Antibodies Targeting Claudin 18.2 and Their Uses

Abstract

The present invention provides antibodies targeting Claudin18.2, as well as multispecific antibodies, chimeric antigen receptors, antibody-drug conjugates, pharmaceutical compositions and kits comprising the same, and their uses in the diagnosis / treatment / prevention of diseases associated with Claudin18.2 expression.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy. More specifically, the present invention relates to an antibody targeting Claudin18.2, and its use in preventing and / or treating and / or diagnosing diseases. Background Art

[0002] There exists a tight junction (TJ) complex between epithelial tissue cells of a normal organism. This complex has functions such as cell adhesion, maintaining cell polarity and permeability, and assisting in transmitting and regulating cell proliferation and differentiation signals, and is extremely important for maintaining the normal structure and function of epithelial tissue cells. Claudin protein is the main component of TJ, participating in maintaining various functions of TJ, and its abnormal expression is closely related to various tumors. Among them, there are two splice variants of Claudin18 protein, Claudin18.1 (also known as CLDN18.1) and Claudin18.2 (also known as CLDN18.2). The former is selectively expressed in normal epithelial cells of the lung, while the latter is only expressed in the differentiated gastric mucosal epithelial cells in normal tissues and is highly expressed in gastric cancer and pancreatic ductal carcinoma in tumor tissues. These characteristics make Claudin18.2 a target with outstanding clinical value in the treatment of gastric cancer and other Claudin18.2-positive tumors.

[0003] Therefore, it is of great value and significance to develop drugs and antibodies targeting the Claudin18.2 target, and to conduct research on new treatment regimens and combination treatment regimens. The present invention aims to provide an antibody targeting Claudin18.2, and its use in disease prevention and / or treatment and / or diagnosis. Summary of the Invention

[0004] In a first aspect, the present invention provides an antibody targeting Claudin18.2 or an antigen-binding fragment thereof, which comprises CDR-L1 shown in SEQ ID NO: 1, CDR-L2 shown in SEQ ID NO: 2, CDR-L3 shown in SEQ ID NO: 3, CDR-H1 shown in SEQ ID NO: 4, CDR-H2 shown in SEQ ID NO: 5, and CDR-H3 shown in SEQ ID NO: 6.

[0005] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NOs: 8, 11, 14, 17, 20, and 23, or has one or several amino acid modifications (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) compared to an amino acid sequence selected from SEQ ID NOs: 8, 11, 14, 17, 20, and 23; the light-chain variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NOs: 7, 10, 13, 16, 19, and 22, or has one or several amino acid modifications (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) compared to an amino acid sequence selected from SEQ ID NO: 7. Preferably, the modifications are conservative modifications, such as conservative substitutions, additions, and deletions of amino acids. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy-chain variable region selected from SEQ ID NOs: 8, 11, 14, 17, 20, and 23 and a light-chain variable region selected from SEQ ID NOs: 7, 10, 13, 16, 19, and 22.

[0006] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy-chain variable region and a light-chain variable region selected from the following:

[0007] (a) a heavy-chain variable region as shown in SEQ ID NO: 8 and a light-chain variable region as shown in SEQ ID NO: 7;

[0008] (b) a heavy-chain variable region as shown in SEQ ID NO: 11 and a light-chain variable region as shown in SEQ ID NO: 10;

[0009] (c) a heavy-chain variable region as shown in SEQ ID NO: 14 and a light-chain variable region as shown in SEQ ID NO: 13;

[0010] (d) a heavy-chain variable region as shown in SEQ ID NO: 17 and a light-chain variable region as shown in SEQ ID NO: 16;

[0011] (e) a heavy-chain variable region as shown in SEQ ID NO: 20 and a light-chain variable region as shown in SEQ ID NO: 19;

[0012] (f) a heavy-chain variable region as shown in SEQ ID NO: 23 and a light-chain variable region as shown in SEQ ID NO: 22;

[0013] Optionally, the heavy chain variable region and the light chain variable region have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity compared to the heavy chain variable region and the light chain variable region of any one of (a)-(f);

[0014] Optionally, the heavy chain variable region and the light chain variable region have modifications of one or several amino acids compared to the heavy chain variable region and the light chain variable region of any one of (a)-(f), for example, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications; preferably, the modifications are conservative modifications, such as conservative substitutions, additions, and deletions of amino acids.

[0015] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the amino acid sequences selected from SEQ ID NO: 9, 12, 15, 18, 21, and 24, or has modifications of one or several amino acids (such as up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) compared to the amino acid sequences selected from SEQ ID NO: 9, 12, 15, 18, 21, and 24. Preferably, the modifications are conservative modifications, such as conservative substitutions, additions, and deletions of amino acids. Preferably, the amino acid sequence of the antibody or antigen-binding fragment thereof of the present invention is selected from SEQ ID NO: 9, 12, 15, 18, 21, and 24.

[0016] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody, preferably a humanized antibody.

[0017] The present invention also provides a nucleic acid molecule encoding the above antibody or antigen-binding fragment thereof. Thus, in one embodiment, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity with the nucleotide sequences selected from SEQ ID NO: 25-30, and the antibody or antigen-binding fragment thereof encoded by it can specifically bind to the Claudin18.2 antigen. Preferably, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof is selected from SEQ ID NO: 25-30.

[0018] In another aspect, the present invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising the anti-Claudin18.2 antibody or antigen-binding fragment thereof as described above, and one or more second antibodies or antigen-binding portions thereof that specifically bind to other antigens.

[0019] In one embodiment, the second antibody or its antigen-binding portion can be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv, microantibody, diabody, or sdAb.

[0020] The present invention also provides a vector comprising a nucleic acid molecule encoding the above-mentioned anti-Claudin18.2 antibody or its antigen-binding fragment or multispecific antibody, and a host cell expressing the anti-Claudin18.2 antibody or its antigen-binding fragment or multispecific antibody.

[0021] In another aspect, the present invention also provides a chimeric antigen receptor comprising the anti-Claudin18.2 antibody or its antigen-binding fragment as described in the present invention, a transmembrane domain, and an intracellular signaling domain. Preferably, the chimeric antigen receptor further comprises one or more co-stimulatory domains. More preferably, the chimeric antigen receptor comprises the anti-Claudin18.2 antibody or its antigen-binding fragment or multispecific antibody as provided herein, a CD8α or CD28 transmembrane region, a CD28 or 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0022] The present invention also provides a nucleic acid molecule encoding a chimeric antigen receptor targeting Claudin18.2 as defined above, and a vector comprising the nucleic acid molecule.

