells displaying green fluorescence in the total number of the cells. The percentage of non-specific control wells minus the corresponding percentage of antibody gives the percentage of cells in total cells that have undergone specific cell lysis in the presence of the antibody, from which value the activity of the anti-CLDN 18.2 antibody to induce ADCC is determined, a lower percentage representing a poorer ability of the anti-CLDN 18.2 antibody to induce ADCC, whereas a higher percentage represents a better ability of the anti-CLDN 18.2 antibody to induce ADCC. Data analysis used GraphPad.

The results of the determination of ADCC activity induced by anti-CLDN 18.2 antibodies are shown in figures 3A-3C, using the logarithm of the antibody concentration on the abscissa and the corrected percentage of specific lysis on the ordinate. The EC50 for the anti-CLDN 18.2 chimeric antibody to induce ADCC was further fitted to a curve, the results of which are shown in table 8. The results show that the detected antibodies all induce the killing effect of PBMC on cells expressing human CLDN18.2, and are significantly better than the reference antibody 175D10.

Table 8: measurement result of ADCC Activity induced by chimeric antibody

Note: n.a. indicates that the EC50 is greater than the assay concentration range, and a blank indicates that the antibody was not measuring this data.

Example 5: evaluation of CDC-inducing Activity of anti-CLDN 18.2 chimeric antibody

HEK 293T-hLDN 18.2 or a human gastric cancer tumor cell line KATO-III naturally expressing hLDN 18.2 is used as a target cell, and fresh human serum is used as an effector cell. Target cells were harvested, washed 2 times with PBS, and the viable cell dye Calcein AM (50. Mu.g of Calcein AM dry powder dissolved by addition of 50. Mu.l DMSO, life Technologies, cat # C3100 MP) was diluted to 3. Mu.M at 5% CO% ₂ The target cells were stained at 37 ℃ for 30 minutes. Staining was complete, washed 2 times with PBS, 8000 target cells plated in 25 μ L DMEM/well using flat bottom 96-well plates. A3-fold gradient dilution was performed by diluting 1/3 volume (100. Mu.L) in 200. Mu.L DMEM. mu.L of diluted antibody (25. Mu.L of LDMEM medium was added to each well of the cell plate) was added to each well of the plate, and 50. Mu.L of freshly isolated 20% human serum (diluted with DMEM) was added to each well as effector cells after incubation with target cells at 5% CO2 for 30 minutes at 37 ℃. Centrifuge at 1000rpm for 3 minutes to allow the cells to settle to the bottom of the plate. The whole field cell scanning analyzer (Nexcelom, model) was used at different time points for 0 hour, 1 hour, 2 hours, 3 hours, and 4 hoursImage Cytometer) take assay readings on the plates. During measurement, calcein AM corresponding to a green fluorescence channel and a bright field channel are selected to simultaneously carry out high-speed scanning imaging on cells in the hole, live cells in the hole are counted according to the imaging obtained by the green fluorescence channel and the fluorescence intensity setting parameters of the cell with a fluorescence label, and total cells in the hole are counted according to the imaging obtained by the bright field channel and the cell shape setting parametersAnd then the two sets of data were divided to obtain the percentage of live cells showing green fluorescence to the total number of cells, the percentage of cells undergoing specific cell killing in the presence of the antibody was subtracted from the percentage of non-specific control wells to obtain the percentage of total cells, and the activity of inducing CDC by the anti-CLDN 18.2 antibody was judged based on this value, a lower percentage indicates a poorer ability of the measured anti-CLDN 18.2 antibody to induce CDC, whereas a higher percentage indicates a better ability of the measured anti-CLDN 18.2 antibody to induce CDC. Data analysis used GraphPad.

The results of the determination of CDC activity induced by anti-CLDN 18.2 antibodies are shown in fig. 4A-4B, using the logarithm of antibody concentration on the abscissa and corrected specific cell killing percentage on the ordinate. The EC50 for CDC induction by anti-CLDN 18.2 antibodies was further fitted to a curve, the results of which are shown in table 9. The results show that the detected antibodies can induce the killing effect of the complement in human serum on the cells expressing human CLDN18.2, and are obviously better than the reference antibody 175D10.

Table 9: measurement result of CDC Activity induced by chimeric antibody

Note: blank indicates that the antibody was not measuring this data.

Example 6: evaluation of CLDN18.2 antibody-induced CLDN18.2 internalization activity

This example detects the level of CLDN18.2 internalization by anti-CLDN 18.2 antibodies mediated cell (HEK 293T-hcldn 18.2) surface by flow cytometry. Two cells were incubated with 10. Mu.g/mL chimeric antibody for 1 and 4 hours at 37 ℃. After washing several times with PBS containing 2% FBS, 10. Mu.g/mL of the secondary antibody was added and stained at 4 ℃ for 30 minutes. The expression level of cell surface CLDN18.2 was then analyzed by flow cytometry.

