CLDN18.2-Targeting Antibodies, Bispecific Antibodies, and Uses Thereof

Abstract

The present invention discloses CLDN18.2 targeting antibodies, bispecific antibodies, and their uses. CLDN18.2 targeting antibodies are single domain heavy chain antibodies that have high affinity to tumor cells that endogenously express CLDN18.2 and can induce high endocytosis activity. Bispecific antibodies can target CLDN18.2 and CD3 and retain the binding effect of Fc to FcRn; meanwhile, mutant Fc is preferred that binds to FcgR and thus reduces non-specific T cell activation caused by cross-linking of FcgR. CD3 terminal activity is optimized so that it can reduce the release of common cytokines in CRS, such as IL6 and TNFα.

Description

[Technical field]

[0001] The present invention relates to the biomedical field, in particular to CLDN18.2-targeting antibodies, bispecific antibodies, and uses thereof. [Background technology]

[0002] Cancer is one of the most fatal diseases of humans today. According to the World Health Organization (WHO) Report 2018, there are approximately 18.07 million new cases of cancer every year. Approximately 9.55 million people die from cancer every year. According to WHO estimates, gastric cancer ranks as the fifth most commonly diagnosed cancer in the world. Gastric cancer ranks as the third leading cause of cancer-related death (for men) and the fourth leading cause (for women). There are one million new cases of gastric cancer every year worldwide. Approximately 35% of patients diagnosed with primary gastric cancer in the United States are people with metastatic gastric cancer. The five-year survival rate of people diagnosed with advanced gastric cancer is 5%, with a median survival time of about six months. First-line drug administration for treating patients with metastatic / recurrent gastric cancer is divided into two cases: (1) for HER2-neu positive patients, trastuzumab is used in combination with chemotherapy drugs; (2) for HER2-neu negative patients, treatment is limited to chemotherapy drugs; however, the treatment outcomes are poor (Front Pharmacol. 2018 Sep 13;9:404).

[0003] The splice variant 1 (CLD18A1, i.e. CLDN18.1, based on Genbank accession numbers NP_057453, NM016369) and splice variant 2 (CLD18A2, i.e. CLDN18.2, based on Genbank accession numbers NM_001002026, NP_001002026) of the CLDN18 (Claudin18) molecule are integral transmembrane proteins with molecular weights of approximately 27.9 / 27.72 kD. Claudins are integral membrane proteins located within epithelial and endothelial tight junctions. The other two major proteins of the tight junction family are occludin and junctional adhesion molecules (JAMs). Claudins are important components of tight junctions and play important roles in maintaining epithelial cell polarity, controlling paracellular diffusion, and regulating cell growth and differentiation. Claudins are largely inaccessible to antibodies in well-organized epithelia, but are presumed to be exposed in tumor cells. Claudin molecules cross the cell membrane four times in the cytoplasm, both at the N-terminus and C-terminus. Human CLDN18.2 (claudin 18.2) protein is a transmembrane protein with a total length of 261 amino acids, of which 1-23 form a signal peptide; the protein has two extramembrane regions after the signal peptide, extracellular loop 1 (ECL1) of about 55 amino acids and ECL2 of about 23 amino acids. CLDN18.1 (claudin 18.1) and CLDN18.2 differ in the first 21 amino acids at the N-terminus, including the first TM and loop 1 (i.e., ECL1), but have identical primary protein sequences at the C-terminus. The ECL1 regions of human CLDN18.2 and human CLDN18.1 are highly similar, and the ECL2 regions of human CLDN18.2 and human CLDN18.1 are identical. Therefore, development of antibodies against the human CLDN18.2 protein target requires the search for antibodies targeted at the ECL1 domain or the spatial structure of the human CLDN18.2 protein, which makes research in this aspect more difficult.CLDN18.1 is selectively expressed in normal lung and gastric epithelium (Mol Cell Biol. 2001 Nov;21(21):7380-90). Expression of CLDN18.2 in normal tissues is highly restricted to differentiated cells of the gastric epithelium and is absent in the gastric stem cell region. However, it is highly expressed in several types of cancer, including gastric, esophageal, pancreatic, and lung tumors, as well as human cancer cell lines. The molecular weight of the protein varies in some cancers and adjacent normal tissues. The high molecular weight proteins observed in healthy tissues can be converted to the same molecular weight observed in cancers by treating tissue lysates with the deglycosylation compound PNGaseF. This suggests that claudins are less N-glycosylated in cancers than in their normal tissue counterparts. This structural difference may result in altered epitopes. A classical N-glycosylation motif is located within amino acid position 116 in the loop D3 domain of the molecule (CN103509110B).

[0004] At present, research on monoclonal antibodies against CLDN18.2 is limited to phase II and phase III clinical trials of Claudiximab (IMAB362) antibody (see WO 2014 / 146672). IMAB362 can induce ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) effects and mediate tumor killing. IMAB362 has shown promising effects in phase I and II clinical trials for the treatment of advanced gastroesophageal cancer (Eur J Cancer. 2018 Sep;100:17-26). However, since IMAB362 is a human or mouse chimeric antibody, it involves immunogenicity risks and does not have high affinity. Due to unmet medical needs for a number of malignant tumors, other CLDN18.2 antibodies with more desirable pharmaceutical characteristics are needed. Therefore, the art lacks effective antibodies targeting human CLDN18.2 protein, especially fully human monoclonal antibodies, and monoclonal antibodies with better cell binding activity.

[0005] At present, CLDN18.2×CD3 bispecific antibodies in clinical development include Amgen's AMG910. AMG910 can induce the TDCC (T-cell dependent cytotoxicity) effect that mediates tumor killing. However, prior art antibodies may have problems such as short half-life, insufficient drug effect, and causing cytokine release syndrome (CRS). Therefore, there is an urgent need to develop safer and more effective bispecific antibodies that can target both human CLDN18.2 and CD3 and bind to cynomolgus monkey CLDN18.2 and CD3. Summary of the Invention [Means for solving the problem]

[0006] In order to solve the technical problem in the prior art of lacking safe and effective monoclonal antibodies targeting human CLDN18.2 and bispecific antibodies that target both human CLDN18.2 and CD3 and can bind to cynomolgus monkey CLDN18.2 and CD3, the present invention provides monoclonal antibodies targeting CLDN18.2, bispecific antibodies targeting CLDN18.2 and CD3, and uses thereof.

[0007] In order to solve the above-mentioned technical problems, a first aspect of the present invention provides a CLDN18.2-targeting antibody comprising a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence shown in any one of SEQ ID NOs: 16 to 18, HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NOs: 42 to 46 and SEQ ID NOs: 48 to 54, and HCDR3 comprises an amino acid sequence shown in any one of SEQ ID NOs: 77 to 82.

[0008] In a preferred embodiment of the invention, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 42, and SEQ ID NO: 77, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:43, and SEQ ID NO:78, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:44, and SEQ ID NO:79, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:17, SEQ ID NO:45, and SEQ ID NO:80, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:43, and SEQ ID NO:80, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:43, and SEQ ID NO:78, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:43, and SEQ ID NO:81, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:46, and SEQ ID NO:82, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:48, and SEQ ID NO:78, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:49, and SEQ ID NO:78, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:50, and SEQ ID NO:78, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:51, and SEQ ID NO:78, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:52, and SEQ ID NO:78, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:53, and SEQ ID NO:79, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:54, and SEQ ID NO:78, respectively.

[0009] The above combinations of amino acid sequences for HCDR1, HCDR2, and HCDR3 are detailed in the table below.

[0010] [Table 1-a]

[0011] In a preferred embodiment of the present invention, the heavy chain variable region further comprises a framework region, in which HFR1 comprises the amino acid sequence shown in SEQ ID NO: 6 or 7, HFR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 28 to 34, HFR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 63 to 68, and HFR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 84 and 86 to 89.

[0012] In a preferred embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NOs: 150 to 157 and 159 to 165. For details, see Table b below.

[0013] [Table 1-b]

[0014] In a preferred embodiment of the invention, the antibody further comprises a heavy chain constant region. More preferably, the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3, and hIgG4, and variants thereof. Even more preferably, the heavy chain constant region is hIgG1.

[0015] In a preferred embodiment of the invention, the antibody is a full length antibody, a Fab, a Fab', a F(ab')2, an Fv, a scFv, a bispecific antibody, a multispecific antibody, a heavy chain antibody, or a single domain antibody, or a monoclonal or polyclonal antibody prepared from the above mentioned antibodies.

[0016] In a more preferred embodiment of the present invention, the antibody is a single domain antibody comprising the amino acid sequence shown in any one of SEQ ID NOs: 182 to 189 and 191 to 197. For details, see Table c below.

[0017] [Table 1-c-1]

[0018] [Table 1-c-2]

[0019] In the present invention, a "Fab fragment" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments including the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. A "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH and CH1 domains, as well as a region between the CH1 and CH2 domains where an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to provide a F(ab')2 molecule. A "F(ab')2 fragment" contains two light chains and two heavy chains including a portion of the constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacks the constant region.

[0020] In the present invention, the scFv (single chain antibody fragment) may be a conventional single chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids. In the scFv, the VL domain and the VH domain are paired to form a monovalent molecule via a linker that allows the generation of a single polypeptide chain [see, for example, Bird et al, Science 242:423-426 (1988) and Huston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of a repeated G4S amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (G4S)4 or (G4S)3 may be used, although variants thereof may also be used.

[0021] The term "multispecific antibody" is used in its broadest sense and includes antibodies with multi-epitope specificity, including, but not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), where the VH-VL unit has multi-epitope specificity, antibodies with two or more VL and VH regions, where each VH-VL unit binds to a different target or a different epitope on the same target, antibodies with two or more single variable regions, where each single variable region binds to a different target or a different epitope on the same target, full length antibodies, antibody fragments, bispecific antibodies, triabodies, antibody fragments that are covalently or non-covalently linked together, etc.

[0022] The antibodies of the present invention include monoclonal antibodies. A monoclonal antibody or mAb or Ab of the present invention refers to an antibody obtained from a single clonal cell line, including but not limited to a eukaryotic, prokaryotic, or phage clonal cell line.

[0023] In the present invention, a "heavy chain antibody" (also called HCAb) refers to an antibody that comprises only one heavy chain variable region (VHH), and two conventional CH2 and CH3 regions.

[0024] In the present invention, a "single domain antibody" (also called "nanobody") refers to a VHH structure cloned from a heavy chain antibody. It is the smallest unit known capable of binding to a target antigen.

[0025] In order to solve the above technical problem, a second aspect of the present invention provides a bispecific antibody comprising a first protein functional region targeting CD3 and a second protein functional region targeting CLDN18.2; the first protein functional region is in the form of a Fab and the second protein functional region is in the form of a VH, preferably comprising two or three VHs; if the second protein functional region comprises three tandemly linked VHs, the first protein functional region and the second protein functional region are each linked to two chains of an Fc; if the second protein functional region comprises two tandemly linked VHs, the first protein functional region and the second protein functional region are each linked to two chains of an Fc; if the second protein functional region comprises three VHs and one of the three VHs is linked to the first protein functional region, the remaining two VHs are linked in tandem and the first protein functional region and the two tandemly linked VHs of the second protein functional region are each linked to two chains of an Fc; Alternatively, the first protein functional domain is in the form of a Fab and the second protein functional domain is in the form of an HCAb; Alternatively, the first protein functional domain is in the form of a Fab and the second protein functional domain is in the form of a VH-HCAb, the second protein functional domain preferably comprising a total of four VHs.

[0026] In the present invention, the "first" and "second" in the first protein functional region and the second protein functional region have no practical meaning and are only used to distinguish antigen-binding domains for different targets. A protein functional region may contain multiple antigen-binding domains in the same or different forms; antigen-binding domains of different protein functional regions can be operatively linked together, and different antigen-binding domains of the same protein functional region cannot be linked to each other.

[0027] For example, in the present invention, the first protein functional region can be a CD3-targeting antigen-binding domain, and the second protein functional region can be a CLDN18.2-targeting antigen-binding domain.

[0028] In a preferred embodiment of the present invention, the second protein functional region comprises a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 16 to 18, HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 42 to 46 and 48 to 54, and HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 77 to 82.

[0029] More preferably, HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 42 and SEQ ID NO: 77, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 43 and SEQ ID NO: 78, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 44 and SEQ ID NO: 79, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 48 and SEQ ID NO: 78, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 49 and SEQ ID NO: 78, respectively. or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 50 and SEQ ID NO: 78, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 51 and SEQ ID NO: 78, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 52 and SEQ ID NO: 78, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 53 and SEQ ID NO: 79, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 54 and SEQ ID NO: 78, respectively. For details, see Table d below.

[0030] [Table 1-d]

[0031] More preferably, the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 150 to 152 and 159 to 165, all of which are listed in Table B.

[0032] In certain embodiments of the invention, the first protein functional region comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:101, SEQ ID NO:116, and SEQ ID NO:131, respectively, and a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:11, SEQ ID NO:38, and SEQ ID NO:72, respectively.

[0033] Preferably, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:149 or SEQ ID NO:144, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:168.

[0034] The amino acid sequence of the first protein functional domain described above is shown in Table e below.

[0035] [Table 1-e]

[0036] Preferably, the bispecific antibody comprises three polypeptide chains in the following form: The two N-termini of Fc are linked to Fab and VH, respectively; preferably, the bispecific antibody has the formula: VH CLDN18.2- VH CLDN18.2 - Hinge-CH2-CH3 or VH CLDN18.2 -VH CLDN18.2 -VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CD3 -CH1-hinge-CH2-CH3, and a second polypeptide chain of the formula: VL CD3 -having a third polypeptide chain designated CL; Alternatively, the C-terminus of one of the HCAbs is linked to the VH or VL of the Fab; preferably, the bispecific antibody has the formula: VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2 - Hinge-CH2-CH3-VH CD3 - a second polypeptide chain represented by the formula: VL CD3 Alternatively, the bispecific antibody may have a third polypeptide chain of the formula: VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2 - Hinge-CH2-CH3-VL CD3 -CL, and a second polypeptide chain of the formula: VH CD3 Alternatively, the bispecific antibody may have a third polypeptide chain of the formula: VH CLDN18.2- VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2- VH CLDN18.2 - Hinge-CH2-CH3-VH CD3 - a second polypeptide chain represented by the formula: VL CD3 Alternatively, the bispecific antibody may have a third polypeptide chain of the formula: VH CLDN18.2- VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2- VH CLDN18.2 - Hinge-CH2-CH3-VL CD3 -CL, and a second polypeptide chain of the formula: VH CD3 -having a third polypeptide chain designated CH1; In addition, the N-terminus of the heavy chain of Fab contains one VH CLDN18.2 and the C-terminus of the heavy chain is linked to one N-terminus of Fc and the C-terminus of the tandemly linked VH is linked to the other N-terminus of Fc; preferably the bispecific antibody has the formula: VH CLDN18.2- VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2 -VH CD3-CH1-hinge-CH2-CH3, and a second polypeptide chain of the formula: VL CD3 -CL.