[0023] The present invention also provides a cell comprising a chimeric antigen receptor targeting Claudin18.2 as defined above, preferably an immune cell, such as a T cell, NK cell, NKT cell, macrophage, dendritic cell. In a preferred embodiment, the engineered immune cell further comprises a second chimeric antigen receptor targeting other tumor antigens.

[0024] In another aspect, the present invention also provides an antibody conjugate comprising the anti-Claudin18.2 antibody or its antigen-binding fragment as defined in the present invention and a second functional moiety, wherein the second functional moiety is selected from Fc, a radioisotope, a structure for extending the half-life, a detectable label, and a drug.

[0025] In one embodiment, the half-life extending structural moiety is selected from: the binding structure of albumin, the binding structure of transferrin, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and white polypeptides (including antibodies) that bind human serum albumin. In one embodiment, the detectable label is selected from fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents. In one embodiment, the drug is selected from cytotoxins and immunomodulators.

[0026] In another aspect, the present invention also provides a detection kit, which comprises the anti-Claudin18.2 antibody or its antigen-binding fragment, multispecific antibody, antibody conjugate or chimeric antigen receptor as described in the present invention.

[0027] In another aspect, the present invention also provides a pharmaceutical composition, which comprises the anti-Claudin18.2 antibody or its antigen-binding fragment, chimeric antigen receptor, multispecific antibody, engineered immune cell or antibody conjugate as described in the present invention, and one or more pharmaceutically acceptable excipients.

[0028] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing a disease associated with Claudin18.2 expression, comprising administering to a subject the anti-Claudin18.2 antibody or its antigen-binding fragment, chimeric antigen receptor, multispecific antibody, antibody conjugate, engineered immune cell or pharmaceutical composition as described above. Detailed Description of the Invention

[0030] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0031] Anti-Claudin18.2 antibody or its antigen-binding fragment

[0032] As used herein, the term "antibody" has the broadest meaning understood by those skilled in the art, and includes monoclonal antibodies (including intact antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that can exhibit the desired biological activity. The antibodies of the present invention can be of any class (such as IgG, IgE, IgM, IgD, IgA, etc.) or subclass (such as IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.).

[0033] As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of antibody fragments in the present invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragment, Fd′, Fv fragment, single-chain antibody (scFv), disulfide-linked Fv (sdFv), the heavy-chain variable region (VH) or the light-chain variable region (VL) of an antibody, linear antibody, "diabody" having two antigen-binding sites, single-domain antibody, nanobody, the natural ligand of the antigen or a functional fragment thereof, etc. Thus, unless the context clearly indicates otherwise, "antibody" in the present invention encompasses antibody fragments or antigen-binding fragments as defined above.

[0034] Generally, a complete antibody comprises two heavy chains and two light chains linked together by disulfide bonds, with each light chain linked to its respective heavy chain by a disulfide bond, presenting a "Y" - shaped structure. Each heavy chain contains a heavy-chain variable region (VH) and a heavy-chain constant region, wherein the heavy-chain variable region contains three complementarity-determining regions (CDRs): CDR-H1, CDR-H2, and CDR-H3, and the heavy-chain constant region contains three constant domains: CH1, CH2, and CH3. Each light chain contains a light-chain variable region (VL) and a light-chain constant region, wherein the light-chain variable region contains three CDRs: CDR-L1, CDR-L2, and CDR-L3, and the light-chain constant region contains a constant domain CL. In the heavy-chain / light-chain variable regions, the CDRs are separated by more conserved framework regions (FRs). The variable regions of the heavy chain / light chain are responsible for the recognition and binding of the antigen, while the constant regions can mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system.

[0035] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of numbering schemes well known in the art, including: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745" ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, January 2003; 27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, June 8, 2001; 309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme).

[0036] The boundaries of the given CDRs or FRs may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the length of the most common antibody region sequences, where insertions are provided by inserting letters (e.g., "30a") and deletions occur in some antibodies. These two schemes place certain insertions and deletions (indels) at different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects. The AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the scheme used in the AbM antibody modeling software of Oxford Molecular.

[0037] Thus, unless otherwise specified, it should be understood that the "CDR" of a given antibody or its region (such as its variable region) encompasses the CDRs defined by any of the above-mentioned schemes or other known schemes. For example, in the case where a specific CDR (e.g., CDR3) is specified as containing a given amino acid sequence, it should be understood that such a CDR may also have the sequence of the corresponding CDR (e.g., CDR3) defined by any of the above-mentioned schemes or other known schemes. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or its region (such as its variable region) encompasses the FRs defined by any of the above-mentioned schemes or other known schemes. Unless otherwise indicated, the numbering scheme used herein to define the boundaries of CDRs and FRs is the Kabat scheme.

[0038] "Single-chain antibody" and "scFv" are used interchangeably herein and refer to an antibody formed by connecting the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an antibody through a linker. The optimal length and / or amino acid composition of the linker can be selected. The length of the linker significantly affects the folding and interaction of the variable regions of the scFv. In fact, if a shorter linker (e.g., between 5 - 10 amino acids) is used, intra-chain folding can be prevented. For selection of the size and composition of the linker, see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444 - 6448; U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794; and PCT Publication Nos. WO2006 / 020258 and WO2007 / 024715, which are incorporated herein by reference in their entireties. The scFv can contain VH and VL connected in any order, such as VH-linker-VL or VL-linker-VH.

[0039] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody, preferably a humanized antibody.

[0040] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of each heavy and light chain amino acid sequence is homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to the corresponding sequences from another species or belonging to another class. Generally, the variable regions of both the light and heavy chains are from the variable regions of an antibody of one species, while the constant regions are homologous to antibody sequences from another species. An obvious advantage of this chimeric form is that the variable regions can be conveniently generated from currently known sources using readily available B cells or hybridomas from non-human hosts, while the constant regions combined therewith are from, for example, human cells. The variable regions have the advantage of being easy to prepare, and the specificity is not affected by the source, and since the constant regions are from humans, the possibility of the antibody eliciting a human immune response upon injection will be lower than when the constant regions are from non-human sources.

[0041] As used herein, a "humanized" antibody means an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. A "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has been humanized to generally reduce its immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0042] Humanized antibodies and methods for their preparation are well known to those skilled in the art, see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008). Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method; framework regions derived from the consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions; human mature (somatic mutated) framework regions or human germline framework regions; and framework regions obtained by screening FR libraries.