MFI _4H MFI for samples after 4 hours incubation; MFI _1H For the MFI of the samples after 1 hour incubation, assuming that under these conditions the antibody has completed binding and endocytosis has not yet occurred, MFI _Background MFI as secondary antibody only, antiThe percent of body-mediated cell surface CLDN18.2 endocytosis was calculated from the following equation:

percent% internalized CLDN18.2 =100-100 × (MFI) _4H -MFI _1H )/(MFI _1H -MFI _Background )

The results are shown in table 10, and these antibodies mediated endocytosis of CLDN18.2 on the surface of HEK293T-hcldn18.2 cells to varying degrees.

Table 10: assay results for chimeric antibody-induced internalization of CLDN18.2

Example 7: humanization and Activity evaluation of anti-CLDN 18.2 antibodies

To improve sequence homology of candidate antibodies to human antibodies and reduce immunogenicity of the antibodies to humans, murine antibodies provided in the above examples can be humanized by inserting murine CDR regions into human framework sequences using Methods known in the art (see, e.g., U.S. Pat. No.5,225,539 to Winter; U.S. Pat. Nos.5,530,101;5,585,089, 5,693,762 and 6,180,370 to Queen et al; and Lo, benny, K.C., edition, in Antibody Engineering: methods and Protocols, volume 248, humana Press, new Jers, 2004). Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody).

Based on this, the inventors prepared humanized antibodies of 3 murine antibodies 44F7, which were designated 7004-09hu09, 7004-09hu10, and 7004-09hu15, respectively, and the amino acid sequences thereof are shown in the following table.

Table 11: light and heavy chain variable region amino acid sequence of humanized antibody

The gene sequences encoding the heavy and light chain variable regions of the above humanized antibody (see SEQ ID NOs: 123-128) were ligated to sequences encoding the heavy chain constant region (SEQ ID NO: 81) and light chain constant region (SEQ ID NO: 82), respectively, of a human antibody and recombinant expression was performed. Binding of the humanized antibody to HEK293T-hcldn18.2 cell surface CLDN18.2 was detected by a cell scanning analyzer using the method described in example 2.2. The results are shown in fig. 5, using the logarithm of the antibody concentration on the abscissa and the percentage of cells showing green fluorescence (% fluorescent cells) to the total number of adherent cells bound to CLDN18.2 antibody on the ordinate. The EC50 of the anti-CLDN 18.2 humanized antibody binding antigen was further fitted from a curve and the results are shown in table 12. The results show that the humanized antibodies tested all bind to cells expressing hcldn18.2 with an affinity of the same order of magnitude as the parental chimeric antibody.

Table 12: measurement of binding Activity of humanized antibody to CLDN18.2

Further, the inventors examined the ability of the humanized antibody to induce ADCC, and the evaluation method was as described in example 4. Human gastric cancer tumor cells KATO-III were used as target cells, and human peripheral blood mononuclear cells isolated by Ficoll were used as effector cells. The results of the assay are shown in figure 6, using the logarithm of the antibody concentration on the abscissa and the corrected percentage of specific cell lysis on the ordinate. The EC50 for the anti-CLDN 18.2 chimeric antibody to induce ADCC was further fitted to a curve, the results of which are shown in table 13. The results show that the antibodies detected all induce the killing effect of PBMC on cells expressing human CLDN18.2, have the same order of magnitude of ADCC induction capability as the parental chimeric antibody, and are significantly better than the reference antibody 175D10.

Table 13: measurement result of ADCC Activity induced by humanized antibody

The inventors also examined the ability of humanized antibodies to induce CDC, as evaluated in example 5. Human gastric cancer tumor cells KATO-III were used as target cells, and fresh human serum was used as effector cells. The results of the assay are shown in figure 7, using the logarithm of the antibody concentration on the abscissa and the corrected specific cell killing percentage on the ordinate. The EC50 for CDC induction by the anti-CLDN 18.2 chimeric antibody was further fitted to a curve, and the results are shown in table 14. The results show that the detected antibodies can induce the killing effect of human serum complement on cells expressing human CLDN18.2, the CDC induction capability of the antibodies is the same order of magnitude as that of the parent chimeric antibody, and the antibodies are obviously superior to the reference antibody 175D10.

Table 14: measurement results of CDC Activity induced by humanized antibody

The above results indicate that the humanized antibody of the present invention not only has a higher degree of humanization, capable of reducing the possibility of immune rejection, but also exhibits anti-tumor activity comparable to that of the parent chimeric antibody and superior to known antibodies.