[0037] More preferably, different functional units, such as VH, CH2-CH3, and VL, are operably linked by a linker peptide, preferably comprising an amino acid sequence as shown in any one of SEQ ID NOs: 244 to 248, preferably a sequence as shown in SEQ ID NO: 246. For details, see Table f.

[0038] [Table 1-f]

[0039] In one embodiment of the invention, the two N-termini of Fc are linked to Fab and VH, respectively; preferably, the bispecific antibody has the formula: VH CLDN18.2- Linker peptide-VH CLDN18.2 - Hinge-CH2-CH3 or VH CLDN18.2 -Linker peptide-VH CLDN18.2 -Linker peptide-VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CD3 -CH1-hinge-CH2-CH3, and a second polypeptide chain of the formula: VL CD3 -CL (for specific examples, see structures (1) and (7) in Figure 4); Alternatively, the C-terminus of one of the HCAbs is linked to the VH or VL of the Fab; preferably, the bispecific antibody has the formula: VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2 -hinge-CH2-CH3-linker peptide-VH CD3 - a second polypeptide chain represented by the formula: VL CD3 Alternatively, the bispecific antibody may have a third polypeptide chain of the formula: VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VHCLDN18.2 -hinge-CH2-CH3-linker peptide-VL CD3 -CL, and a second polypeptide chain of the formula: VH CD3 -having a third polypeptide chain designated CH1 (for specific examples, see structures (2) and (3) in Figure 4); Alternatively, one C-terminus of the VH-HCAb is linked to the VH or VL of the Fab; preferably, the bispecific antibody has the formula: VH CLDN18.2- Linker peptide-VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2- Linker peptide-VH CLDN18.2 -hinge-CH2-CH3-linker peptide-VH CD3 - a second polypeptide chain represented by the formula: VL CD3 Alternatively, the bispecific antibody may have a third polypeptide chain of the formula: VH CLDN18.2- Linker peptide-VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2- Linker peptide-VH CLDN18.2 -hinge-CH2-CH3-linker peptide-VL CD3 -CL, and a second polypeptide chain of the formula: VH CD3 -having a third polypeptide chain designated CH1 (for specific examples, see structures (4) and (5) in Figure 4); In addition, the N-terminus of the heavy chain of Fab contains one VH CLDN18.2 and the C-terminus of the heavy chain is linked to one N-terminus of Fc and the C-terminus of the tandemly linked VH is linked to the other N-terminus of Fc; preferably the bispecific antibody has the formula: VH CLDN18.2- Linker peptide-VH CLDN18.2 - a first polypeptide chain represented by hinge-CH2-CH3, the formula: VH CLDN18.2 -Linker peptide-VH CD3 -CH1-hinge-CH2-CH3, and a second polypeptide chain of the formula: VL CD3-CL (for a specific example, see structure (6) in Figure 4).

[0040] In certain embodiments of the invention, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:214, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:219, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:220, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:221, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:222, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:223, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:224, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:225, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:226, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:227, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:228, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:227, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:230, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:229; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:219, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:231, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:219, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:232, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:229; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:219, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:233, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:234, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:213, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:219, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:209, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:221, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:209, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:236, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:235, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:236, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:237, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:238, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:235, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:239, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:235, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:240, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:235, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:241, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:235, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Alternatively, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:242, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:235, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200; Additionally, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:243, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:235, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:200.

[0041] Sequence information for certain of the above mentioned embodiments is provided below in Table g.

[0042] [Table 1-g-1]

[0043] [Table 1-g-2]

[0044] [Table 1-g-3]

[0045] [Table 1-g-4]

[0046] [Table 1-g-5]

[0047] To solve the above mentioned technical problem, a third aspect of the present invention provides an isolated nucleic acid encoding an antibody according to the first aspect of the invention or a bispecific antibody according to the second aspect of the invention.

[0048] The method for preparing the nucleic acid is a conventional preparation method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned antibody by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned antibody by artificial complete sequence synthesis.

[0049] It is known to those skilled in the art that substitutions, deletions, modifications, insertions, or additions that provide polynucleotide homologs can be appropriately introduced into the base sequence encoding the amino acid sequence of the above-mentioned antibody. The polynucleotide homologs of the present invention can be generated by substituting, deleting, or adding one or more bases of the gene encoding the antibody sequence within the range in which the activity of the antibody is maintained.

[0050] In order to solve the above technical problem, the fourth aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid according to the third aspect of the present invention. The recombinant expression vector can be obtained by using conventional methods in the art, i.e., by linking the nucleic acid molecule of the present invention to various expression vectors. The expression vector can be any conventional vector in the art, as long as it can carry the above-mentioned nucleic acid molecule.

[0051] Preferably, the recombinant expression vector is a plasmid, cosmid, phage, or viral vector, which viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0052] In order to solve the above-mentioned technical problems, a fifth aspect of the present invention provides a transformant comprising a recombinant expression vector according to the fourth aspect of the present invention in a host cell; preferably, the host cell is an E. coli TG1, BL21 cell, or a CHO-K1 cell.

[0053] The recombinant expression transformant can be prepared by using a conventional method in the art, for example, by transforming the above-mentioned recombinant expression vector into a host cell. The host cell can be any conventional host cell in the art, provided that it allows stable replication of the above-mentioned recombinant expression vector and the carried nucleic acid can be efficiently expressed. Preferably, the host cell is E. coli TG1 or BL21 cell (expressing single chain antibody or Fab antibody), or CHO-K1 cell (expressing full length IgG antibody). The preferred recombinant expression transformant of the present invention can be obtained by transforming the above-mentioned recombinant expression plasmid into a host cell. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method, or an electric transformation method.

[0054] In the present invention, a CLDN18.2 targeting antibody can be used to prepare a chimeric antigen receptor (CAR) or the like on a cell, such as a T cell or an NK cell, to modify the same. Thus, a sixth aspect of the present invention provides a chimeric antigen receptor (CAR) comprising an antibody according to the first aspect of the invention or a bispecific antibody according to the second aspect of the invention.

[0055] For example, the chimeric antigen receptor may comprise the following structure: (a) an extracellular binding domain, an scFv, that specifically recognizes CLDN18.2; (b) a hinge domain; (c) a transmembrane domain; (d) a costimulatory intracellular domain; and (e) a signaling domain; the extracellular binding domain comprises a CLDN18.2-targeting antibody according to the first aspect of the present invention.

[0056] In order to solve the above mentioned technical problem, a seventh aspect of the present invention provides a genetically modified cell comprising an antibody according to the first aspect of the invention or a bispecific antibody according to the second aspect of the invention. Preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell, more preferably an immune cell, such as a T cell or a NK cell.

[0057] In order to solve the above-mentioned technical problems, an eighth aspect of the present invention provides a method for preparing a bispecific antibody, the method comprising culturing a transformant according to the fifth aspect of the present invention, and obtaining an antibody or bispecific antibody from the culture.

[0058] In order to solve the above mentioned technical problems, a ninth aspect of the present invention provides an antibody-drug conjugate (ADC) comprising a cytotoxic agent and an antibody according to the first aspect of the invention or a bispecific antibody according to the second aspect of the invention; preferably, the cytotoxic agent is MMAF or MMAE.

[0059] The preparation method of the antibody-drug conjugate may be a conventional method in the art, preferably the preparation method described in Doronina, 2006, Bioconjugate Chem. 17:114-124. Preferably, the preparation method produces an antibody-drug conjugate with a minimal low conjugate fraction (LCF) of less than 10%.

[0060] The antibody-drug conjugate may be in any physical form known in the art, preferably as a clear solution.

[0061] In order to solve the above mentioned technical problems, a tenth aspect of the present invention provides a pharmaceutical composition comprising an antibody according to the first aspect of the invention or a bispecific antibody according to the second aspect of the invention and a pharma- ceutically acceptable carrier.

[0062] Preferably, the pharmaceutical composition further comprises one or more of the group consisting of a hormonal agent, a small molecule targeted agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

[0063] In order to solve the above mentioned technical problem, an eleventh aspect of the present invention provides the use of an antibody according to the first aspect of the invention, a bispecific antibody according to the second aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention in the manufacture of a medicament for the prevention or treatment of a CD3- and / or CLDN18.2-related disease; The disease is preferably cancer, and the cancer is preferably breast cancer, ovarian cancer, uterine cancer, kidney cancer, melanoma, lung cancer, gastric cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumors, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, or colorectal cancer; preferably, the cancer is breast cancer, ovarian cancer, uterine cancer, kidney cancer, or bile duct cancer; more preferably, the cancer is breast cancer.

[0064] In order to solve the above-mentioned technical problem, a twelfth aspect of the present invention provides a kit comprising an antibody according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a chimeric antigen receptor according to the sixth aspect of the present invention, a genetically modified cell according to the seventh aspect of the present invention, an antibody-drug conjugate according to the ninth aspect of the present invention, or a pharmaceutical composition according to the tenth aspect of the present invention; Preferably, the kit further comprises: (i) a device for administering the antibody or antigen-binding fragment thereof, antibody-drug conjugate, or pharmaceutical composition; and / or (ii) instructions.

[0065] In order to solve the above technical problem, a thirteenth aspect of the present invention provides a kit-of-parts, including kit A and kit B: Kit A comprises an antibody according to the first aspect of the invention, a bispecific antibody according to the second aspect of the invention, a chimeric antigen receptor according to the sixth aspect of the invention, a genetically modified cell according to the seventh aspect of the invention, an antibody-drug conjugate according to the ninth aspect of the invention, and / or a pharmaceutical composition according to the tenth aspect of the invention; Kit B includes other anti-tumor antibodies, or pharmaceutical compositions including other anti-tumor antibodies, and / or one or more of the group consisting of hormonal agents, small molecule targeted agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0066] Kit A and Kit B may be used simultaneously, Kit A may be used before Kit B, or Kit B may be used before Kit A. The order of use may be determined according to the actual requirements of a particular application.

[0067] In order to solve the above mentioned technical problems, a fourteenth aspect of the present invention provides a method for diagnosing, treating and / or preventing a CLDN18.2 mediated disease or disorder, comprising administering to a patient in need thereof a therapeutically effective amount of an antibody according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a chimeric antigen receptor according to the sixth aspect of the present invention, an antibody-drug conjugate according to the ninth aspect of the present invention or a pharmaceutical composition according to the tenth aspect of the present invention, or treating a patient in need thereof with a kit-of-parts according to the thirteenth aspect of the present invention.

[0068] Preferably, the disease or disorder is a tumor, preferably a CLDN18.2 positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma, and / or leukemia.

[0069] "CLDN18.2 positive" as used herein refers to overexpression of CLDN18.2 protein, for example the CLDN18.2 positive cell NUGC4_D8 cell line; otherwise it is referred to as "CLDN18.2 negative".

[0070] In order to solve the above-mentioned technical problems, a fifteenth aspect of the present invention provides a method for immunodetection or determination of CLDN18.2, comprising using an antibody according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a chimeric antigen receptor according to the sixth aspect of the present invention, an antibody-drug conjugate according to the ninth aspect of the present invention, or a pharmaceutical composition according to the tenth aspect of the present invention. Preferably, the detection is for non-diagnostic and / or therapeutic purposes.

[0071] In order to solve the above-mentioned technical problems, a sixteenth aspect of the present invention provides a combination therapy comprising administering to a patient in need thereof an antibody according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a chimeric antigen receptor according to the sixth aspect of the present invention, an antibody-drug conjugate according to the ninth aspect of the present invention, or a pharmaceutical composition according to the tenth aspect of the present invention, and a second therapeutic agent; the second therapeutic agent preferably comprises another anti-tumor antibody, or a pharmaceutical composition comprising another anti-tumor antibody, and / or one or more of the group consisting of a hormonal agent, a small molecule targeted agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

[0072] In this application, all the amino acid sequences of CDRs listed are shown according to the Chothia scheme (sequences in the claims of the present invention are also shown according to the Chothia scheme). However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art using various methods, such as the Kabat scheme based on sequence variability (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health (US), Bethesda, Maryland (1991)), and the Chothia scheme based on the position of structural loop regions (see J Mol Biol 273:927-48, 1997). In this application, a combined scheme including the Kabat scheme and the Chothia scheme can also be used to determine amino acid residues in the variable domain order. The combined scheme combines the Kabat scheme with the Chothia scheme to obtain a larger range. For details, see Table 1. It will be understood by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be construed to encompass the complementarity determining regions defined by any one of the above known schemes described herein. Although the scope of what is claimed in the claims of the present invention are sequences shown based on the Chothia scheme, amino acid sequences corresponding to other CDR definition schemes are also intended to fall within the scope of the present invention.

[0073] [Table 1-1]

[0074] In the Chothia scheme, Laa to Lbb may refer to the amino acid sequence from position aa to position bb starting from the N-terminus of the light chain of an antibody; Haa to Hbb may refer to the amino acid sequence from position aa to position bb starting from the N-terminus of the heavy chain of an antibody. For example, L24 to L34 may refer to the amino acid sequence from position 24 to position 34 according to the Chothia scheme starting from the N-terminus of the light chain of an antibody; H26 to H32 may refer to the amino acid sequence from position 26 to position 32 according to the Chothia scheme starting from the N-terminus of the heavy chain of an antibody. It is well known to those skilled in the art that there are positions within the numbering CDRs according to the Chothia scheme where insertion sites exist.

[0075] In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are routine procedures that are widely used in the corresponding fields.Meanwhile, in order to more fully understand the present invention, the definitions and explanations of relevant terms are provided below.

[0076] The three-letter and one-letter codes for amino acids used in the present invention are known to those skilled in the art or are described in J. Biol. Chem, 243, p3558 (1968).

[0077] As used herein, the terms "include / includes / including" or "comprise / comprises / comprising" are intended to mean that the compositions and methods include the recited elements, but do not exclude other elements; however, "consist / consists / consisting of" are also included, depending on the context.

[0078] The term "CLDN18.2" includes isotypes, mammalian (e.g., human) CLDN18.2, species homologs of human CLDN18.2, and analogs that contain at least one common epitope with CLDN18.2. The amino acid sequence of CLDN18.2 (e.g., human CLDN18.2) is known in the art, as shown in the NCBI database.

[0079] The term "CLDN18.1" includes isotypes, mammalian (e.g., human) CLDN18.1, species homologs of human CLDN18.1, and analogs that contain at least one common epitope with CLDN18.1. The amino acid sequence of CLDN18.1 (e.g., human CLDN18.1) is known in the art, as shown in the NCBI database.