[0043] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions from human germline immunoglobulin sequences. The human antibodies of the present invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis or by in vivo somatic mutation).

[0044] In one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that targets Claudin 18.2 and comprises CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, CDR-L3 as shown in SEQ ID NO: 3, CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6.

[0045] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy-chain variable region and a light-chain variable region. The heavy-chain variable region has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with an amino acid sequence selected from SEQ ID NOs: 8, 11, 14, 17, 20 and 23, or has one or several (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared with an amino acid sequence selected from SEQ ID NOs: 8, 11, 14, 17, 20 and 23; the light-chain variable region has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with an amino acid sequence selected from SEQ ID NOs: 7, 10, 13, 16, 19 and 22, or has one or several amino acids (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) modifications compared with an amino acid sequence selected from SEQ ID NOs: 7, 10, 13, 16, 19 and 22. Preferably, the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy-chain variable region selected from SEQ ID NOs: 8, 11, 14, 17, 20 and 23 and a light-chain variable region selected from SEQ ID NOs: 7, 10, 13, 16, 19 and 22. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy-chain variable region and a light-chain variable region selected from the following:

[0046] (g) A heavy-chain variable region as shown in SEQ ID NO: 8 and a light-chain variable region as shown in SEQ ID NO: 7;

[0047] (h) A heavy-chain variable region as shown in SEQ ID NO: 11 and a light-chain variable region as shown in SEQ ID NO: 10;

[0048] (i) The heavy chain variable region as shown in SEQ ID NO: 14 and the light chain variable region as shown in SEQ ID NO: 13;

[0049] (j) The heavy chain variable region as shown in SEQ ID NO: 17 and the light chain variable region as shown in SEQ ID NO: 16;

[0050] (k) The heavy chain variable region as shown in SEQ ID NO: 20 and the light chain variable region as shown in SEQ ID NO: 19;

[0051] (l) The heavy chain variable region as shown in SEQ ID NO: 23 and the light chain variable region as shown in SEQ ID NO: 22;

[0052] Optionally, the heavy chain variable region and the light chain variable region have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity compared to the heavy chain variable region and the light chain variable region of any one group of (a)-(f);

[0053] Optionally, the heavy chain variable region and the light chain variable region have one or several amino acid modifications compared to the heavy chain variable region and the light chain variable region of any one group of (a)-(f), such as up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications; preferably, the modification is a conservative modification, such as conservative substitution, addition and deletion of amino acids.

[0054] In one embodiment, the antibody or its antigen-binding fragment of the present invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the amino acid sequences selected from SEQ ID NO: 9, 12, 15, 18, 21 and 24, or has one or several amino acid modifications compared to the amino acid sequences selected from SEQ ID NO: 9, 12, 15, 18, 21 and 24, such as up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications. Preferably, the modification is a conservative modification, such as conservative substitution, addition and deletion of amino acids. Preferably, the amino acid sequence of the antibody or its antigen-binding fragment of the present invention is selected from SEQ ID NO: 9, 12, 15, 18, 21 and 24.

[0055] As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include conservative substitutions, additions, and deletions of amino acids. The modifications can be introduced into the chimeric antigen receptors of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved.

[0056] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies having exactly the same sequence have 100% identity. Those skilled in the art will appreciate that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and ClustalW.

[0057] In one aspect, the present invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising the anti-Claudin18.2 antibody or an antigen-binding fragment thereof as described above, which further comprises one or more second antibodies that specifically bind to other antigens.

[0058] As used herein, the term "multispecific" refers to an antigen-binding protein having multi-epitope specificity (i.e., capable of specifically binding two, three, or more different epitopes on a single biomolecule or capable of specifically binding epitopes on two, three, or more different biomolecules). As used herein, the term "bispecific" indicates that the antigen-binding protein has two different antigen-binding specificities.

[0059] In one embodiment, the second antibody can be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv, minibody, diabody, or sdAb.

[0060] Thus, in one embodiment, the second antibody targets an antigen selected from the group consisting of: BCMA, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, angiogenic factors, VEGF, PIGF, ED-B fibronectin, oncogenes, oncogene products, CD66a-d, necrotic antigens, Ii, IL-2, T101, TAC, IL-6, ROR1, DR4, DR5, tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, ErbB dimer, EGFR vIII, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, GPRC5D, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, κ light chain, Lewis Y, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A6, PRAME, survivin, EGP2, EGP40, TAG72, B7-H6, IL-13Ra2, CA9, CD171, G250 / CAIX, HLA-A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, bispecific antigen, antigen associated with a universal tag, cancer-testis antigen, MUC1, MUC16, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, VEGF-R2, CEA, prostate specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, OGD2, CE7, WT-1, cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, activin, AFP, p53, cyclin (such as cyclin A1 (CCNA1)) and / or pathogen-specific antigen, biotinylated molecule, molecule expressed by HIV, HCV, HBV, and / or other pathogens; and / or neoepitope or neoantigen.

[0061] Nucleic acids, vectors, host cells

[0062] In another aspect, the present invention relates to nucleic acid molecules encoding the anti-Claudin18.2 antibodies or multispecific antibodies of the present invention. The nucleic acids of the present invention can be RNA, DNA or cDNA. According to one embodiment of the present invention, the nucleic acid of the present invention is a substantially isolated nucleic acid.

[0063] In one embodiment, the nucleic acid molecule encoding the anti-Claudin18.2 antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity with a nucleotide sequence selected from SEQ ID NOs: 25-30, and the anti-Claudin18.2 antibody encoded thereby is capable of specifically binding to Claudin18.2 (i.e., hardly binds to non-target antigens such as Claudin18.1). Preferably, the nucleic acid molecule encoding the anti-Claudin18.2 antibody is as shown in SEQ ID NOs: 25-30.

[0064] The nucleic acids of the present invention can also be in the form of vectors, can be present in vectors and / or can be part of vectors, such as plasmids, cosmid plasmids or YACs. The vector can especially be an expression vector, i.e., a vector that can provide the expression of the Claudin18.2 antibody in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector usually contains at least one nucleic acid molecule of the present invention, which is operably linked to one or more suitable expression regulatory elements (such as promoters, enhancers, terminators, etc.). The selection of the regulatory elements and their sequences for expression in a specific host is well known to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the Claudin18.2 antibody of the present invention include, but are not limited to, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0065] In another aspect, the present invention also provides host cells expressing the Claudin18.2 antibodies, multispecific antibodies and / or containing the nucleic acids or vectors of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.