Example 8: evaluation of in vivo antitumor Activity of anti-CLDN 18.2 chimeric antibody

To investigate the antitumor effects of the anti-CLDN 18.2 antibody in animals, nude mice (SCID, tokyo Wintolite laboratory animal technology Co., ltd.) were inoculated subcutaneously with 1X 10 ⁷ HEK 293T-hCLDNN 18.2 cells were administered twice weekly at 10mg/kg each time for four weeks, starting on the day of inoculation, and the tumor size of the mice was measured by continued observation after discontinuation. Figure 8A shows the change in tumor volume in mice after treatment. The results show that the 44F7-chIgG1 antibody treatment not only significantly inhibits the growth of tumors, but also completely eliminates the tumors of mice in the administration group, and the anti-tumor effect of the antibody is significantly better than that of the reference antibody 175D10. In addition, fig. 8B shows that 44F7-chIgG1 significantly prolonged the overall survival of tumor-bearing mice, demonstrating excellent anti-tumor activity. It can be seen that 44F7-chIgG1 also shows a clear advantage in the prolongation of survival over the reference antibody 175D10.Such technical effect is remarkable and surprising.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail are possible in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof having the ability of specifically binding to CLDN18.2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising: VH CDR1 shown as SEQ ID NO. 75, VH CDR2 shown as SEQ ID NO. 76, and VH CDR3 shown as SEQ ID NO. 77; and, the VL comprises: VLCDR1 as shown in SEQ ID NO:78 or 96, VLCDR2 as shown in SEQ ID NO:79, and VLCDR3 as shown in SEQ ID NO: 80.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO. 73 and VL having the sequence shown as SEQ ID NO. 74.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is humanized.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO.91 and VL having the sequence shown as SEQ ID NO. 92.

5. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof comprises: VH having a sequence shown as SEQ ID NO. 99 and VL having a sequence shown as SEQ ID NO. 100.

6. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown as SEQ ID NO. 101 and VL having the sequence shown as SEQ ID NO. 102.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and a light chain constant region (CL) derived from a human immunoglobulin.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain constant region is an IgG heavy chain constant region.

9. The antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain constant region is an IgG1, igG2, igG3, or IgG4 heavy chain constant region.

10. The antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) set forth in SEQ ID NO: 81.

11. The antibody or antigen-binding fragment thereof of claim 7, wherein the light chain constant region is a kappa light chain constant region.

12. The antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) represented by SEQ ID NO. 82.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', (Fab') ₂ Fv, diabodies (diabodies) and single domain antibodies (sdabs).

14. The antibody or antigen-binding fragment thereof of claim 13, wherein the Fv is a disulfide-linked Fv.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antigen-binding fragment is an scFv.

16. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the multispecific antibody is a bispecific antibody.

18. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof is labeled.

19. The antibody or antigen-binding fragment thereof of claim 18, wherein the antibody or antigen-binding fragment thereof carries a detectable label.

20. The antibody or antigen-binding fragment thereof of claim 19, wherein the detectable label is selected from the group consisting of an enzyme, a radionuclide, a luminescent substance, and biotin.

21. The antibody or antigen-binding fragment thereof of claim 19, wherein the detectable label is a fluorescent dye.

22. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-21, or a heavy chain variable region and a light chain variable region thereof.

23. A vector comprising the isolated nucleic acid molecule of claim 22.

24. The vector of claim 23, wherein the vector is a cloning vector or an expression vector.

25. A host cell comprising the isolated nucleic acid molecule of claim 22 or the vector of claim 23 or 24.

26. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-21, comprising culturing the host cell of claim 25 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

27. A bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1-21.

28. The bispecific or multispecific molecule of claim 27, wherein the bispecific or multispecific molecule specifically binds to CLDN18.2 and additionally specifically binds to one or more other targets.

29. The bispecific or multispecific molecule of claim 27, wherein the bispecific or multispecific molecule further comprises at least one second antibody having a second binding specificity for a second target.

30. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-21 and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

31. The immunoconjugate of claim 30, wherein the therapeutic agent is selected from cytotoxic agents.

32. The immunoconjugate of claim 30, wherein said therapeutic agent is selected from an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, and any combination thereof.

33. The immunoconjugate of any one of claims 30-32, wherein said immunoconjugate is an antibody-drug conjugate (ADC).

34. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-21, the bispecific or multispecific molecule of any one of claims 27-29, or the immunoconjugate of any one of claims 30-33, and a pharmaceutically acceptable carrier and/or excipient.

35. The pharmaceutical composition of claim 34, wherein the pharmaceutical composition further comprises an additional pharmaceutically active agent.

36. The pharmaceutical composition of claim 35, wherein the additional pharmaceutically active agent is a drug having anti-tumor activity.