[0080] The term "epitope" refers to the portion of an antigen (e.g., human CLDN18.2) that specifically interacts with an antibody molecule. The term "compete" in the present invention refers to the ability of an antibody molecule to interfere with the binding of an anti-CLDN18.2 antibody molecule to a target (e.g., human CLDN18.2). The interference with binding may be direct or indirect (e.g., via allosteric modulation of the antibody molecule or the target). Competitive binding assays (e.g., FACS assays, ELISA, or BIACORE assays) can be used to determine the extent to which an antibody molecule can interfere with the binding of another antibody molecule to its target.

[0081] The term "antibody" as used in the present invention includes immunoglobulins of tetrapeptide chain structure formed by the binding between two identical heavy chains and two identical light chains by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and arrangement of their heavy chain constant regions, and therefore in their antigenicity. Thus, immunoglobulins can be classified into five classes or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Igs of the same class can be divided into different subclasses according to the differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chains; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ or λ chains according to the differences in the constant regions. Each of the five classes of Igs can have κ or λ chains.

[0082] In the present invention, the light chain variable region of the antibody of the present invention may further comprise a light chain constant region comprising a human κ or λ chain, or a variant thereof.In the present invention, the heavy chain variable region of the antibody of the present invention may further comprise a heavy chain constant region comprising a human IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0083] Approximately 110 amino acid sequences near the N-terminus of the heavy and light chains of an antibody are referred to as the variable region (V region) because they vary considerably; the remaining amino acid sequences near the C-terminus are relatively stable and therefore referred to as the constant region (C region). The variable region contains three hypervariable regions (HVRs) and four framework regions (FWRs) whose sequences are relatively conserved. The three hypervariable regions are also known as complementarity determining regions (CDRs) because they determine the specificity of the antibody. Each light chain variable region (VL) or heavy chain variable region (VH) consists of three CDR regions and four FWR regions arranged in the following order from amino-terminus to carboxy-terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0084] In the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain further includes 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 three domains (CH1, CH2, and CH3). 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 region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)), with conserved regions called framework regions (FWRs) distributed between them. Each VH and VL consists of three CDRs and four FWRs arranged in the following order from amino-terminus to carboxy-terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The corresponding variable regions of each heavy / light chain (VH and VL), respectively, form the antibody binding site. In particular, the heavy chain may comprise three or more CDRs, for example, 6, 9, or 12 CDRs. For example, in a bispecific antibody of the invention, the heavy chain may be an ScFv with the N-terminus of the heavy chain of an IgG antibody linked to another antibody, in which case the heavy chain comprises nine CDRs.

[0085] The term "human antibody" includes antibodies having variable and constant regions from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted into human framework sequences (i.e., "humanized antibodies").

[0086] The term "specific" as used herein with respect to an antibody means that the antibody recognizes a particular antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, such species cross-reactivity does not, in and of itself, change the classification of the antibody according to its specificity. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in and of itself, change the classification of the antibody according to its specificity. In some cases, the term "specificity" or "specific binding" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical entity, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) within the chemical entity; for example, antibodies generally recognize and bind to a particular protein structure, not a protein. If an antibody is specific for epitope "A", then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will pull down the amount of labeled A bound by the antibody.

[0087] The term "chimeric antigen receptor" or "CAR" as used herein includes an extracellular domain (extracellular binding domain), a hinge domain, a transmembrane domain (transmembrane region) that can bind antigens and polypeptides to generate a path of a cytoplasmic signal to the domain (i.e., the intracellular signal domain). The hinge domain can be considered as a part that confers flexibility to the extracellular antigen binding region. The intracellular signal domain refers to a protein that transmits information into the cell through a defined signaling pathway by generating a second messenger to regulate the activity of the cell, or a protein that functions as an effector by responding to such a messenger. It generates a signal that can promote the immune effector function of the cell of the CAR (e.g., a CART cell). The intracellular signal domain includes a signaling domain and can also include a costimulatory intracellular domain derived from a costimulatory molecule.

[0088] "Homology", "variant sequence", or "mutation" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences. If all positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared times 100%. For example, if 6 out of 10 positions in the two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. In general, the comparison is made when the two aligned sequences give the highest percentage of identity. "Optimization" refers to mutations that maintain or improve the binding of the antibody to the antigen. In the present invention, it refers to mutations that maintain, preserve, or improve the binding to CLDN18.2.

[0089] The terms "polypeptide", "peptide" and "protein" (if single-chain) are used interchangeably herein. The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably.

[0090] The term "mutation" includes amino acid or nucleotide substitutions, additions, and / or deletions. An "amino acid substitution" and a "conservative amino acid substitution" are the replacement of an amino acid residue with another amino acid residue and the replacement of an amino acid residue with an amino acid residue having a similar side chain, respectively.

[0091] "Lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant horizontal gene transfer in vivo.

[0092] The term "vector" as used herein refers to a composition that contains an isolated nucleic acid and is useful for delivering the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and others.

[0093] As used herein, the expressions "cell" and "cell line" are used interchangeably and all such designations include progeny. The term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell, such as E. coli, a fungal cell, such as a yeast cell, or an animal cell, such as a fibroblast, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK 293 cell, or a human cell.

[0094] The term "transfection" refers to the introduction of foreign nucleic acid into a eukaryotic cell. Transfection can be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0095] The term "immune cell" refers to a cell capable of eliciting an immune response. "Immune cell" and other grammatical variations can refer to immune cells of any origin. Examples of "immune cells" include white blood cells and lymphocytes (T cells, B cells, and natural killer (NK) cells) derived from hematopoietic stem cells (HSCs) generated in the bone marrow, and cells derived from bone marrow (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). The term "immune cell" can also refer to human or non-human immune cells.

[0096] The term "T cells" as used herein refers to a class of lymphocytes that mature in the thymus. T cells play a key role in cell-mediated immunity and differ from other lymphocytes (e.g., B cells) in that they have a T cell receptor on their cell surface. "T cells" include all types of immune cells that express CD3, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs), and gamma-delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses. The term "NK cells" as used herein refers to a class of lymphocytes that originate from the bone marrow and play a key role in the innate immune system. NK cells provide rapid immune responses against virus-infected, tumor, or other stressed cells, even in the absence of antibodies and major histocompatibility complexes on the cell surface.

[0097] For example, immune cells may be derived from blood, e.g., autologous T cells, allogeneic T cells, autologous NK cells, and allogeneic NK cells, or may be derived from cell lines such as NK cell lines prepared by infection with the EBV virus, NK cells obtained by induced differentiation of embryonic stem cells and iPSCs, and the NK92 cell line.

[0098] The terms "optionally," "optionally," "either," or "any one of" mean that the subsequently described event or circumstance may, but does not necessarily, occur, and the description includes examples in which the event or circumstance does or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a particular sequence may, but does not necessarily, be present. As used herein, "a" and "an" are used in the present invention to refer to one or more grammatical objects. Unless specifically stated in the context, the term "or" may be used in the present invention to mean, and is interchangeable with, the term "and / or." "About" and "approximately" are generally intended to mean an acceptable degree of error in the amount measured, given the nature or precision of the measurement. Exemplary degrees of error are typically within 10%, more typically within 5% thereof. The methods and compositions disclosed herein encompass polypeptides and nucleic acids having a designated sequence, a variant sequence, or a sequence that is substantially identical or similar thereto, e.g., a sequence that is at least 85%, 90%, 95%, 99% or more identical to a designated sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence.

[0099] The term EC as used herein 50 refers to the concentration of 50% of the maximal effect, i.e., the concentration capable of producing 50% of the maximal effect.

[0100] As used herein, the terms "antibody-drug conjugate" and "ADC" are used interchangeably.

[0101] Auristatins are fully synthetic drugs whose chemical structure is relatively easy to modify to optimize their physical properties and druggability. Auristatin derivatives used for conjugation with antibodies mainly include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), the former of which is synthesized by adding 2-amino-1-phenylpropyl-1-ol to the C-terminus of a synthetic pentapeptide derived from the natural tubulin polymerase inhibitor dolastatin-10. The inhibitory activity of MMAE against various human tumor cell lines is less than 1 nanomolar. To reduce the cytotoxic activity of MMAE, phenylalanine is added to the C-terminus of dolastatin-10 in MMAF. The cell membrane translocation ability of MMAF is poor due to the introduced carboxyl in the structure. Thus, the biological activity of cells is significantly reduced, but the inhibitory activity against cells is greatly improved after conjugation with antibodies (US Pat. No. 7,750,116).

[0102] In some embodiments, the antibody cytotoxic drug conjugate or pharma- ceutically acceptable salt or solvate thereof comprises an antibody of the invention conjugated to one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that disable tubulin by inhibiting its polymerization. Maytansine was originally isolated from the West African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol vinegar (U.S. Pat. No. 4,151,042). Maytansinoid drug modules are attractive drug modules in antibody-drug conjugates because: (i) they are relatively easy to prepare by fermentation or chemical modification or derivatization of fermentation products; (ii) they are easily derivatized with functional groups suitable for conjugation to antibodies via non-disulfide linkers; (iii) they are stable in plasma; and (iv) they are effective against a variety of tumor cell lines. Maytansine compounds suitable for use as maytansinoid drug modules are well known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973). Maytansinol and maytansinol analogues can also be prepared synthetically according to known methods. Exemplary embodiments of maytansinoid drug modules include: DM1, DM3, and DM4, as disclosed herein.

[0103] The methods, compositions, and combination therapies of the invention may be combined with other active agents or therapeutic modalities, and the methods include administering to a subject an anti-CLDN18.2 antibody molecule of the invention in an amount effective to treat or prevent a disease (e.g., cancer), optionally in combination with one or more inhibitors of PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, Tim-3 antibodies (immunotherapy), or other tumor therapeutic antibodies, Her-2, EGFR, VEGF, VEGFR antibodies, etc., as well as ADCs (e.g., T-DM1), bispecific antibodies, chemotherapeutic agents, etc., and further include administering the anti-CLDN18.2 antibody molecule, the additional active agent, or all, in an amount or dosage that is higher, lower, or equal to the amount or dosage of each active agent when used alone (e.g., as monotherapy). The amount or dosage of the anti-CLDN18.2 antibody, additional active agent, or all administered is, for example, at least 20%, at least 30%, at least 40%, or at least 50% lower than the amount or dosage of each active agent when used alone (e.g., as monotherapy).

[0104] Furthermore, as described in the examples of the present invention, anti-CLDN18.2 antibodies and drug conjugates of CLDN18.2 antibodies can achieve the purpose of treating cancer patients by binding to CLDN18.2 to induce apoptosis of target cells (tumor cells), inhibit the proliferation of tumor cells, and increase the in vivo ADCC and CDC killing effect of effector cells on tumor cells. Therefore, in a specific embodiment, the anti-CLDN18.2 antibodies and drug conjugates of CLDN18.2 antibodies described in the present invention show the anti-tumor effect of the antibodies of the present invention, as well as a method for inhibiting the proliferation of tumor cells, comprising administering a therapeutically effective amount of the anti-CLDN18.2 antibodies and drug conjugates of CLDN18.2 antibodies of the present invention to a subject via these mechanisms. The method is suitable for the in vivo treatment of cancer. To achieve a target-specific therapeutic effect, the anti-CLDN18.2 antibody molecule may be administered together with other antibodies. When administering CLDN18.2 antibodies and drug conjugates of CLDN18.2 antibodies in combination with one or more active agents, the combinations can be administered in any order or simultaneously to patients with a type of cancer, particularly patients with tumors with high expression of CLDN18.2. In certain embodiments, treatment (e.g., reduction or remission) of a hyperproliferative symptom or disease (e.g., cancer) in a subject is achieved. The method includes administering to a subject one or more anti-CLDN18.2 antibodies or drug conjugates of CLDN18.2 antibodies of the present invention, alone or in combination with other active agents or therapeutic modalities.

[0105] The anti-CLDN18.2 antibody molecule is used alone or in combination with another immunomodulator (e.g., anti-LAG-3, anti-Tim-3, anti-PD-L, or anti-PD-L1, and anti-CTLA-4 antibody molecules) to treat gastric cancer, pancreatic cancer, lung cancer, esophageal cancer, ovarian cancer, etc. The anti-CLDN18.2 antibody molecule may be administered in combination with one or more of the following: immune-based strategies, targeted agents (e.g., VEGF inhibitors, such as monoclonal antibodies against VEGF); VEGF tyrosine kinase inhibitors, such as sunitinib, sorafenib, and apatinib; RNAi inhibitors or inhibitors of downstream mediators of VEGF signaling, such as inhibitors of the mammalian target of rapamycin (mTOR).

[0106] As used herein, the terms "cancer" and "cancer patient" are intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. Examples include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.

[0107] Non-limiting examples of cancers that may be suitably treated using the CLDN18.2-targeting antibodies disclosed in the present invention include gastric cancer, esophageal cancer, lung cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, glioma, and / or leukemia, etc., or metastatic lesions thereof.

[0108] The above preferred conditions may be arbitrarily combined to obtain preferred embodiments of the present invention based on common knowledge in the art.

[0109] The reagents and starting materials used in the present invention are commercially available.