[0066] Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0067] Suitable fungal cells include cells of species of Trichoderma, Neurospora, and Aspergillus; or include cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0068] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0069] However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins can also be used in the present invention.

[0070] Chimeric Antigen Receptor

[0071] In another aspect, the present invention also provides recombinant receptors comprising the anti-Claudin18.2 antibody as described above, such as recombinant TCR receptors or chimeric antigen receptors. Preferably, the present invention also provides chimeric antigen receptors comprising the anti-Claudin18.2 antibody as described above.

[0072] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes a ligand-binding domain (e.g., the antigen-binding portion of an antibody), a transmembrane domain, an optional co-stimulatory domain, and an intracellular signaling domain, with each domain connected by a linker. The CAR can redirect the specificity and reactivity of T cells and other immune cells to a selected target in a non-MHC-restricted manner using the antigen-binding properties of an antibody.

[0073] In one embodiment, the present invention provides a chimeric antigen receptor comprising an anti-Claudin18.2 antibody or an antigen-binding fragment thereof as described above, or a multispecific antibody containing the anti-Claudin18.2 antibody, a transmembrane domain, and an intracellular signaling domain.

[0074] As used herein, the term "transmembrane domain" refers to a polypeptide structure capable of enabling the chimeric antigen receptor to be expressed on the surface of an immune cell (such as a lymphocyte, NK cell, or NKT cell) and guiding the cellular response of the immune cell against a target cell. The transmembrane domain can be natural or synthetic and can also be derived from any membrane-binding protein or transmembrane protein. When the chimeric antigen receptor binds to a target antigen, the transmembrane domain is capable of signal transduction. Transmembrane domains particularly suitable for use in the present invention can be derived from, for example, the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. Alternatively, the transmembrane domain can be synthetic and can mainly comprise hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the CD8α chain or CD28 and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 31 or 33, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO: 32 or 34.

[0075] As used herein, the term "intracellular signaling domain" refers to the protein portion that transduces effector function signals and directs the cell to perform specified functions. In one embodiment, the intracellular signaling domain comprised by the chimeric antigen receptor of the present invention can be the intracellular region sequences of the T cell receptor and co-receptor, which act together after antigen receptor binding to initiate signal transduction, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar functions. The intracellular signaling domain can comprise a number of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). Non-limiting examples of the intracellular signaling domain of the present invention include, but are not limited to, the intracellular regions of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, Claudin18.29a, Claudin18.29b, and CD66d, etc. In a preferred embodiment, the signaling domain of the CAR of the present invention can comprise the CD3ζ intracellular region, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 39 or 41, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO: 38 or 40.

[0076] In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the antibody. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antibody. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may be wholly or partially derived from a natural molecule, such as the extracellular region of CD8, CD4 or CD28 in whole or in part, or the antibody constant region in whole or in part. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a hinge region portion of CD8α, CD28, FcγRIIIα receptor, IgG4 or IgG1, more preferably the CD8α, CD28 or IgG4 hinge, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 47, 49 or 51, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 48, 50 or 52.

[0077] In one embodiment, the chimeric antigen receptor may further comprise one or more co-stimulatory domains. The co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule, which comprises the entire intracellular portion of the co-stimulatory molecule, or a functional fragment thereof. A "co-stimulatory molecule" refers to a cognate binding partner that specifically binds to a co-stimulatory ligand on a T cell, thereby mediating a co-stimulatory response (such as proliferation) of the T cell. Co-stimulatory molecules include, but are not limited to, class I MHC molecules, BTLA, and Toll ligand receptors. Non-limiting examples of the co-stimulatory domain of the present invention include, but are not limited to, co-stimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, Claudin18.2, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70. Preferably, the co-stimulatory domain of the CAR of the present invention is from 4-1BB, CD28, or 4-1BB + CD28. In one embodiment, the 4-1BB co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 37, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO: 38. In one embodiment, the CD28 co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 35, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO: 36.

[0078] In one embodiment, the CAR of the present invention may further comprise a signal peptide such that when it is expressed in a cell, such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long hydrophobic amino acid segment that has a tendency to form a single α-helix. At the end of the signal peptide, there is usually an amino acid segment that is recognized and cleaved by signal peptidase. Signal peptidase can cleave during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides useful in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, etc. In one embodiment, the signal peptide useful in the present invention is from B2M or CD8α, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 43 or 45, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO: 44 or 46.

[0079] In one embodiment, the CAR contains an anti-Claudin18.2 antibody or an antigen-binding fragment thereof as provided herein, or a multispecific antibody containing the anti-Claudin18.2 antibody, a CD8α or CD28 transmembrane domain, a CD28 or 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. In this embodiment, the CAR may further comprise a signal peptide from B2M, CD8α, IgG1 or GM-CSFRα.

[0080] The present invention also provides a nucleic acid molecule encoding a chimeric antigen receptor targeting Claudin18.2 as defined above, and a vector containing the nucleic acid molecule.

[0081] As used herein, the term "vector" is a nucleic acid molecule used as a vehicle to transfer (exogenous) genetic material into a host cell, in which the nucleic acid molecule can, for example, replicate and / or be expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a vehicle for delivering an isolated nucleic acid into a cell interior, for example, by homologous recombination or by using a heterologous recombinase that acts on sequences at a specific targeting site. An "expression vector" is a vector for the transcription of heterologous nucleic acid sequences (such as those encoding the chimeric antigen receptor polypeptides of the present invention) in a suitable host cell and the translation of their mRNAs. Suitable vectors for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the present invention include, but are not limited to, plasmids, viruses (such as retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyomaviruses, and adeno-associated viruses (AAV), etc.), bacteriophages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). A vector itself is usually a nucleic acid molecule, typically a DNA sequence containing an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Engineered vectors typically also contain an origin of replication that autonomously replicates in a host cell (if stable expression of the polynucleotide is desired), a selection marker, and restriction enzyme cleavage sites (such as a multiple cloning site, MCS). A vector may additionally contain elements such as a promoter, a polyadenylation tail (polyA), a 3' UTR, an enhancer, a terminator, an insulator, an operon, a selection marker, a reporter gene, a targeting sequence, and / or a protein purification tag. In a specific embodiment, the vector is a vector for in vitro transcription.