37. The pharmaceutical composition of claim 36, wherein the drug having anti-tumor activity is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, anti-metabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.

38. The pharmaceutical composition of claim 35, wherein the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.

39. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-21.

40. The kit of claim 39, wherein the antibody or antigen-binding fragment thereof is detectably labeled.

41. The kit of claim 40, wherein said detectable label is selected from the group consisting of an enzyme, a radionuclide, a luminescent substance, and biotin.

42. The kit of claim 40, wherein said detectable label is a fluorescent dye.

43. The kit of claim 39, wherein the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of any one of claims 1-21.

44. The kit of claim 43, wherein said second antibody further comprises a detectable label.

45. The kit of claim 44, wherein said detectable label is selected from the group consisting of an enzyme, a radionuclide, a luminescent substance, and biotin.

46. The kit of claim 44, wherein the detectable label is a fluorescent dye.

47. A chimeric antigen receptor comprising the antigen binding domain of the antibody or antigen binding fragment thereof of any one of claims 1-21.

48. The chimeric antigen receptor of claim 47, wherein the antigen binding domain comprises the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of any one of claims 1-21.

49. The chimeric antigen receptor of claim 47, wherein the antigen binding domain is an scFv.

50. The chimeric antigen receptor of claim 47, wherein the chimeric antigen receptor comprises an antigen-binding fragment of the antibody of any one of claims 1-21.

51. The chimeric antigen receptor of any one of claims 47-50, wherein the chimeric antigen receptor is expressed by an immune effector cell.

52. The chimeric antigen receptor of claim 51, wherein the immune effector cell is a T cell.

53. An isolated nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 47-52.

54. A vector comprising the isolated nucleic acid molecule of claim 53.

55. A host cell comprising the isolated nucleic acid molecule of claim 53 or the vector of claim 54.

56. The host cell of claim 55, wherein the host cell is an immune effector cell.

57. The host cell of claim 56, wherein the immune effector cell is a T cell or NK cell.

58. The host cell of claim 55, wherein the host cell is a chimeric antigen receptor T cell (CAR-T).

59. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-21, or the bispecific or multispecific molecule of any one of claims 27-29, or the immunoconjugate of any one of claims 30-33, or the pharmaceutical composition of any one of claims 34-38, or the chimeric antigen receptor of any one of claims 47-52, or the host cell of any one of claims 55-58 in the manufacture of a medicament for inhibiting growth and/or killing of a tumor cell expressing CLDN18.2.

60. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-21, or the bispecific or multispecific molecule of any one of claims 27-29, or the immunoconjugate of any one of claims 30-33, or the pharmaceutical composition of any one of claims 34-38, or the chimeric antigen receptor of any one of claims 47-52, or the host cell of any one of claims 55-58, in the manufacture of a medicament for preventing and/or treating a CLDN 18.2-expressing tumor in a subject.

61. The use of claim 60, wherein said medicament further comprises an additional pharmaceutically active agent.

62. The use of claim 61, wherein the additional pharmaceutically active agent is a drug with anti-tumor activity.

63. The use of claim 62, wherein said drug having anti-tumor activity is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, anti-metabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.

64. The use of any one of claims 60-63, wherein the tumor is involved in tumor cells expressing CLDN18.2.

65. The use of claim 64, wherein said CLDN18.2 is expressed on the surface of said tumor cells.

66. The use of any one of claims 60-63, wherein the tumor is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, otorhinolaryngological (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastatic cancers thereof.

67. The use of claim 66, wherein the metastatic cancer is gastric cancer metastasis.

68. The use of claim 67, wherein the gastric cancer metastasis is Krukenberg tumor, peritoneal metastasis or lymph node metastasis.

69. The use of any one of claims 60-63, wherein the subject is a mammal.

70. The use of any one of claims 60-63, wherein the subject is a human.

71. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-21 in the preparation of a kit for detecting the presence or amount of CLDN18.2 in a sample; wherein the detecting comprises the steps of:

(1) Contacting the sample with the antibody or antigen-binding fragment thereof;

72. The use of claim 71, wherein said CLDN18.2 is human CLDN18.2.

73. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-21 in the preparation of a kit for detecting whether a tumor is treatable by an anti-tumor therapy targeting CLDN18.2.

74. The use of claim 73, wherein said CLDN18.2 is human CLDN18.2.

75. The use of claim 73, wherein the tumor is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, bronchial cancer, non-small cell lung cancer, breast cancer, otorhinolaryngological (ENT) cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, cancer of the gallbladder, and metastatic cancers thereof.

76. The use of claim 75, wherein the metastatic cancer is gastric cancer metastasis.

77. The use of claim 76, wherein the gastric cancer metastasis is Krukenberg tumor, peritoneal metastasis or lymph node metastasis.