[0110] The beneficial effects of the present invention are as follows: 1. The present invention describes an anti-CLDN18.2 HCAb antibody with superior affinity, specificity, and endocytosis activity. The antibody is a novel fully human antibody containing a "heavy chain" with a molecular weight of only about half that of conventional IgG antibodies. Due to the absence of light chains, the antibody can be used for the development of bispecific antibodies, and the common problems of light chain mismatch and heterodimerization in the development of bispecific antibodies are avoided. It also has the potential to be developed into ADCs. In certain preferred embodiments, the HCAb antibody has greater affinity for tumor cells that endogenously express CLDN18.2 and can induce greater endocytosis activity compared to IMAB362 analogs. 2. The present invention also describes a CLDN18.2×CD3 bispecific antibody with excellent in vitro TDCC activity and in vivo drug effect. The bispecific antibody of the present invention has the activity of specifically binding to CLDN18.2 and has a better tumor cell killing effect than the bispecific antibody analog of Amgen's patent. In a particular preferred embodiment, the bispecific antibody has an Fc fragment, so that it retains the binding effect of Fc to FcRn; meanwhile, a mutant Fc is preferred that binds to FcgR and thus reduces the non-specific T cell activation caused by cross-linking of FcgR. The CD3 terminus activity is optimized so that it can reduce the release of common cytokines in CRS, such as IL6 and TNFα. The CLDN18.2 terminus is in the form of a tandemly linked VHH that avoids the common problem of mismatching of light and heavy chains, retains excellent hydrophilicity, and improves selectivity for tumor cells with high expression of CLDN18.2. The antibodies have good in vivo stability and long in vivo half-life, and exhibit great in vivo anti-tumor activity. [Brief description of the drawings]

[0111] [Figure 1a-c] 1a-f show the binding affinity of HCAB antibodies to (a-b) NUGC4_D8, (c) SNU601, (d) HEK293 / hCLDN18.2, and (e-f) HEK293 / hCLDN18.1 cells. [Fig. 1d-f]1a-f show the binding affinity of HCAB antibodies to (a-b) NUGC4_D8, (c) SNU601, (d) HEK293 / hCLDN18.2, and (e-f) HEK293 / hCLDN18.1 cells. [Diagram 2] 2a-b show the competitive binding activity of HCAB antibodies against (a) PR000400 and (b) PR004549 on HEK293 / hCLDN18.2 cells. [Diagram 3] 3a-b show the viability of target cells when co-cultured with test antibodies and MMAF-conjugated anti-human IgG antibodies. [Figure 4a-b] 4a-h show the structure of the CLDN18.2xCD3 bispecific antibody. [Figure 4c-d] 4a-h show the structure of the CLDN18.2xCD3 bispecific antibody. [Figure 4e-f] 4a-h show the structure of the CLDN18.2xCD3 bispecific antibody. [Figure 4g-h] 4a-h show the structure of the CLDN18.2xCD3 bispecific antibody. [Figure 5a-c] 5a-s show the binding affinity of the CLDN18.2 x CD3 bispecific antibody to (a-c) HEK293 / hCLDN18.2, (d-i) NUGC4_D8, (j-o) Jurkat, and (p-s) HEK293 / hCLDN18.1 cells. [Fig. 5d-f] 5a-s show the binding affinity of the CLDN18.2 x CD3 bispecific antibody to (a-c) HEK293 / hCLDN18.2, (d-i) NUGC4_D8, (j-o) Jurkat, and (p-s) HEK293 / hCLDN18.1 cells. [Figure 5g-i] 5a-s show the binding affinity of the CLDN18.2 x CD3 bispecific antibody to (a-c) HEK293 / hCLDN18.2, (d-i) NUGC4_D8, (j-o) Jurkat, and (p-s) HEK293 / hCLDN18.1 cells. [Figure 5j-l]5a-s show the binding affinity of the CLDN18.2 x CD3 bispecific antibody to (a-c) HEK293 / hCLDN18.2, (d-i) NUGC4_D8, (j-o) Jurkat, and (p-s) HEK293 / hCLDN18.1 cells. [Figure 5m-o] 5a-s show the binding affinity of the CLDN18.2 x CD3 bispecific antibody to (a-c) HEK293 / hCLDN18.2, (d-i) NUGC4_D8, (j-o) Jurkat, and (p-s) HEK293 / hCLDN18.1 cells. [Figure 5p-r] 5a-s show the binding affinity of the CLDN18.2 x CD3 bispecific antibody to (a-c) HEK293 / hCLDN18.2, (d-i) NUGC4_D8, (j-o) Jurkat, and (p-s) HEK293 / hCLDN18.1 cells. [Figure 5s] 5a-s show the binding affinity of the CLDN18.2 x CD3 bispecific antibody to (a-c) HEK293 / hCLDN18.2, (d-i) NUGC4_D8, (j-o) Jurkat, and (p-s) HEK293 / hCLDN18.1 cells. [Figure 6a-b] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6c-d] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6e-f] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6g-h]6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6i-j] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6k-l] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6m-n] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6o-p] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6q-r] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6s-t] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6u-v] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 6w] 6a-w show (a) the expression yield of CLDN18.2 by IM95 cells and the TDCC activity of the bispecific antibodies against (b-k) NUGC4_D8, (l-o) IM95, (p-s) HEK293 / hCLDN18.1, and (t-w) SNU620 cells. [Figure 7] FIG. 7 shows the binding affinity of the CLDN18.2×CD3 bispecific antibody to cells overexpressing human CLDN18.2 paralogous family proteins CLDN1, CLDN2, CLDN3, CLDN4, CLDN6, and CLDN9. [Figure 8] 8a-c show the binding affinity of the CLDN18.2×CD3 bispecific antibody to (a) HEK293 / cynoCLDN18.1, (b) HEK293 / cynoCLDN18.2, and (c) cynomolgus monkey CD3+ T cells. [Figure 9] FIG. 9 shows the TDCC activity of CLDN18.2×CD3 bispecific antibody against HEK293 / cynoCLDN18.2 cells by cyno T cells. [Figure 10] Figure 10a-b shows the cytokine release induced in vitro by CLDN18.2xCD3 bispecific antibody: (a) TNFα and (b) IL-6. [Figure 11] 11a-b show the ADCC activity of CLDN18.2×CD3 bispecific antibody against (a) Jurkat and (b) NUGC4_D8 cells. [Figure 12] 12a-b show the CDC activity of the CLDN18.2×CD3 bispecific antibody against (a) HEK293 / hCLDN18.2 and (b) Jurkat cells. [Figure 13]Figures 13a-b show the competitive binding activity of CLDN18.2 x CD3 bispecific antibodies to (a) PR000400 and (b) PR004549 on HEK293 / hCLDN18.2 cells. [Figure 14] FIG. 14 shows the pharmacokinetics of the CLDN18.2×CD3 bispecific antibody. [Figure 15a-b] Figures 15a-e show in vivo pharmacodynamic studies in (a-b) NUGC4_D8 PBMC, (c) SNU620 PBMC, and (d) HuP-T4 PBMC tumor models, and (e) in vivo cytokine storm studies for the CLDN18.2xCD3 bispecific antibody. [Fig. 15c-d] Figures 15a-e show in vivo pharmacodynamic studies in (a-b) NUGC4_D8 PBMC, (c) SNU620 PBMC, and (d) HuP-T4 PBMC tumor models, and (e) in vivo cytokine storm studies for the CLDN18.2xCD3 bispecific antibody. [Figure 15e] Figures 15a-e show in vivo pharmacodynamic studies in (a-b) NUGC4_D8 PBMC, (c) SNU620 PBMC, and (d) HuP-T4 PBMC tumor models, and (e) in vivo cytokine storm studies for the CLDN18.2xCD3 bispecific antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0112] The present invention is further illustrated by the following examples, which are not intended to limit the present invention. Experimental procedures in the following examples, for which conditions are not specified, are carried out according to conventional procedures and conditions or in accordance with instructions. EXAMPLES

[0113] Example 1: Preparation of expression vectors and stably transfected cell lines, and immunization of mice 1.1. Preparation of expression vectors for immunizing mice A human CLDN18.2 expression vector for immunizing fully humanized transgenic mice was prepared as follows: a cDNA sequence encoding human CLDN18.2 (Uniprot ID P56856-iso2) was synthesized, and the coding sequence of the human CLDN18.2 gene was cloned into pCAGGS plasmid (YOUBIO, VT1076) by enzymatic digestion.

[0114] 1.2. Preparation of stably transfected cell lines HEK293 (ATCC, Cat#: CRL-1573) cell lines stably expressing human CLDN18.1 or CLDN18.2 were specifically constructed as follows: Plasmids encoding human CLDN18.1 (GenScript, OHu29174D) or CLDN18.2 (GenScript, OHu03374D) were transfected into HEK293 cells to generate stable cell lines overexpressing human CLDN18.1 or CLDN18.2. Expression of CLDN18.1 and CLDN18.2 was detected by fluorescence-activated cell sorting (FACS). Specifically, 20,000 transfected cells were plated into each well of a 96-well plate, followed by the addition of a commercially available rabbit anti-human CLDN18 antibody (LifeSpan Bio, LS-C168812-400). After 1 hour of incubation at 4°C, the cells were washed twice with PBS before adding AF-680-conjugated secondary goat anti-rabbit IgG antibody (Invitrogen, A21109). After 1 hour of incubation at 4°C, the cells were washed three times with PBS before the fluorescence intensity of the cells was monitored using a FACS instrument (IntelliCytiQue Plus BR).

[0115] 1.3. Immunization of mice Fully humanized transgenic mice (commercially available Harbour HCAB 1.0 mice, purchased from Harbour BioMed) were immunized with the previously prepared human CLDN18.2 expression vector and CLDN18.2-expressing HEK293 cells (HEK293 / hCLDN18.2 cells). Bullets for gene guns were prepared with human CLDN18.2 expression vector and gold powder. Mice were immunized at multiple sites in the abdomen using a gene gun. Mice were immunized with expression vector DNA (50 μg each time) at 2-week intervals. After three immunizations, mice were immunized with HEK293 / hCLDN18.2 cells at 2-week intervals (4 × 10 cells per mouse per immunization). 6 After two immunizations, blood was collected for titration. Mouse sera were assayed for binding affinity by FACS using human CLDN18.2-expressing CHOK1 cells (kyinno, kc-1180). Mice were selected according to the titration results to screen HCAB monoclonal antibodies. Mice were immunized with HEK293 / hCLDN18.2 cells (4 × 10 per mouse) as immunogen. 6 The mice were given a booster immunization with IgG1-associated IgG1 (IgG1-associated IgG1) 3 days before screening.

[0116] Example 2: Generation and screening of anti-CLDN18.2 HCAB single domain antibodies Mice with high anti-CLDN18.2 antibody serum titers obtained in Example 1 were selected. Spleens of these mice were collected and B cells were isolated. CD138 (BD, 558626) positive plasma cells were sorted using a BD FACS AriaIII cell sorter, and CLDN18.2 (CHOK1 / hCLDN18.2, kyinno, KC-1180) positive B cell population was enriched using magnetic beads (Thermofisher, 11206D). RNA of B cells was extracted and reverse transcribed into cDNA (SuperScript IV First-Strand Synthesis System, Invitrogen, 18091200), and human VH genes were amplified by PCR using specific primers. PCR primers: 5'-GGTGTCCAGTGTSAGGTGCAGCTG-3' (SEQ ID NO: 249) 5'-AATCCCTGGGCACTGAAGAGACGGTGACC-3' (SEQ ID NO: 250)

[0117] The amplified VH gene fragment was assembled into a mammalian cell expression plasmid pCAG vector encoding the sequence of the heavy chain Fc domain of human IgG1 antibody.

[0118] The constructed plasmids were transfected into HEK293 mammalian host cells (ATCC, CRL-1573) to obtain expression supernatants of HCAb antibodies. Primary screening was performed by Mirrorball using CHOK1 / hCLDN18.2 expressing human CLDN18.2. Positive clones were selected for secondary screening. Secondary screening was performed by FACS using HEK293 / hCLDN18.1 and HEK293 / hCLDN18.2 cells.

[0119] 295 monoclonal antibodies that specifically bind to human CLDN18.2 were obtained, while the nucleotide sequences encoding the variable domains and corresponding amino acid sequences of the antibody molecules were obtained using conventional sequencing methods. After removing repetitive sequences, 211 fully human CLDN18.2 HCAb monoclonal antibodies with unique sequences and specifically binding to human CLDN18.2 were obtained. 54 antibodies with the top overall ranking were selected for recombinant expression according to the primary and secondary screening results. The purified monoclonal antibodies were further screened by flow cytometry for the ability to bind to tumor cells that endogenously express human CLDN18.2, and the top 8 antibody sequences with the top overall ranking were selected as candidate molecules, as shown in Table 3.

[0120] It is well known to those skilled in the art that the CDRs of an antibody can be defined in various ways in the art, for example, the Kabat scheme based on sequence variability (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health (US), Bethesda, Maryland (1991)), and the Chothia scheme based on the location of structural loop regions (see J Mol Biol 273:927-48, 1997). In the present application, a combined scheme including the Kabat scheme and the Chothia scheme can also be used to determine the amino acid residues in the variable domain order. The combined scheme combines the Kabat scheme with the Chothia scheme to obtain a larger range. This is detailed in Table 1 of the Summary of the Invention. Germline gene analysis and PTM site analysis obtained after sequencing from this example are shown in Table 2 below. The SEQ ID NOs of the antigen binding proteins are shown in Table 3 below.

[0121] [Table 2]

[0122] [Table 3]

[0123] Example 3: Removal of post-translational modification sites of anti-CLDN18.2 HCAB antibody Both PR004533 and PR004536 have isomerization and deamidation sites in the CDR2 region of the heavy chain. For post-translational modification sites in the CDR2 region, four amino acids, NS and DG, were randomly mutated by PCR. The PCR products were electrotransfected into E. coli to establish a random mutation library of four amino acid sites. The mutant library was screened by Mirrorball using CHOK1 / hCLDN18.1 and CHOK1 / hCLDN18.2 cells, and positive molecules that specifically bind to CLDN18.2 were selected for sequencing. The sequence numbers of the molecules are shown in Table 4 below.

[0124] [Table 4]

[0125] Example 4: Preparation and characterization of full-length anti-CLDN18.2 HCAb single domain antibodies 4.1. Preparation of recombinant HCAb single domain antibodies After obtaining the sequence of the heavy chain variable domain encoding the HCAb single domain antibody molecule, the sequence of the heavy chain variable domain can be fused with the corresponding sequence of the heavy chain constant domain of a human antibody and expressed using conventional recombinant DNA technology to obtain a recombinant HCAb single domain antibody molecule. In this example, the sequence of the heavy chain variable domain (VH) of the antibody was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the sequence of the heavy chain constant domain of a human IgG1 antibody so as to encode a full-length sequence generating an HCAb single domain antibody. In this example, the sequence of the variable domain of the monoclonal antibody molecule obtained from the immunized Harbour HCAb mouse was a human antibody sequence, so that a fully human anti-CLDN18.2 recombinant HCAb antibody was also obtained from this example.

[0126] The plasmid encoding the recombinant HCAb single domain antibody can be transfected into mammalian host cells (e.g., Chinese Hamster Ovary (CHO) cells) to obtain the corresponding purified recombinant antibody using conventional recombinant protein expression and purification techniques. Specifically, ExpiCHO-S™ cells (Gibco, A29127) were grown in ExpiCHO™ Expression Medium (Gibco, A2910001). Prior to transient transfection, cells were grown at a concentration of 3×10 6 ~4×10 6 The cells were then cultured at 37°C in an 8% CO2 shaker for 24 hours at a concentration of 7 × 10 6 ~10×10 6 The cells were then diluted to a concentration of 6 × 10 6 A 10 mL cell culture was prepared by diluting to 1000 cells / mL. 8 μg of the above mentioned plasmid (ratio of plasmid to cells is 0.8 μg:1 mL) encoding HCAb single domain antibody was dissolved in 0.4 mL of OptiPRO™ SFM medium (Gibco, 12309019). The resulting mixture was filtered through a 0.22 μm sterile filter. Then, 32 μL of ExpiFectamine™ CHO reagent (Gibco, A29129) was added to 0.37 mL of OptiPRO™ SFM medium (Gibco, 12309019). The ExpiFectamine™ CHO reagent solution was immediately and slowly added to the plasmid solution. The mixture was inverted and mixed thoroughly. The flask was shaken while the mixture of plasmid and transfection reagent was slowly added dropwise. The cells were cultured at 37°C in an 8% CO2 shaker for 8-9 days. Cell viability was observed after 8 days.