[0082] Engineered Immune Cells

[0083] In one aspect, the present invention also provides engineered immune cells that express the CARs of the present invention.

[0084] As used herein, the term "immune cell" refers to any cell of the immune system having one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC). For example, immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells, and / or NKT cells. In one embodiment, the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, etc. Preferably, the immune cells are T cells. The T cells can be any T cells, such as T cells cultured in vitro, e.g., primary T cells, or T cells from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified. The T cells can be at any stage of development, including but not limited to, CD4+ / CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. A variety of techniques known to those skilled in the art, such as Ficoll separation, can be used to obtain T cells from the blood of a subject.

[0085] The nucleic acid sequence encoding the chimeric antigen receptor can be introduced into immune cells by conventional methods known in the art (such as by transduction, transfection, transformation, etc.). "Transfection" is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. An example is RNA transfection, which is the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. This term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells usually involves opening transient pores or "holes" in the cell membrane to allow uptake of materials. Transfection can be carried out using calcium phosphate, by electroporation, by cell squeezing, or by mixing cationic lipids with the material to produce liposomes that fuse with the cell membrane and deposit their cargo inside. Exemplary techniques for transfection of eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation. The term "transformation" is used to describe non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and also into non-animal eukaryotic cells (including plant cells). Thus, transformation is a genetic alteration of bacteria or non-animal eukaryotic cells that results from direct uptake from their surroundings and subsequent incorporation of foreign genetic material (nucleic acid molecules) through the cell membrane. Transformation can be achieved by artificial means. For transformation to occur, the cells or bacteria must be in a competent state. For prokaryotic transformation, techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation. After introducing the nucleic acid or vector into immune cells, those skilled in the art can amplify and activate the resulting immune cells by conventional techniques.

[0086] In one embodiment, to reduce the risk of graft-versus-host disease, the engineered immune cells further comprise the expression of at least one gene selected from the following being inhibited or silenced: CD52, GR, dCK, TCR / CD3 genes (such as TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA), and immune checkpoint genes, such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3. Preferably, the engineered immune cells further comprise the expression of at least one gene selected from the following being inhibited or silenced: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA.

[0087] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art. For example, antisense RNA, RNA decoys, RNA aptamers, siRNA, shRNA / miRNA, trans-dominant negative proteins (TNPs), chimeric / antibody conjugates, chemokine ligands, anti-infectious cell proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors can be used to inhibit gene expression. Additionally, gene silencing can also be achieved by mediating DNA cleavage, for example, by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system.

[0088] In one embodiment, the engineered immune cell further comprises a second chimeric antigen receptor targeting other tumor antigens. The other tumor antigens targeted by the second chimeric antigen receptor can be selected from, for example, BCMA, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, CD138, B7, MUC-1, HM1.24, angiogenic factors, VEGF, PIGF, ED-B fibronectin, CD66a-d, IL-2, T101, TAC, IL-6, ROR1, DR4, DR5, tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, ErbB dimer, EGFR vIII, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, GPRC5D, HMW-MAA, IL-22R-α, IL-13R-α2, κ light chain, Lewis Y, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A6, PRAME, survivin, EGP2, EGP40, TAG72, B7-H6, IL-13Ra2, CA9, CD171, G250 / CAIX, HLA-A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, MUC1, MUC16, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, VEGF-R2, CEA, prostate specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, WT-1, cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, activin, AFP, p53, D1, CS-1, BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecule, MAGE-A1, MAGEA3, CCNA1 and / or pathogen-specific antigens, biotinylated molecules, molecules expressed by HIV, HCV, HBV, and / or other pathogens.

[0089] In one embodiment, a plurality of immune cells are provided, each immune cell being engineered to express one or more chimeric antigen receptors. For example, in some embodiments, one immune cell is engineered to express a chimeric antigen receptor that binds to and / or targets Claudin18.2 (e.g., a CAR comprising the anti-Claudin18.2 antibody described in the present invention), and another cell is engineered to express a chimeric antigen receptor that binds to and / or targets other antigens. In one embodiment, the immune cells may also express a multispecific chimeric antigen receptor that targets one or more antigens including Claudin18.2. For example, such a multispecific chimeric antigen receptor may comprise a multispecific antibody that targets Claudin18.2, or may comprise both the anti-Claudin18.2 antibody described in the present invention and an antibody that targets other antigens. In such embodiments, the plurality of engineered immune cells may be administered together or separately. In one embodiment, the plurality of immune cells may be in the same composition or in different compositions. Exemplary compositions of the cells include the compositions described in the following sections of this application.

[0090] Antibody Conjugates

[0091] In one aspect, the present invention provides an antibody conjugate comprising an anti-Claudin18.2 antibody as defined in the present invention and a second functional moiety, wherein the second functional moiety is selected from the group consisting of Fc, a radioisotope, a half-life extending moiety, a detectable label, and a drug.

[0092] In one embodiment, the present invention provides an antibody conjugate comprising an anti-Claudin18.2 antibody as defined in the present invention and an Fc. As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which includes native Fc and variant Fc. "Native Fc" refers to a molecule or sequence comprising a non-antigen-binding fragment, whether in monomeric or multimeric form, produced by digestion of a complete antibody. The immunoglobulin source from which the native Fc is produced is preferably of human origin. The native Fc fragment consists of monomeric polypeptides that can be joined in dimeric or multimeric form by covalent linkages (e.g., disulfide bonds) and non-covalent linkages. Depending on the class (e.g., IgG, IgA, IgE, IgD, IgM) or subtype (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2), there are 1-4 intermolecular disulfide bonds between the monomer subunits of the native Fc molecule. An example of native Fc is the disulfide-linked dimer produced by digestion of IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). The term "native Fc" as used herein generally refers to monomeric, dimeric, and multimeric forms. "Variant Fc" refers to an amino acid sequence that is different from the amino acid sequence of "native" or "wild-type" Fc due to at least one "amino acid modification" as defined herein, also referred to as "Fc variant". Thus, "Fc" also includes single-chain Fc (scFc), i.e., a single-chain Fc consisting of two Fc monomers linked by a polypeptide linker, which can naturally fold into a functional dimeric Fc region. In one embodiment, the Fc is preferably the Fc of a human immunoglobulin, more preferably the Fc of human IgG1.

[0093] In one embodiment, the present invention provides an antibody conjugate comprising an anti-Claudin18.2 antibody as defined in the present invention and a radioisotope. Examples of radioisotopes that can be used in the present invention include, but are not limited to, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , 99m Tc, 123 I, 18 F and 68 Ga.

[0094] In one embodiment, the present invention provides an antibody conjugate comprising an anti-Claudin18.2 antibody as defined in the present invention and a half-life extending moiety selected from albumin binding structures, transferrin binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin binding polypeptides (including antibodies).

[0095] In one embodiment, the present invention provides an antibody conjugate comprising an anti-Claudin18.2 antibody as defined in the present invention and a detectable label. The term "detectable label" as used herein means a compound that produces a detectable signal. For example, the detectable label can be an MRI contrast agent, a scintigraphic contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, an optical imaging contrast agent. Examples of detectable labels include fluorophores (such as fluorescein, Alexa, or cyanine), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as horseradish peroxidase, glucose-6-phosphatase, β-galactosidase), antibiotic (e.g., kanamycin, ampicillin, chloramphenicol, tetracycline, etc.) resistance genes, and contrast agents (such as nanoparticles or gadolinium). Those skilled in the art can select a suitable detectable label according to the detection system used.

[0096] In one embodiment, the present invention provides an antibody conjugate comprising an anti-Claudin18.2 antibody as defined by the present invention and a drug conjugated to the anti-Claudin18.2 antibody, such as a cytotoxin or an immunomodulator (i.e., an antibody-drug conjugate). Typically, the drug is covalently linked to the antibody and generally relies on a linker. In one embodiment, the drug is a cytotoxin. In another embodiment, the drug is an immunomodulator. Examples of cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozocin, mitomycin, cis-dichlorodiamine platinum (II) (DDP), cisplatin, carboplatin, zorubicin, doxorubicin, detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, actinomycin D, bleomycin, calicheamicin, mithramycin, anthramycin (AMC), vincristine, vinblastine, paclitaxel, ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, mitoxantrone, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), interferons, and combinations thereof. Examples of immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, ciclosporin, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxin, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., G-CSF and (GM-CSF)), interferons (e.g., interferon-α, interferon-β, and interferon-γ), stem cell growth factor designated "S1 factor", erythropoietin, and thrombopoietin, or combinations thereof.

[0097] Kits and Pharmaceutical Compositions

[0098] In another aspect, the present invention also provides a detection kit comprising the humanized antibody, multispecific antibody, antibody conjugate, or chimeric antigen receptor described in the present invention.

[0099] In another aspect, the present invention also provides a pharmaceutical composition comprising the humanized antibody, chimeric antigen receptor, multispecific antibody, engineered immune cell or antibody conjugate described in the present invention, and one or more pharmaceutically acceptable excipients.

[0100] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible, in pharmacology and / or physiology, with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesives, glidants, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators. Those skilled in the art know to select suitable excipients to prepare the desired pharmaceutical composition of the present invention. Exemplary excipients for use in the pharmaceutical composition of the present invention include saline, buffered saline, glucose and water. Generally, the selection of suitable excipients depends especially on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

[0101] The pharmaceutical composition according to the present invention is applicable to administration by various routes. Generally, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

[0102] The pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, especially in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. Processes known in the art for producing pharmaceuticals can include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, entrapping or lyophilizing processes. A pharmaceutical composition comprising, for example, immune cells as described herein is generally provided in solution form and preferably contains a pharmaceutically acceptable buffer.

[0103] The pharmaceutical composition according to the present invention can also be administered in combination with one or more other medicaments suitable for the treatment and / or prevention of the disease to be treated. Preferred examples of the medicaments suitable for combination include known anti-cancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNase and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibody or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides. In addition, the pharmaceutical composition of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0104] Therapeutic / Preventive / Diagnostic Uses

[0105] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing a disease associated with Claudin18.2 expression, comprising administering to a subject the humanized antibody, chimeric antigen receptor, multispecific antibody, antibody conjugate, engineered immune cell or pharmaceutical composition as described above.

[0106] In one embodiment, the diseases associated with Claudin18.2 expression include, but are not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (such as non-small cell lung cancer), liver cancer, gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, cholangiocarcinoma, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (such as multiple myeloma), sarcoma, glioblastoma, leukemia, teratoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, adrenal cancer, brain cancer, colon cancer, head and neck cancer, lymph node cancer, ear, nose and throat (ENT) cancer, and metastatic, recurrent or refractory lesions of these cancers.

[0107] In a preferred embodiment, the diseases associated with Claudin18.2 expression are selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, and lung cancer.

[0108] The present invention will be described in detail below with reference to the accompanying drawings and in combination with examples. It should be noted that those skilled in the art should understand that the accompanying drawings and embodiments of the present invention are only for illustrative purposes and do not constitute any limitation to the present invention. Without contradiction, the embodiments in this application and the features in the embodiments can be combined with each other. Brief Description of the Drawings

[0109] Figure 1 : Shows the expression levels of Claudin18.1 or Claudin18.2 in various cells overexpressing Claudin18.1 (A) or Claudin18.2 (B) constructed.

[0110] Figure 2 : Shows the GFP expression levels in various cells containing luciferase-GFP constructed.

[0111] Figure 3 : Shows the binding of cloned BH232 to Claudin18.1 (A) and Claudin18.2 (B).

[0112] Figure 4 : Shows the scFv expression levels in BH232-CAR T cells (A) and NT cells (B).

[0113] Figure 5 : Shows the killing effects of BH232-CAR T cells (A) and NT cells (B) on different target cells and non-target cells at various effector-to-target ratios.

[0114] Figure 6 : Shows the degranulation of BH232-CAR T cells and NT cells after co-culture with various target cells and non-target cells.

[0115] Figure 7 : Shows the release levels of IL2 (A) and IFN-γ (B) of BH232-CAR T cells and NT cells after co-culture with various target cells and non-target cells.

[0116] Figure 8 : Shows the expression level of Claudin18.2 scFv in hCAR-T cells constructed with a humanized antibody.

[0117] Figure 9: It shows the killing effects of hCAR-T cells on target cells NUGC4-18.2-luci (A), non-target cells NUGC4-luci (B), and 293T-18.1-luci (C) at various effector-to-target ratios.

[0118] Figure 10 : It shows the degranulation effects after co-culture of hCAR-T cells with target cells NUGC4-18.2-luci and non-target cells NUGC4-luci, 293T-18.1-luci.