[0127] The culture was harvested and centrifuged at 3,300g for 10 min, and then the supernatant was collected and centrifuged at high speed to remove impurities. A gravity column (Bio-Rad, #7311550) containing MabSelect™ (GE Healthcare Life Science, 71-5020-91 AE) was equilibrated with PBS (pH 7.4) and rinsed with 2-5 column volumes of PBS. The supernatant sample was loaded onto the column. The column was rinsed with 5-10 column volumes of PBS. The target protein was eluted with 0.1 M glycine (pH 3.5). The eluate was adjusted to neutral with Tris-HCl (pH 8.0), concentrated, and buffer exchanged into PBS buffer by ultrafiltration tube (Millipore, UFC901024) to obtain a purified antibody solution. The purified antibody solution was then subjected to concentration determination using a NanoDrop (Thermo Scientific™ NanoDrop™ One) and subpackaged and stored for later use.

[0128] 4.2. Antibody Characterization by SEC-HPLC, HIC-HPLC, and DSF An appropriate amount of the above purified sample was loaded onto an analytical SEC column TSKgel G3000SWxl (HPLC system model: Agilent 1260 Infinity II) for the measurement of purity. The following parameters and conditions were used in this method: mobile phase: 1×PBS, pH 7.4 (Sangon, E607016); room temperature; flow rate: 1.0 mL / min; sample concentration: 1 mg / mL; injection volume: 20 μL; detection wavelength: 280 nm. After recording, the chromatogram was integrated using ChemStation software to calculate the relevant data. Analysis was generated and retention times were reported for different components in the sample.

[0129] An appropriate amount of the above purified sample was loaded onto an analytical HIC column TSKgel Buty1-NPR 4.6*35 (HPLC system model: Agilent 1260 Infinity II) for the measurement of purity and hydrophobicity. The method consisted of a linear gradient from 100% mobile phase A (20 mM PB, 1.8 M (NH4)2SO4, pH 6.0) to 100% mobile phase B (20 mM PB, pH 6.0) within 16 min. The flow rate was set at 0.7 mL / min. The sample concentration was 1 mg / mL. The injection volume was 20 μL. The detection wavelength was 280 nm. After recording, the chromatogram was integrated using ChemStation software to calculate the relevant data. The analysis was generated and the retention times were reported for the different components in the sample.

[0130] In this example, the thermal denaturation temperature (Tm) of protein molecules was measured by differential scanning fluorescence measurement (DSF). 10 μg of protein was added to a 96-well PCR plate (Thermo, AB-0700 / W), followed by 2 μL of 100× diluted dye SYPRO™ (Invitrogen, 2008138), followed by buffer to obtain a final volume of 40 μL per well. The PCR plate was sealed and placed in a real-time fluorescent quantitative PCR instrument (Bio-Rad CFX96 PCR System) and incubated at 25° C. for 5 minutes, then gradually heated from 25° C. to 95° C. with a gradient of 0.2° C. / 0.2 minutes, and cooled to 25° C. at the end of the experiment. Data analysis was performed using Bio-Rad CFX Maestro software to calculate the Tm of the samples using the FRET scanning mode. The results of the above characterization are shown in Table 5 below.

[0131] [Table 5]

[0132] Example 5: Binding affinity of anti-CLDN18.2 HCAB antibodies to cells The binding affinity of the antibodies was detected by FACS using human CLDN18.1-expressing HEK293 cells, human CLDN18.2-expressing HEK293 cells, NUGC4_D8 cells, and SNU601 cells that endogenously express human CLDN18.2 (Cobioer, CBP60507). Subcloned NUGC4_D8 cells were screened by limiting dilution using NUGC4 cells (JCRB, JCRB0834). Binding affinity was determined as follows: cells were centrifuged at 300g for 5 min and then resuspended in FACS buffer (PBS containing 2% FBS). The cell density was adjusted to 10 6 The cells / mL were adjusted and 50 μL of the cell suspension was added to each well of a 96-well plate. The antibodies were diluted to various concentrations in FACS buffer and 50 μL of the antibody dilutions were added to each well of a 96-well plate. After 2 hours of incubation at 4° C., the plate was washed twice with FACS buffer. Then, FACS buffer containing APC-conjugated goat anti-human IgG secondary antibody (Jackson, 109-605-098) was added. After 1 hour of incubation at 4° C., the plate was washed twice with FACS buffer. The cells were resuspended in fixative and then the fluorescence of the cells was monitored using a FACS instrument (ACEA NovoCyte). PR000400, an IMAB362 analog (generated in-house, see WO2014 / 146672, has the same variable regions as IMAB362 and differs from IMAB362 by only a few amino acids in the constant region) was used as a positive control for CLDN18.2 binding. PR004549, a CL-1xI2C scFc analog (generated in-house, see WO2020025792A1, has the same variable regions as CL-1xI2C scFc and differs from CL-1xI2C scFc by only a few amino acids in the constant region) was used as a positive control for CLDN18.2 binding. PR002725 antibody was used as a positive control for CLDN18.1 binding. See CN2020 / 118650 (Tables 6, 7, and 8). Iso hIgG1 (CrownBio, C0001-4) antibody was used as a negative control.

[0133] Figure 1(a-b), Table 9, and Table 10 show the binding affinity of the antibodies to NUGC4_D8 cells that endogenously express CLDN18.2. The tested antibodies were able to bind to NUGC4_D8 cells in a dose-dependent manner. The results show that: PR004533, PR004949, PR004950, PR004952, PR004953, PR007242, PR007243, PR007244, PR007245, PR007246, and PR007248 antibodies show higher affinity to NUGC4_D8 cells that endogenously express CLDN18.2 than PR000400. Figure 1c and Table 11 show the binding affinity of the antibodies to SNU601 cells that endogenously express CLDN18.2. The tested antibodies were able to bind to SNU601 cells in a dose-dependent manner. The results show that: PR004227, PR004533, PR004536, PR004540, PR004949, PR004950, and PR004952 antibodies show higher affinity to SNU601 cells that endogenously express CLDN18.2 than PR000400. Figure 1d and Table 12 show the binding affinity of the antibodies to HEK293 cells overexpressing human CLDN18.2 (HEK293 / hCLDN18.2). Figure 1(e-f) shows the binding affinity of the antibodies to HEK293 cells overexpressing human CLDN18.1 (HEK293 / hCLDN18.1). The tested antibodies have low binding affinity to HEK293 / hCLDN18.1 cells. From the above results, it can be inferred that the tested antibody binds to the human CLDN18.2 protein at ECL1 (extracellular loop 1) and not at ECL2.

[0134] [Table 6]

[0135] [Table 7]

[0136] [Table 8]

[0137] [Table 9]

[0138] [Table 10]

[0139] [Table 11]

[0140] [Table 12]

[0141] Example 6: Competitive binding activity of anti-CLDN18.2 HCAb antibodies This example studies the binding of anti-human CLDN18.2 HCAb monoclonal antibodies to the epitope region of human CLDN18.2 antigen. Competitive binding experiments were performed at the cellular level using HEK293 / hCLDN18.2 cells overexpressing human CLDN18.2. Briefly, anti-human CLDN18.2 antibodies PR000400 and PR004549 were biotinylated using a biotinylation kit (ThermoFisher, A35358) according to the instructions. 50 μL of biotinylated anti-human CLDN18.2 antibodies PR000400 or PR004549 were each thoroughly mixed with 50 μL of corresponding serially diluted non-biotinylated anti-human CLDN18.2 antibodies in a 96-well V-bottom plate (Corning, 3894). Then, a suspension of HEK293 / hCLDN18.2 cells overexpressing human CLDN18.2 was added to the wells at 3 × 10 6The cells were adjusted to 1000 cells / mL and seeded at 50 μL / well. The cells were co-incubated at 4°C for 2 hours. The cells in each well were washed twice with 200 μL pre-chilled FACS buffer (2% FBS in PBS) and centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. After washing twice, fluorescent secondary antibody (BD, 554060, 1 μg / mL final concentration) was added and incubated in the dark at 4°C for 1 hour. The cells in each well were washed twice with 200 μL pre-chilled FACS buffer (2% FBS in PBS) and centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL pre-chilled FACS buffer, and the fluorescent signal value was read using a BD FACS CANTOII. The inhibition rate was calculated using the formula: inhibition rate (%) = (AB) / A × 100 (Note: A: fluorescence signal after interaction of biotinylated antibody with ISO hIgG1 (Crownbio, C0001-4); B: fluorescence signal after interaction of biotinylated antibody with non-biotinylated antibody).

[0142] As shown in Figure 2(a-b) and Table 13, all of the anti-CLDN18.2 HCAb antibodies of the present invention can block the binding of PR000400 or PR004549 to human CLDN18.2, and the detected blocking ability of the antibodies increases in positive correlation with the antibody concentration; the inhibition rate can reach >80%. This indicates that the tested HCAbs have epitopes that are highly similar to those of PR000400 and PR004549. The tested antibodies have low binding affinity to HEK293 / hCLDN18.1 cells. From the above results, it can be inferred that the tested antibodies bind to human CLDN18.2 protein at ECL1 (extracellular loop 1) rather than ECL2.

[0143] [Table 13]

[0144] Example 7: Endocytic activity of anti-CLDN18.2 HCAB antibodies Antibodies were assayed for their ability to induce cytotoxic killing against NUGC4_D8 cells when co-cultured with MMAF-conjugated anti-human IgG antibody (Moradec, Cat#: AH-102-AF) using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, G7573). NUGC4_D8 cells were centrifuged at 300g for 5 min and then resuspended in culture medium (RPMI1640 + 10% FBS) to a density of 2 × 10 cells. 4 The concentration of cells was adjusted to 100 μL / mL. 50 μL of cell suspension was added to each well of a 96-well plate. Cells were incubated overnight at 37° C. Antibodies were diluted to various concentrations in medium and 25 μL of antibody dilution was added to each well of a 96-well plate. MMAF-conjugated anti-human IgG antibodies were diluted in medium and 25 μL of antibody dilution was added to each well of a 96-well plate to a final concentration of 6.6 nM. Cells were incubated with the antibodies for 3 days at 37° C. The 96-well plate was left undisturbed for 30 minutes at room temperature and 100 μL of CellTiter-Glo color development solution was added to each well at room temperature. Samples were then incubated in the dark for 10 minutes at room temperature. Plates were read out on a PE Enspire. Cell viability (%)=[(luminescent sample) / (luminescent mock control)]×100. PR000400 (IMAB362 analog) was used as a positive control and Iso hIgG1 (CrownBio, C0001-4) antibody was used as a negative control. Figure 3(a-b) shows the viability of target cells. When co-cultured with MMAF-conjugated anti-human IgG antibody, the test antibody shows better cytotoxic effect against NUGC4_D8 cells in a dose-dependent manner compared to PR000400.

[0145] Example 8: Structure and design of CLDN18.2 x CD3 bispecific antibodies A bispecific antibody was prepared using the selected anti-CLDN8.2 and anti-CD3 antibodies. The prepared CLDN18.2×CD3 bispecific antibody can bind to two targets simultaneously, one end can recognize CLDN18.2 specifically expressed on the surface of tumor cells, and the other end can bind to CD3 molecules on T cells. After binding to the surface of tumor cells, the CLDN18.2×CD3 bispecific antibody molecule can kill tumor cells by recruiting and activating T cells around the tumor cells.

[0146] As shown in Figure 4, structure (1) is a molecule with a "2+1" Fab-Fc-Dual VH asymmetric structure; for the molecule with the "2+1" asymmetric structure, the structure includes three protein chains, each including the heavy and light chains of the corresponding anti-CD3 antibody and the Dual VH polypeptide chain of the anti-CLDN18.2 antibody; structures (2) and (3) were molecules with a "2+1" HCAb-Fc-Fab asymmetric structure, which includes three protein chains, each including the heavy and light chains of the corresponding anti-CD3 antibody and the HCAb polypeptide chain of the anti-CLDN18.2 antibody.

[0147] Structure (4) is a molecule with a "4+1" VH-VH_HC-Fab asymmetric structure, which encompasses three protein chains, each including the heavy and light chains of the corresponding anti-CD3 antibody and the tetravalent VH polypeptide chain of the anti-CLDN18.2 antibody.

[0148] Structure (5) is a molecule with a "4+1" VH-VH_LC-Fab asymmetric structure, which encompasses three protein chains, each including the heavy and light chains of the corresponding anti-CD3 antibody and the tetravalent VH polypeptide chain of the anti-CLDN18.2 antibody.

[0149] Structure (6) is a molecule with a "3+1" VH_HC-Fab-Fc-Dual VH asymmetric structure, which includes three protein chains, each including a heavy and light chain of an anti-CD3 antibody with a corresponding N-terminus linked to a monovalent VH of an anti-CLDN18.2 antibody, and a bivalent VH polypeptide chain of an anti-CLDN18.2 antibody.

[0150] Structure (7) is a molecule with a "3+1" Fab-Fc-VH-VH-VH asymmetric structure, which comprises three protein chains, including the heavy and light chains of the corresponding anti-CD3 antibody and the trivalent VH polypeptide chain of the anti-CLDN18.2 antibody, respectively.

[0151] Structure (8) is a molecule with a "1+1" Fab-Fc-scFv asymmetric structure, which comprises two protein chains including the heavy and light chains of the corresponding anti-CD3 antibody and an scFv polypeptide chain of an anti-lysozyme or anti-CLDN18.1 antibody, respectively.