[0119] Figure 11 : It shows the inhibitory effects of CAR-T cells on mouse tumors. Detailed implementation manners

[0120] Example 1. Construction of cell lines overexpressing Claudin18.1 or Claudin18.2

[0121] The complete coding sequences of claudin18.1 (GeBank: NM_016369) and claudin18.2 (GeBank: NM_001002026) were synthesized respectively and cloned into the vector pGEM-T Easy (Promega, product number A1360) to obtain pLV-claudin18.1 and pLV-claudin18.2 plasmids.

[0122] Using liposome transfection reagent (Roche, product number 06366546001), 293T cells and CHO cells were transfected with pLV-claudin18.1 plasmid, and monoclonal cells were isolated by limited dilution method to obtain 293T-18.1 and CHO-18.1 monoclonal cell lines; 293T cells, NUGC4 cells and CHO cells were transfected with pLV-claudin18.2 plasmid, and monoclonal cells were isolated by limited dilution method to obtain 293T-18.2, NUGC4-18.2 and CHO-18.2 monoclonal cell lines. Then, using antibodies CLDN18.2 antibody (Sanyou Biotech, product number SY11-362) and CLDN18 antibody (biorbyt, product number orb39924), the expression of Claudin18.1 or Claudin18.2 in the above monoclonal cell lines was detected by flow cytometry, and the results are as Figure 1 shown.

[0123] It can be seen that 293T-18.1 and CHO-18.1 cells can bind to anti-CLDN18 antibody but hardly bind to anti-CLD18.2 antibody, indicating their specific expression of CLDN18.1 ( Figure 1A). CHO-18.2, 293T-18.2 and NUGC4-18.2 cells strongly bind to anti-CLDN18.2 antibodies ( Figure 1 B).

[0124] The pLVX-acGFP-N1-Fluc plasmid (PPL, catalog number PPL00157-4a) containing the nucleic acid encoding the luciferase-GFP fusion protein was packaged into lentivirus and infected 293T cells, 293T-18.1 cells, 293T-18.2 cells, NUGC4 cells and NUGC4-18.2 cells, respectively, to obtain 293T-luci, 293T-18.1-luci, 293T-18.2-luci, NUGC4-luci, and NUGC4-18.2-luci cell lines. The GFP signal in the cell lines was detected by flow cytometry, and the results were as follows: Figure 2 As shown, it can be seen that the five prepared cell lines can efficiently express luciferase-GFP fusion protein and can be used for subsequent experiments.

[0125] Example 2. Screening of anti-Claudin18.2 antibodies

[0126] The pLV-Claudin18.2 plasmid was administered to Balb / c mice of appropriate age by intramuscular injection, and then the immunization injection was repeated every 2 to 3 weeks for a total of 4 times. Then, the mouse spleen lymphocytes were taken, mixed with SP2 / 0 myeloma cells and PEG was added to mediate cell fusion to prepare hybridoma cells. Claudin18.2-positive cell lines (293T-18.2 cells, NUGC4-18.2 cells) were used to screen hybridoma clones that bind to Claudin18.2 by ELISA or flow cytometry, while Claudin18.1-positive cell lines (293T-18.1 cells) were used to screen hybridoma clones that do not bind to Claudin18.1 by ELISA or flow cytometry. After multiple rounds of screening, an antibody clone that can specifically bind to Claudin18.2 but not to Claudin18.1 was obtained, named BH232 clone.

[0127] To detect the binding of BH232 to Claudin18.1 and Claudin18.2, CHO-18.1 cells or CHO-18.2 cells were stained with the supernatant of the BH232 clone and detected by flow cytometry. The results are shown in Figure 3 As shown. It can be seen that the binding of this clone to Claudin18.1 is very weak ( Figure 3 A, less than 10%), while the binding to Claudin18.2 was as high as 98.5% ( Figure 3B), indicating that the screened BH232 clone can specifically bind to Claudin18.2.

[0128] Total RNA of the BH232 clone was extracted and reverse-transcribed into cDNA using a cDNA synthesis kit (Novoprotein, product number R211-01). Using the cDNA as a template, degenerate primers were used for PCR (Zhou H et al., Nucleic Acids Research 22:888-889 (1994); Chardes T et al., FEBS Letters 452:386-394 (1999)). The PCR product was recovered, cloned into the pMD18-T vector (Takara, product number 6011), and then transformed into the competent strain JM109 (General Biology, product number CS03020). The next day, clones were selected for sequencing, and the amino acid sequence of the BH232 clone was obtained as shown in Table 1 below.

[0129] Table 1. Amino acid sequence of the BH232 clone

[0130] CDR-L1 SEQ ID NO: 1 CDR-L2 SEQ ID NO: 2 CDR-L3 SEQ ID NO: 3 CDR-H1 SEQ ID NO: 4 CDR-H2 SEQ ID NO: 5 CDR-H3 SEQ ID NO: 6 VL SEQ ID NO: 7 VH SEQ ID NO: 8 scFv(aa) SEQ ID NO: 9 scFv(nt) SEQ ID NO: 25

[0131] Example 3. Preparation of CAR-T cells targeting Claudin18.2 and verification of their function

[0132] 3.1 Preparation of CAR-T Cells

[0133] The sequence encoding the following protein was synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), product number: PPL00157-4a): CD8α signal peptide (SEQ ID No: 45), anti-claudin18.2 single-chain antibody (SEQ ID No: 9), CD8α hinge region (SEQ ID No: 47), CD8α transmembrane region (SEQ ID No: 31), 4-1BB intracellular region (SEQ ID No: 37), and CD3ζ intracellular region (SEQ ID No: 39), and the correct insertion of the target sequence was confirmed by sequencing.

[0134] Add 3 ml of Opti-MEM (Gibco, catalog number 31985-070) to a sterile tube to dilute the above plasmid, and then add the packaging vector psPAX2 (Addgene, catalog number 12260) and the envelope vector pMD2.G (Addgene, catalog number 12259) according to the ratio of plasmid:virus packaging vector:virus envelope vector = 4:2:1. Then, add 120 μl of X-treme GENE HP DNA transfection reagent (Roche, catalog number 06366236001), mix immediately, incubate at room temperature for 15 min, and then add the plasmid / vector / transfection reagent mixture drop by drop to the culture flask of 293T cells. Collect the virus at 24 hours and 48 hours, combine them, and obtain concentrated lentivirus by ultracentrifugation (25000 g, 4 °C, 2.5 hours).