[0152] To minimize the formation of by-products with mismatched heavy chains (e.g., mismatch of the two heavy chains of an anti-CD3 antibody), a mutant heterodimeric Fc region was used, which has "knob-hole" mutations and modified disulfide bonds as described in WO2009080251 and WO2009080252. The CLDN18.2xCD3 bispecific antibody has an IgG1 Fc and has L234A, L235A, or L234A, L235A, and G237A (numbered according to the EU index) mutations on CH3 of the Fc. Each bispecific antibody was generated by co-transfecting three or four different mammalian expression vectors encoding: 1) the heavy chain of the corresponding anti-CLDN18.2 antibody with "hole" mutations in the Fc region to generate a heterodimeric antibody (CH3 of Fc has L234A, L235A, or L234A, L235A, and G237A mutations); 2) the heavy chain of the corresponding anti-CD3 antibody with "knob" mutations in the Fc region to generate a heterodimeric antibody (CH3 of Fc has L234A, L235A, or L234A, L235A, and G237A mutations); and 3) the light chain of the corresponding anti-CD3 antibody. The "knob" mutations in the Fc region of human IgG1 consist of: T366W, and the "hole" mutations consist of: T366S, L368A, and Y407V. In addition, one may include S354C in the "knob" Fc region, and Y349C in the "hole"; these form disulfide bond pairs to increase the stability and yield of the heterodimeric antibody.

[0153] Specific information regarding the CLDN18.2×CD3 bispecific antibody constructed in the present invention is shown in Tables 14, 15, and 16.

[0154] [Table 14]

[0155] [Table 15]

[0156] [Table 16]

[0157] Example 9: Preparation and characterization of CLDN18.2 x CD3 bispecific antibodies 9.1 Preparation of recombinant CLDN18.2 x CD3 bispecific antibodies Multiple plasmids encoding recombinant CLDN18.2xCD3 bispecific antibodies can be transfected into mammalian host cells (e.g., Chinese Hamster Ovary (CHO) cells) according to a specific ratio to obtain the corresponding purified recombinant antibodies using conventional recombinant protein expression and purification techniques. Specifically, ExpiCHO-S™ cells (Gibco, A29127) were grown in ExpiCHO™ Expression Medium (Gibco, A2910001). Prior to transient transfection, cells were grown at a concentration of 3×10 6 ~4×10 6 The cells were then cultured at 37°C in an 8% CO2 shaker for 24 hours at a concentration of 7 × 10 6 ~10×10 6 The cells were then diluted to a concentration of 6 × 10 6A 10 mL cell culture was prepared by diluting to 10000 cells / mL. A total of 8 μg of the above-mentioned plasmids encoding the CLDN18.2×CD3 bispecific antibody (the ratio of plasmid to cells is 0.8 μg:1 mL) was dissolved in 0.4 mL of OptiPRO™ SFM medium (Gibco, 12309019). The resulting mixture was filtered through a 0.22 μm sterile filter. Then, 32 μL of ExpiFectamine™ CHO Reagent (Gibco, A29129) was added to 0.37 mL of OptiPRO™ SFM medium (Gibco, 12309019). The ExpiFectamine™ CHO Reagent solution was immediately and slowly added to the plasmid solution. The mixture was inverted and mixed thoroughly. The flask was shaken while the mixture of plasmid and transfection reagent was slowly added dropwise. The cells were cultured at 37°C in an 8% CO2 shaker for 8-9 days. Cell viability was observed after 8 days.

[0158] The culture was harvested and centrifuged at 3300g for 10 min, and then the supernatant was collected and centrifuged at high speed to remove impurities. A gravity column (Bio-Rad, 7311550) containing MabSelect™ (GE Healthcare Life Science, 71-5020-91 AE) was equilibrated with PBS (pH 7.4) and rinsed with 2-5 column volumes of PBS. The supernatant sample was loaded onto the column. The column was rinsed with 5-10 column volumes of PBS. The target protein was eluted with 0.1 M glycine (pH 3.5). The eluate was adjusted to neutral with Tris-HCl (pH 8.0), concentrated, and buffer exchanged into PBS buffer by ultrafiltration tube (Millipore, UFC901024) to obtain a purified antibody solution. The purified antibody solution was then subjected to concentration determination using a NanoDrop (Thermo Scientific™ NanoDrop™ One) and subpackaged and stored for later use.

[0159] 9.2. Antibody Characterization by SEC-HPLC, HIC-HPLC, and DSF An appropriate amount of the above purified sample was loaded onto an analytical SEC column TSKgel G3000SWxl (HPLC system model: Agilent 1260 Infinity II) for the measurement of purity. The following parameters and conditions were used in this method: mobile phase: 1×PBS, pH 7.4 (Sangon, E607016); room temperature; flow rate: 1.0 mL / min; sample concentration: 1 mg / mL; injection volume: 20 μL; detection wavelength: 280 nm. After recording, the chromatogram was integrated using ChemStation software to calculate the relevant data. Analysis was generated and retention times were reported for different components in the sample.

[0160] An appropriate amount of the above purified sample was loaded onto an analytical HIC column TSKgel Buty1-NPR 4.6*35 (HPLC system model: Agilent 1260 Infinity II) for the measurement of purity and hydrophobicity. The method consisted of a linear gradient from 100% mobile phase A (20 mM PB, 1.8 M (NH4)2SO4, pH 6.0) to 100% mobile phase B (20 mM PB, pH 6.0) within 16 min. The flow rate was set at 0.7 mL / min. The sample concentration was 1 mg / mL. The injection volume was 20 μL. The detection wavelength was 280 nm. After recording, the chromatogram was integrated using ChemStation software to calculate the relevant data. The analysis was generated and the retention times were reported for the different components in the sample.

[0161] In this example, the thermal denaturation temperature (Tm) of protein molecules was measured by differential scanning fluorescence measurement (DSF). 10 μg of protein was added to a 96-well PCR plate (Thermo, AB-0700 / W), followed by adding 2 μL of 100× diluted dye SYPRO™ (Invitrogen, 2008138), followed by adding buffer to obtain a final volume of 40 μL per well. The PCR plate was sealed and placed in a real-time fluorescent quantitative PCR instrument (Bio-Rad CFX96 PCR System) and incubated at 25° C. for 5 minutes, then gradually heated from 25° C. to 95° C. with a gradient of 0.2° C. / 0.2 minutes, and cooled to 25° C. at the end of the experiment. Data analysis was performed using Bio-Rad CFX Maestro software to calculate the Tm of the samples using the FRET scanning mode. The results of the above characterization are shown in Table 17 below.

[0162] [Table 17]

[0163] Example 10: Binding affinity of CLDN18.2 x CD3 bispecific antibodies to cells Antibodies were assayed for binding affinity by FACS. Test cells included HEK293 / hCLDN18.2, HEK293 / hCLDN18.1, NUGC4_D8, and Jurkat cells. Binding affinity was determined as follows: cells were centrifuged at 300 g for 5 min and then resuspended in FACS buffer (PBS containing 2% FBS). Cell density was adjusted to 10 6The cells / mL were adjusted and 50 μL of the cell suspension was added to each well of a 96-well plate. The antibodies were diluted to various concentrations in FACS buffer and 50 μL of the antibody dilution was added to each well of a 96-well plate. After 2 hours of incubation at 4° C., the plate was washed twice with FACS buffer. Then, FACS buffer containing APC-conjugated goat anti-human IgG secondary antibody (final concentration of 1.5 μg / mL, Jackson, 109-605-098) was added. After 1 hour of incubation at 4° C., the plate was washed twice with FACS buffer. The cells were resuspended in fixative and then the fluorescence of the cells was monitored using a FACS instrument (ACEA NovoCyte). Figure 5 and Table 18 show the binding affinity of the antibodies to the cells. All the tested antibodies can bind to CLDN18.2 and CD3 expressing cells, but not to CLDN18.1 expressing cells.

[0164] [Table 18]

[0165] Example 11: TDCC activity of CLDN18.2 x CD3 bispecific antibodies CLDN18.2xCD3 bispecific antibodies were assayed for TDCC efficacy against NUGC4_D4, SNU620, IM95, and HEK293 / hCLDN18.1 cells using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). T cells were isolated from human PBMCs using a Human Total T Cell Isolation Kit (Miltenyi, 130-096-535). Human T cells and target cells were resuspended in culture medium (RPMI1640 + 5% FBS). Target cell density was 3x10 5 Adjust the T cell density to 1.2 x 10 cells / mL. 6The cells were adjusted to 50 μL / mL. 50 μL of each type of cell was added to wells of a 96-well plate (4:1 effector to target ratio). Test antibodies were diluted to various concentrations in culture medium (RPMI1640+5% FBS) and 50 μL was added to wells of a 96-well plate. Samples were incubated at 37°C for 48 hours, and then 10x Triton-X 100 lysate (RPMI1640+5% FBS+10% Triton-X 100) was added to the target cell maximum LDH release control wells and the volume compensation control wells. The mixture was mixed thoroughly and incubated at 37°C for 0.5 hours. The 96-well plate was centrifuged at 400g for 4 minutes. 50 μL of the supernatant was removed and then LDH color development solution was added at a concentration of 50 μL / well. The mixture was left to stand for 20 minutes at room temperature in the absence of light, after which the plate was transferred to MD StakMax (OD 490) was read out. PR004549 was used as a positive control for CLDN18.2 target cells, PR004313 as a positive control for CLDN18.1 target cells, and PR004312 antibody was used as a negative control. For calculation of results, the corrected readout was calculated first. The readout of the medium background control well was subtracted from the readout of the experimental wells, the target cell spontaneously released LDH control wells, and the effector cell spontaneously released LDH control wells, and then the readout of the volume correction control well was subtracted from the readout of the target cell maximum LDH release control wells. TDCC activity (%) = (corrected readout of the experimental wells - corrected readout of the effector cell spontaneously released LDH control wells - corrected readout of the target cell spontaneously released LDH control wells) / (corrected readout of the target cell maximum LDH release control wells - corrected readout of the target cell spontaneously released LDH control wells) x 100. FIG. 6 shows the TDCC activity of the test antibodies, and the values ​​are detailed in Table 19. In NUGC4_D8 cells, which endogenously express CLDN18.2 at a high level, the test antibodies can induce higher or comparable TDCC activity compared to PR004549. Meanwhile, in IM95 cells, which express CLDN18.2 at a low level, the test antibodies induce lower TDCC activity compared to PR004549. SNU620 cells have a methionine to leucine mutation at amino acid 149 of CLDN18.2. This represents a subset of gastric cancer patients with CLDN18.2 mutations. The test antibodies can induce higher or comparable TDCC activity compared to PR004549. The test antibodies could not induce TDCC effect on HEK293 / hCLDN 18.1 cells.

[0166] [Table 19]

[0167] Example 12: Binding affinity of antibodies to human Fc receptor proteins by BLI method The binding kinetics between proteins and antibodies was analyzed by Biolayer Interferometry (BLI) technique using an Octet Red 96e (Fortebio) system. The rotation speed was set at 1000 rpm for the system. 10x kinetics buffer (ForteBio, Cat#18-1105) was diluted to 1x kinetics buffer for affinity assay and dilution of samples. An in-line FAB2G sensor (Fortebio, 18-5125) was equilibrated with test buffer for 10 minutes and then used to capture antibodies (PR002725, PR005397, PR006384, PR005411, PR006292, PR006023, and PR006293) at a capture height of 1 nm. After equilibration in buffer for 120 seconds, the FAB2G sensor was bound to two-fold serially diluted human Fc receptor proteins. Protein concentration and binding dissociation time are shown in Table 20. Finally, the FAB2G sensor was immersed in 10 mM glycine-HCl solution at pH 1.5 for regeneration to elute the protein bound to the sensor. The affinity assay of the antibody against FcRn was carried out under both pH 6.0 and pH 7.4 buffer conditions. For PR004549, the capture sensor is ProL (Fortebio, 18-5085). When data analysis was carried out using Octet Data Analysis software (Fortebio, version 11.0), 0 nM was used as the reference hole to perform reference subtraction; the "1:1 global fitting" method was selected to fit the data to calculate the kinetic parameters of protein binding to the antibody, and the kon(1 / Ms), kdis(1 / s) and KD(M) values ​​were obtained. For fast binding and fast dissociation interactions, the "steady state" method was selected to fit the data to obtain the KD(M) values. The binding affinities of the antibodies to human Fc receptor proteins are shown in Table 21.

[0168] [Table 20]

[0169] [Table 21-1]

[0170] [Table 21-2]

[0171] Example 13: Binding affinity of antibodies to CD3 proteins of various species by BLI method The binding kinetics between proteins and antibodies was analyzed by Biolayer Interferometry (BLI) technique using Octet Red 96e (Fortebio) system. Human CD3E (Acro, CDE-H5223), Cynomolgus CD3E (Acro, CDE-C5226), and mouse CD3E (Acro, CDE-M5256) were mixed with biotin (Thermo Scientific, A39257) at a molar ratio of 1:3. The mixture was incubated overnight at 4°C, and then excess biotin was removed to obtain biotinylated CD3E. The rotation speed was set at 1000 rpm for the system. 10x kinetics buffer (ForteBio, Cat#18-1105) was diluted into 1x kinetics buffer for affinity assay and dilution of samples. An in-line SA sensor (Fortebio, 18-5019) was equilibrated with the test buffer for 10 minutes and then used to capture biotin-labeled or biotinylated CD3 at a capture height of 0.2 nm. After equilibration in the buffer for 120 seconds, the SA sensor with captured CD3 was bound to antibodies serially diluted 2-fold. The antibody concentrations are shown in Table 22, and the binding and dissociation times were set at 180 and 300 seconds. Finally, the SA sensor was immersed in a 10 mM glycine-HCl solution at pH 1.5 for regeneration, and the antibodies bound to the sensor were eluted. The antibody anti-CD3e 48-2B (santa cruz biotechnology,s SC-1174) is a mouse positive antibody. Data analysis was performed using Octet Data Analysis software (Fortebio, version 11.0), where 0 nM was used as the reference hole to perform baseline subtraction; the "1:1 global fitting" method was selected to fit the data to calculate the kinetic parameters of protein binding to the antibody, and k(1 / Ms), kdis(1 / s), and KD(M) values ​​were obtained. For fast-binding and fast-dissociating interactions, the "steady state" method was selected to fit the data to obtain KD(M) values. The binding affinities of the antibodies to CD3 proteins of various species are shown in Table 23.PR002199 was derived from the anti-BCMA (TNB308902) x CD3 (TNB_F2B) bispecific antibody from Teneobio's WO 2018052503. PR004931 was derived from the anti-CEA x CD3 bispecific antibody from Roche's WO 2017055389A1.

[0172] [Table 22]

[0173] [Table 23]

[0174] Example 14: Binding affinity of CLDN18.2 x CD3 bispecific antibodies to human CLDN18.2 paralogous proteins Antibodies were assayed for binding affinity to human CLDN18.2 paralogous proteins by FACS (ACEA NovoCyte). Human CLDN18.2 paralogous family CLDN1, CLDN2, CLDN3, CLDN4, CLDN6, and CLDN9 genes were transiently transfected into HEK293 cells. Plasmid information is shown in Table 24. Binding affinity was determined as follows: cells were centrifuged at 300g for 5 min and then resuspended in FACS buffer (PBS containing 2% FBS). Cell density was adjusted to 10 6Adjusted to cells / mL, 50 μL of cell suspension was added to each well of a 96-well plate. Antibodies were diluted to 60 nM in FACS buffer, and 50 μL of antibody dilution was added to each well of a 96-well plate. After 2 hours of incubation at 4° C., the plate was washed twice with FACS buffer. FACS buffer containing secondary antibody was then added. After 1 hour of incubation at 4° C., the plate was washed twice with FACS buffer. Cells were resuspended in fixative before running FACS. Information on positive control antibody, negative control antibody, and secondary antibody is shown in Table 25. PR005080 was clone 1A2 antibody (generated in-house, see EP 3567053 A1) used as a positive control for CLDN2 binding. FACS results are shown in FIG. 7. This indicates that there was no non-specific binding of PR006384 and PR006292 to the human CLDN18.2 paralogous family proteins CLDN1, CLDN2, CLDN3, CLDN4, CLDN6, and CLDN9.

[0175] [Table 24]

[0176] [Table 25]

[0177] Example 15: Binding affinity of CLDN18.2 x CD3 bispecific antibodies to cynomolgus monkey target protein Antibodies were assayed for binding affinity by FACS. Test cells included cynomolgus CLDN18.2-expressing HEK293 cells (HEK293 / cynoCLDN18.2), cynomolgus CLDN18.1-overexpressing HEK293 cells (HEK293 / cynoCLDN18.1), and cynomolgus CD3-positive T cells. CD3-positive T cells were isolated from cynomolgus PBMCs using a non-human primate CD3 cell isolation kit (Miltenyi, 130-092-012). Binding affinity was determined as follows: cells were centrifuged at 300 g for 5 min and then resuspended in FACS buffer (PBS containing 2% FBS). Cell density was adjusted to 10 6 The cells / mL were adjusted and 50 μL of the cell suspension was added to each well of a 96-well plate. The antibodies were diluted to various concentrations in FACS buffer and 50 μL of the antibody dilution was added to each well of a 96-well plate. After 2 hours of incubation at 4° C., the plate was washed twice with FACS buffer. Then, FACS buffer containing APC-conjugated goat anti-human IgG secondary antibody (1.5 μg / mL final concentration, Jackson, 109-605-098) was added. After 1 hour of incubation at 4° C., the plate was washed twice with FACS buffer. The cells were resuspended in fixative and then run on a FACS (ACEA NovoCyte). Figure 8 and Table 26 show the binding affinity of the antibodies to CD3-positive T cells in HEK293 cells overexpressing cynomolgus CLDN18.1 and CLDN18.2, and in cynomolgus PBMCs. All tested antibodies are able to bind to CLDN18.2 and CD3 expressing cells, but not to CLDN18.1 expressing cells.

[0178] [Table 26]

[0179] Example 16: TDCC activity of CLDN18.2 x CD3 bispecific antibody against cynomolgus monkey CLDN18.2-expressing cells The CLDN18.2xCD3 bispecific antibody was assayed for its activity in inducing TDCC efficacy against HEK293 / cynoCLDN18.2 using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). CD3 positive T cells were isolated from cynomolgus monkey PBMCs using a non-human primate CD3 cell isolation kit (Miltenyi, 130-092-012). Cynomolgus monkey T cells and target cells were resuspended in culture medium (RPMI1640 + 5% FBS). Target cell density was 3x10 5 Adjust the T cell density to 1.2 x 10 cells / mL. 6 The cells were adjusted to 50 μL / mL. 50 μL of each type of cell was added to wells of a 96-well plate (4:1 effector to target ratio). Test antibodies were diluted to various concentrations in culture medium (RPMI1640+5% FBS) and 50 μL was added to wells of a 96-well plate. Samples were incubated at 37°C for 24 hours, and then 10x Triton-X 100 lysate (RPMI1640+5% FBS+10% Triton-X 100) was added to the target cell maximum LDH release control wells and the volume compensation control wells. The mixture was mixed thoroughly and incubated at 37°C for 0.5 hours. The 96-well plate was centrifuged at 400g for 4 minutes. 50 μL of the supernatant was removed and then LDH color development solution was added at a concentration of 50 μL / well. The mixture was left to stand for 20 minutes at room temperature in the absence of light, after which the plate was transferred to MD StakMax (OD 490) for the calculation of the results. The corrected readings were calculated first. The readings of the medium background control wells were subtracted from the readings of the experimental wells, the target cell spontaneously released LDH control wells, and the effector cell spontaneously released LDH control wells, and then the readings of the volume corrected control wells were subtracted from the readings of the target cell maximum LDH release control wells. TDCC activity (%) = (corrected readings of the experimental wells - corrected readings of the effector cell spontaneously released LDH control wells - corrected readings of the target cell spontaneously released LDH control wells) / (corrected readings of the target cell maximum LDH release control wells - corrected readings of the target cell spontaneously released LDH control wells) x 100. Figure 9 shows the TDCC activity induced by the antibody against HEK293 / cynoCLDN18.2. The specific values ​​are shown in Table 27.

[0180] [Table 27]

[0181] Example 17. In vitro cytokine release assay The CLDN18.2xCD3 bispecific antibody was assayed for in vitro induced cytokine release using human PBMCs to predict the safety of the antibody. PBMCs were incubated with the antibody in the presence or absence of HEK293 / hCLDN18.1 cells. PBMCs and HEK293 / hCLDN18.1 were resuspended in culture medium (RPMI1640+10% FBS). The density of HEK293 / hCLDN18.1 cells was increased to 1.5x10 6 Adjust the cell density of PBMC to 2 x 10 cells / mL. 6The concentration of the antibody was adjusted to 100 μL / mL. 100 μL of HEK293 / hCLDN18.1 cells and 200 μL of PBMCs were added to wells of a 48-well plate. Test antibodies were diluted to various concentrations in culture medium (RPMI1640+10% FBS) and 100 μL was added to wells of a 48-well plate to a final volume of 400 μL. LPS (Sigma, L6529) was used as a positive control. Iso hIgG1 (CrownBio, C0001-4) antibody was used as a negative control. Samples were incubated for 24 hours at 37° C. The supernatant was centrifuged at 300 g for 10 minutes and 300 μL of the supernatant was collected. The concentrations of IL-6 (Invitrogen, 88-7066) and TNF-α (Invitrogen, 88-7346) in the supernatant were quantified by ELISA. Figure 10 shows the cytokine release induced by antibodies in vitro. The release of IL-6 and TNF-α induced by PR004549 was higher than that induced by PR006292 in the absence of CLDN18.2 target cells, indicating a better safety profile of PR006292.

[0182] Example 18: ADCC activity of CLDN18.2 x CD3 bispecific antibodies The CLDN18.2xCD3 bispecific antibody was assayed for its activity in inducing ADCC effects on Jurkat cells and HEK293 / hCLDN 18.2 using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). Human PBMCs were centrifuged at 300g for 10 min and cultured overnight in medium (RPMI1640+10%FBS). NK cells were isolated from human PBMCs using a human NK cell isolation kit (Miltenyi, 130-092-657). Jurkat cells were centrifuged at 300g for 5 min and human NK cells at 300g for 10 min. The cells were then resuspended in medium (RPMI1640+5%FBS). The target cell density was 3×10 5 Adjust the NK cell density to 1.8 x 10 cells / mL. 6The cells were adjusted to 50 μL / mL. 50 μL of each type of cell was added to wells of a 96-well plate (effector to target ratio of 6:1). Test antibodies were diluted to various concentrations in culture medium (RPMI1640+5% FBS) and 50 μL was added to wells of a 96-well plate. Samples were incubated for 5 hours at 37°C, and then 10x Triton-X 100 lysate (RPMI1640+5% FBS+10% Triton-X 100) was added to the target cell maximum LDH release control wells and the volume compensation control wells. The mixture was mixed thoroughly and incubated for 0.5 hours at 37°C. The 96-well plate was centrifuged at 300 g for 5 minutes. 50 μL of the supernatant was removed and then LDH color development solution was added at a concentration of 50 μL / well. The mixture was left to stand for 20 minutes at room temperature in the absence of light, after which the plate was transferred to MD StakMax (OD 490 ) was read out. PR003767 was used as a positive control and Iso hIgG1 (CrownBio, C0001-4) antibody was used as a negative control. For calculation of results, corrected readouts were first calculated. The readouts of medium background control wells were subtracted from the readouts of experimental wells, target cell spontaneously released LDH control wells, and effector cell spontaneously released LDH control wells, and then the readouts of volume corrected control wells were subtracted from the readouts of target cell maximum LDH release control wells. ADCC activity (%) = (corrected readouts of experimental wells - corrected readouts of effector cell spontaneously released LDH control wells - corrected readouts of target cell spontaneously released LDH control wells) / (corrected readouts of target cell maximum LDH release control wells - corrected readouts of target cell spontaneously released LDH control wells) x 100. Figure 11 (a) shows the ADCC activity of antibodies against Jurkat cells. PR006292 and PR004549 were unable to induce ADCC effects on Jurkat cells.

[0183] The CLDN18.2xCD3 bispecific antibody was assayed for its ability to induce ADCC effects against NUGC4_D8 using NK92 / CD16a cells. NUGC4_D8 and NK92 / CD16a were resuspended in culture medium (RPMI1640+5% FBS). The target cell density was 3x10 5Adjust the NK92 / CD16a cell density to 1.8 x 10 cells / mL. 6 The antibodies were adjusted to 100 / mL. 50 μL of each type of cells were added to wells of a 96-well plate (effector to target ratio of 6:1). Test antibodies were diluted to various concentrations in culture medium (RPMI1640+5% FBS) and 50 μL were added to wells of a 96-well plate. Samples were incubated at 37° C. for 5 hours. PR003197 was used as a positive control. Figure 11(b) shows the ADCC activity of antibodies against NUGC4_D8. PR006292 and PR004549 could not induce ADCC effect against NUGC4_D8 cells.

[0184] Example 19: CDC activity of CLDN18.2 x CD3 bispecific antibodies CLDN18.2 antibodies were assayed for CDC effects on HEK293 / hCLDN18.2 and Jurkat cells using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, G7573). Target cells were centrifuged at 300g for 5 min and then resuspended in RPMI1640 serum-free medium. Target cell density was 2×10 5Cells / mL was adjusted and 25 μL of cell suspension was added to each well of a 96-well plate. Antibodies were diluted to various concentrations in serum-free medium and 25 μL of antibody dilution was added to each well of a 96-well plate. 50 μL of normal human serum (Access cell culture, 515) was added to a final concentration of 50% and the resulting mixture was incubated at 37° C. for 24 hours. The 96-well plate was left undisturbed for 30 minutes at room temperature and 100 μL of CellTiter-Glo color development solution was added to each well at room temperature. The samples were then incubated for 10 minutes at room temperature in the absence of light. The plate was read by PE Enspire. CDC activity (%)=[1-(luminescent sample) / (luminescent mock control)]×100. IMAB362 analog was used as a positive control and Iso hIgG1 (CrownBio, C0001-4) antibody was used as a negative control. Figure 12 shows the CDC activity of PR006292 antibody on Jurkat cells and human CLDN18.2 overexpressing HEK293 cells. PR006292 induced a greater CDC effect in HEK293 / hCLDN18.2 than that induced by PR004549, but no CDC activity was observed in Jurkat cells. The CDC activity of CLDN18.2xCD3 bispecific antibodies is specifically shown in Table 28.

[0185] [Table 28]

[0186] Example 20: Competitive binding activity of CLDN18.2 x CD3 bispecific antibodies This example studies the binding of anti-human CLDN18.2 bispecific antibodies to the epitope region of human CLDN18.2 antigen. Competitive binding experiments were performed at the cellular level using human CLDN18.2 overexpressing HEK293 / hCLDN18.2 cells. Briefly, anti-human CLDN18.2 antibodies PR000400 and PR004549 were biotinylated using a biotinylation kit (ThermoFisher, A35358) according to the instructions. 2×10 cells were plated in a 96-well V-bottom plate (Corning, 3894).6 cells / mL and a suspension of human CLDN18.2 overexpressing HEK293T / hCLDN18.2 cells was added at 50 μL / well, followed by 25 μL of biotinylated anti-human CLDN18.2 antibody PR000400 or PR004549. The mixture was mixed thoroughly and incubated at 4°C for 30 minutes, and 25 μL of corresponding serially diluted non-biotinylated anti-human CLDN18.2 antibody was added. The mixture was mixed thoroughly and incubated at 4°C for 1 hour. The cells in each well were washed twice with 200 μL pre-chilled FACS buffer (2% BSA in DPBS) and centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. A fluorescent secondary antibody (Jackson ImmunoResearch, 016-540-084, 1:500) was added. The mixture was incubated at 4°C for 1 hour in the absence of light. The cells in each well were washed twice with 200 μL pre-chilled FACS buffer (2% BSA in DPBS) and centrifuged at 500 g for 5 min at 4° C., and then the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL pre-chilled FACS buffer, and the fluorescence signal value was read out using ACEA_NovoCyte. The inhibition rate was calculated using the formula: Inhibition rate (%)=(AB) / A×100) (Note: A: fluorescence signal after interaction of biotinylated antibody with ISO(hIgG1) (Crownbio, c0001-4); B: fluorescence signal after interaction of biotinylated antibody with non-biotinylated antibody).

[0187] As shown in Figure 13 and Table 29, all of the anti-CLDN18.2 bispecific antibodies of the present invention can block the binding of PR000400 or PR004549 to human CLDN18.2, and the detected blocking ability of the antibodies increases in positive correlation with the antibody concentration. This indicates that these antibodies have epitopes that are very similar to those of PR000400 and PR004549. The tested antibodies have low binding affinity to HEK293 / hCLDN18.1 cells. From the above results, it can be inferred that the tested antibodies bind to human CLDN18.2 protein at ECL1 (extracellular loop 1) rather than ECL2.

[0188] [Table 29]

[0189] Example 21: Pharmacokinetic study of CLDN18.2 x CD3 bispecific antibodies Pharmacokinetic studies were performed using BALB / c nude mice as follows. Six female BALB / c nude mice weighing 18-22 g were selected to receive antibody treatment at a dose of 5 mg / kg by intravenous injection. Whole blood from three mice in one group was collected before administration and 15 min, 24 h (1 day), 4 days, and 10 days after administration, and whole blood from three mice in the other group was collected before administration and 5 h, 2 days, 7 days, and 14 days after administration. Whole blood was allowed to stand for 30 min to clot and then centrifuged. Isolated serum samples were cryopreserved at -80°C until analysis. Drug concentrations in mouse serum were quantified by ELISA. Total ELISA was performed by capturing human Fc-containing antibodies in mouse serum using goat anti-human Fc polyclonal antibody and detected by HRP-labeled goat anti-human Fc secondary antibody. CLDN18.2 binding domain ELISA (Free X method) was performed by capturing CLDN18.2 binding domain-containing antibodies in mouse serum with CLDN18.2 protein and detected by HRP-labeled goat anti-human Fc secondary antibody. Plasma concentration data were analyzed by noncompartmental analysis (NCA) using Phoenix WinNonlin software (version 8.2) to evaluate pharmacokinetic parameters.

[0190] The pharmacokinetics of PR006292, PR006384, and PR004549 are shown in Figure 14, and the pharmacokinetic parameters are shown in Table 30. PR006292 and PR006384 have better stability in mice than PR004549, longer half-life in mice, and higher drug exposure.

[0191] [Table 30]

[0192] Example 22: In vivo pharmacodynamic studies on CLDN18.2 x CD3 bispecific antibodies NUGC4_D8 tumor model In vivo pharmacodynamics studies were carried out by re-establishing the NUGC4_D8 tumor model of human PBMC immune system using NCG mice. The method is specifically as follows: On the day of cell inoculation, each NCG mouse was subcutaneously inoculated with NUGC4_D8 cells and PBMCs. The average tumor volume of each group of mice was 90 mm 3 When the tumor volume reached 100 mm, the mice were divided into groups and a total of one administration was performed via the tail vein. After the start of administration, body weight and tumor volume were measured twice a week. Tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long axis × tumor short axis 2 Data were analyzed using t-tests.

[0193] The in vivo antitumor effects of PR005397, PR005411, and PR004549 are shown in Figure 15(a). Specifically, the mean tumor volume of the Iso hIgG1 control group of mice 25 days after inoculation was 1897 mm. 3 The mean tumor volume in the group treated with the test drug PR004549 (0.2 mg / kg) on ​​the 25th day after inoculation was 104 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p value < 0.0001), and the tumor growth inhibition rate TGI (%) was 94.48%. The mean tumor volume of the test drug PR004549 (0.04 mg / kg) treatment group on the 25th day after inoculation was 538 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p value < 0.0001), and the tumor growth inhibition rate TGI (%) was 71.61%. The mean tumor volume of the test drug PR005411 (0.2 mg / kg) treatment group on the 25th day after inoculation was 30 mm 3This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p value < 0.0001), and the tumor growth inhibition rate TGI (%) was 98.39%. The mean tumor volume of the test drug PR005411 (0.04 mg / kg) treatment group on the 25th day after inoculation was 263 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p value < 0.0001), and the tumor growth inhibition rate TGI (%) was 86.1%. The mean tumor volume of the test drug PR005397 (0.04 mg / kg) treatment group on the 25th day after inoculation was 327 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p-value <0.0001), and the tumor growth inhibition rate TGI (%) was 82.75%. Throughout the treatment, the animals showed good tolerance to the drug, and no significant weight loss or animal death occurred. The in vivo antitumor effects of PR005397 and PR005411 are superior to that of PR004549.

[0194] The in vivo antitumor effects of PR006292, PR006293, PR006384, and PR004549 are shown in Figure 15(b). Specifically, the mean tumor volume of the Iso hIgG1 control group of mice 25 days after inoculation was 1355 mm. 3 The mean tumor volume in the group treated with the test drug PR004549 (0.2 mg / kg) 25 days after inoculation was 408 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p value = 0.0001), and the tumor growth inhibition rate TGI (%) was 69.83%. The mean tumor volume of the test drug PR004549 (0.04 mg / kg) treatment group on the 25th day after inoculation was 743 mm 3 This was significantly different from the mean tumor volume of the Iso hIgG1 control group (p-value=0.0037), and the tumor growth inhibition rate TGI (%) was 45.15%. The mean tumor volume of the test drug PR006293 (0.2 mg / kg) treatment group on the 25th day after inoculation was 39 mm 3This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p value < 0.0001), and the tumor growth inhibition rate TGI (%) was 97.06%. The mean tumor volume of the test drug PR006293 (0.04 mg / kg) treatment group on the 25th day after inoculation was 190 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p value < 0.0001), and the tumor growth inhibition rate TGI (%) was 85.96%. The mean tumor volume of the test drug PR006384 (0.2 mg / kg) treatment group on the 25th day after inoculation was 81 mm 3 This was significantly different from the mean tumor volume of the Iso hIgG1 control group (p value < 0.0001), and the tumor growth inhibition rate TGI (%) was 94%. The mean tumor volume of the test drug PR006384 (0.04 mg / kg) treatment group on the 25th day after inoculation was 752 mm 3 This was significantly different from the mean tumor volume of the Iso hIgG1 control group (p-value = 0.0071), and the tumor growth inhibition rate TGI (%) was 44.47%. The mean tumor volume of the test drug PR006292 (0.04 mg / kg) treatment group on the 25th day after inoculation was 580 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p-value=0.0006), with a tumor growth inhibition rate (TGI) (%) of 57.15%. Throughout the treatment, the animals showed good tolerance to the drug, and no significant weight loss or animal death occurred. The in vivo antitumor effects of PR006292, PR006293, and PR006384 are superior to that of PR004549.

[0195] SNU620 PBMC tumor model In vivo pharmacodynamics studies were carried out by re-establishing the SNU620 tumor model of human PBMC immune system using NCG mice. The method is specifically as follows: On the day of cell inoculation, each NCG mouse was subcutaneously inoculated with SNU620 tumor cells. The average tumor volume of each group of mice was 70 mm 3When the tumor volume reached 100 mm, the mice were divided into groups and a total of four doses were administered via the tail vein. After the start of administration, body weight and tumor volume were measured twice a week. Tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long axis × tumor short axis 2 Data were analyzed using t-tests.

[0196] The in vivo antitumor effects of PR006292 and PR004549 are shown in Figure 15(c). Specifically, the mean tumor volume of the Iso hIgG1 control group of mice 36 days after inoculation was 847 mm. 3 The mean tumor volume in the group treated with the test drug PR006292 (0.2 mg / kg) on ​​the 36th day after inoculation was 131 mm 3 This was significantly different from the mean tumor volume of the Iso hIgG1 control group (p-value = 0.0076), and the tumor growth inhibition rate TGI (%) was 84.53%. The mean tumor volume of the test drug PR006292 (0.04 mg / kg) treatment group on the 36th day after inoculation was 505 mm 3 This was significantly different from the mean tumor volume of the Iso hIgG1 control group (p-value = 0.3856), and the tumor growth inhibition rate TGI (%) was 40.36%. The mean tumor volume of the test drug PR004549 (0.2 mg / kg) treatment group on day 36 post-inoculation was 858 mm 3 This was significantly different from the mean tumor volume of the Iso hIgG1 control group (p-value > 0.9999), and the tumor growth inhibition rate TGI (%) was -1.28%. The mean tumor volume of the test drug PR004549 (0.04 mg / kg) treatment group on day 36 post-inoculation was 844 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p-value>0.9999), with a tumor growth inhibition rate (TGI) (%) of 0.39%. Throughout the treatment, the animals tolerated the drug well, with no significant weight loss or animal deaths.

[0197] HuP-T4 PBMC tumor model In vivo pharmacodynamics studies were carried out by re-establishing the HuP-T4 tumor model of human PBMC immune system using NCG mice. The method is specifically as follows: On the day of cell inoculation, each NCG mouse was subcutaneously inoculated with HuP-T4 tumor cells. The average tumor volume of each group of mice was 130 mm 3 When the tumor volume reached 100 mm, the mice were divided into groups and a total of four doses were administered via the tail vein. After the start of administration, body weight and tumor volume were measured twice a week. Tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long axis × tumor short axis 2 Data were analyzed using t-tests.

[0198] The in vivo antitumor effect of PR006292 is shown in Figure 15(d). Specifically, the mean tumor volume of the Iso hIgG1 control group of mice 36 days after inoculation was 1059 mm 3 The mean tumor volume in the group treated with the test drug PR006292 (0.5 mg / kg) on ​​the 36th day after inoculation was 129 mm 3 This showed a significant difference from the mean tumor volume of the Iso hIgG1 control group (p-value=0.0022), and the tumor growth inhibition rate TGI (%) was 87.75%. Throughout the treatment, the animals showed good tolerance to the drug, and no significant weight loss or animal death occurred.

[0199] In vivo cytokine release assay in mice In vivo cytokine storm studies were carried out by re-establishing a human PBMC immune system using NCG mice. The method was specifically as follows: 2 × 10 7 Human PBMCs were injected intravenously and PR006292, PR004549 and control IgG1 antibodies were administered intravenously the following day. Blood was drawn and serum collected at 0 hours (pre-dose), 4 and 24 hours post-dose. MSD methods were used to detect the levels of multiple cytokines in serum, including IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8 and TNF-α. Data were analyzed using t-tests.

[0200] Figure 15(e) shows the expression of some cytokines that were detectable in mouse serum 4 hours after injection of the antibody. The results showed that PR006292 induced lower release of cytokines such as IFN-γ, IL-2, and TNF-α compared to the control antibody PR004549, indicating better safety.

Claims

1. An antibody comprising a single chain targeting CLDN18.2, which comprises a heavy chain variable region containing HCDR1, HCDR2, and HCDR3, wherein said HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 16 to 18, said HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 51, 42 to 46, SEQ ID NOs: 48 to 50 or 52 to 54, and said HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 78, 77 or 79 to 82, wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 51, and SEQ ID NO: 78; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 42, and SEQ ID NO: 77; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 43, and SEQ ID NO: 78; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 44, and SEQ ID NO: 79; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 45, and SEQ ID NO: 80; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 43, and SEQ ID NO: 80; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 43, and SEQ ID NO: 78; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 43, and SEQ ID NO: 81; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 46, and SEQ ID NO: 82; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 48, and SEQ ID NO: 78; or wherein said HCDR1, said HCDR2, and said HCDR3 each comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 49, and SEQ ID NO: 78; or Does the CDR1, CDR2, and CDR3 contain the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 50, and SEQ ID NO: 78 respectively; or Does the CDR1, CDR2, and CDR3 contain the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 52, and SEQ ID NO: 78 respectively; or Does the CDR1, CDR2, and CDR3 contain the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 53, and SEQ ID NO: 79 respectively; or The CDR1, CDR2, and CDR3 contain the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 54, and SEQ ID NO: 78 respectively, an antibody.

2. The heavy chain variable region further includes a framework region, in which HFR1 contains the amino acid sequence shown in SEQ ID NO: 6 or 7, HFR2 contains the amino acid sequence shown in any one of SEQ ID NO: 29, 28, or 30-34, HFR3 contains the amino acid sequence shown in any one of SEQ ID NO: 64, 63, or 65-68, and HFR4 contains the amino acid sequence shown in any one of SEQ ID NO: 86, 84, or 87-89. The antibody according to claim 1.

3. The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:

162. The antibody according to claim 2.

4. The antibody further includes a heavy chain constant region. The antibody according to claim 1.

5. A bispecific antibody comprising a first protein functional region targeting CD3 and a second protein functional region comprising a single chain targeting CLDN18.2, The second protein functional region includes a heavy chain variable region containing CDR1, CDR2, and CDR3. The CDR1 contains the amino acid sequence shown in any one of SEQ ID NO: 16-18. The CDR2 contains the amino acid sequence shown in any one of SEQ ID NO: 51, 42-46, SEQ ID NO: 48-50, or 52-54. The CDR3 contains the amino acid sequence shown in any one of SEQ ID NO: 78, 77, or 79-82; Preferably, does the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 51, and SEQ ID NO: 78; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 42, and SEQ ID NO: 77; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 43, and SEQ ID NO: 78; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 44, and SEQ ID NO: 79; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 48, and SEQ ID NO: 78; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 49, and SEQ ID NO: 78; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 50, and SEQ ID NO: 78; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 52, and SEQ ID NO: 78; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 53, and SEQ ID NO: 79; or do the HCDR1, the HCDR2, and the HCDR3 contain the amino acid sequences respectively shown in SEQ ID NO: 16, SEQ ID NO: 54, and SEQ ID NO: 78; More preferably, the heavy chain variable region contains the amino acid sequence shown in any one of SEQ ID NO: 162, 150-152, SEQ ID NO: 159-161 or 163-165, and the bispecific antibody. **Claim 6** The bispecific antibody according to claim 5, wherein the first protein functional region includes a light chain variable region containing LCDR1, LCDR2, and LCDR3 respectively shown in SEQ ID NO: 101, SEQ ID NO: 116, and SEQ ID NO: 131, and a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 respectively shown in SEQ ID NO: 11, SEQ ID NO: 38, and SEQ ID NO:

72. **Claim 7** The bispecific antibody comprises three polypeptide chains in the following form: Formula: VH ＣＬＤＮ１８．２－ Linker peptide-VH ＣＬＤＮ１８．２ - The first polypeptide chain shown by - hinge - CH2 - CH3, formula: VH ＣＤ３ - The second polypeptide chain shown by - CH1 - hinge - CH2 - CH3, and formula: VL ＣＤ３ - The bispecific antibody according to claim 5, having a third polypeptide chain shown by - CL.

8. The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 236, the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 235, and the third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:

200. The bispecific antibody according to claim 7.

9. The bispecific antibody according to claim 5, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

162.

10. The bispecific antibody according to claim 6, wherein the functional region targeting CD3 comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 149 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

168.

11. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 4 or the bispecific antibody according to any one of claims 5 to 10, and a pharmaceutically acceptable carrier.

12. Further comprising one or more of the group consisting of a hormonal agent, a small molecule targeting agent, a proteasome inhibitor, a contrast agent, a diagnostic agent, a chemotherapeutic agent, a tumor lysing agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine. The pharmaceutical composition according to claim 11.

13. The pharmaceutical composition according to claim 11 for use in therapy.

14. The pharmaceutical composition according to claim 11 for use in a method of treating and / or preventing a tumor, preferably a CLDN18.2 positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma, and / or leukemia.

15. A kit of parts for use in the therapy of a CLDN18.2-mediated disease or disorder, comprising Kit A and Kit B: Kit A comprises the antibody according to any one of claims 1 to 4 and / or the bispecific antibody according to any one of claims 5 to 10; Kit B comprises another anti-tumor antibody, or a pharmaceutical composition comprising the another anti-tumor antibody, and / or one or more of the group consisting of a hormonal agent, a small molecule targeting agent, a proteasome inhibitor, a contrast agent, a diagnostic agent, a chemotherapeutic agent, a tumor lysing agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine. Kit of parts. The kit of parts according to claim 15 for use in a therapy. The kit of parts according to claim 15 for use in a method of treating and / or preventing a tumor, preferably a CLDN18.2 positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma, and / or leukemia.

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