[0135] Activate T cells with DynaBeads CD3 / CD28 CTSTM (Gibco, catalog number 40203D) and culture them at 37 °C and 5% CO2 for 1 day. Then, add the concentrated lentivirus and continue to culture for 3 days to obtain BH232-CAR T cells targeting Claudin18.2. Unmodified wild-type T cells (NT) are used as a control.

[0136] After culturing at 37 °C and 5% CO2 for 11 days, use Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab')2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, catalog number 115-065-072) as the primary antibody and APC Streptavidin (BD Pharmingen, catalog number 554067) as the secondary antibody to detect the expression level of the anti-Claudin18.2 single-chain antibody on CAR-T cells by flow cytometry. The results are as Figure 4 shown.

[0137] It can be seen that the anti-Claudin18.2 single-chain antibody in the CAR-T cells prepared by the present invention can be effectively expressed.

[0138] 3.2 Detection of the Killing Effect of CAR-T Cells on Target Cells

[0139] At 1x10 4The target cells (293T-18.2-luci cells, NUGC4-18.2-luci cells) or non-target cells (293T-18.1-luci cells, NUGC4-luci cells, 293T-luci cells) were seeded into 96-well plates at a concentration of Figure 5 cells / well, and then NT cells and BH232-CART cells were seeded into the 96-well plates for co-culture at an effector-to-target ratio of 16:1, 8:1, 4:1, 2:1, 1:1 (i.e., the ratio of effector T cells to target cells). After 16-18 hours, the fluorescence values were measured using a microplate reader. According to the calculation formula: (mean fluorescence of target cells - mean fluorescence of samples) / mean fluorescence of target cells × 100%, the killing efficiency was calculated, and the results are as

[0140] shown.

[0141] 3.3 Detection of the Degranulation of CAR-T Cells

[0142] The target cells (293T-18.2-luci cells, NUGC4-18.2-luci cells, CHO-18.2-luci cells) and non-target cells (293T-luci cells, 293T-18.1-luci cells, NUGC4-luci cells, CHO-18.1-luci cells) were seeded into 96-well plates at a concentration of 1×10 5 cells / well respectively, and BH232-CAR T cells and NT cells (negative control) were added at a ratio of 1:1. Then, 10 μL of PE Mouse anti-human CD107a antibody (BD, catalog number 555801) was added to each well, and the cells were incubated in the dark at 37°C and 5% CO2. After 1 h, 20 μL of Golgi Stop (BD, catalog number 51-2092K2) was added to each well, and the cells were incubated in the dark at 37°C and 5% CO2 for 2.5 h. Then, 10 μL of APC anti-human CD8 (BD, catalog number 555369) was added to each well, and the cells were incubated in the dark at 37°C and 5% CO2 for 0.5 h. The cell samples in each well were detected by flow cytometry, and the proportion of CD107a and CD8 double-positive cells among T cells was analyzed. The results Figure 6 are shown.

[0143] It can be seen that, compared with NT cells, the BH232-CAR T cells prepared by the present invention show significantly increased specific degranulation effects on the three target cells, namely 293T-18.2-luci cells, NUGC4-18.2-luci cells, and CHO-18.2-luci cells, while no significantly increased degranulation effects are observed on non-target cells.

[0144] 3.4 Detection of the Cytokine Release Level of CAR-T Cells

[0145] At a concentration of 1x10 5 cells / well, the target cells (293T-18.2-luci cells, NUGC4-18.2-luci cells, CHO-18.2-luci cells) and non-target cells (293T-luci cells, 293T-18.1-luci cells, NUGC4-luci cells, CHO-18.1-luci cells) were seeded in 96-well plates. BH232-CAR T cells and NT cells (negative control) were added at a ratio of 1:1, and the co-culture supernatant was collected after co-culturing for 18 - 24 hours.

[0146] According to the manufacturer's recommendations, the Human IL-2 DuoSet ELISA Kit (R&D systems, catalog number DY202) and the Human IFN-gamma DuoSet ELISA Kit (R&D systems, catalog number DY285) were used to detect the contents of IL2 and IFN-γ in the co-culture supernatant, and the results are as Figure 7 shown.

[0147] It can be seen that, compared with NT cells, after co-culturing the BH232-CAR T cells of the present invention with the three target cells, the release levels of the cytokines IL2 (A) and IFN-γ (B) are both significantly increased, and this cytokine release is specific.

[0148] Example 4. Preparation of humanized anti-Claudin18.2 antibody

[0149] Using CDR transplantation technology, the murine anti-claudin18.2 antibody was humanized. Specifically, the CDR regions of the BH232 clone were transplanted into the framework region of human heavy chain subtype III and the framework region of light chain κ subtype I (Carter P, PNAS 89:4285-4289 (1992); Presta LG et al., Cancer Research 57:4593-4599 (1997)), and point mutations were introduced into the amino acid residues of the framework region to enhance antibody affinity. Finally, five humanized anti-Claudin18.2 antibodies were obtained, and their sequences are shown in Table 2 below.

[0150] Table 2. Amino acid sequences of humanized anti-Claudin18.2 antibodies

[0151] Clone VL VH scFv(aa) scFv(nt) BH232_V1 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 26 BH232_V2 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 27 BH232_V3 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 28 BH232_V4 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 29 BH232_V5 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 30

[0152] Example 5. Preparation of hCAR-T cells containing humanized anti-Claudin18.2 antibodies and verification of their functions

[0153] Synthesize the sequences encoding the following proteins and clone them into the pLVX vector (Public Protein / Plasmid Library (PPL), catalog number: PPL00157-4a): B2M signal peptide (SEQ ID No: 43), humanized anti-claudin18.2 single-chain antibody (any one of the sequences selected from SEQ ID NO: 12, 15, 18, 21, and 24), CD28 hinge region (SEQ ID No: 49), CD28 transmembrane region (SEQ ID No: 33), CD28 intracellular region (SEQ ID No: 35), and CD3ζ intracellular region (SEQ ID No: 41), and confirm the correct insertion of the target sequence by sequencing.

[0154] Pack the above plasmid into lentivirus according to the method described in 3.1 of Example 3 and infect activated T cells to obtain hCAR-T cells containing humanized anti-Claudin18.2 antibodies. Use Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab')2 Fragment Specific (min X Hu, Bov, Hrs SrProt) (jackson immunoresearch, catalog number 115-065-072) as the primary antibody and PE Streptavidin (Biolegend, catalog number 405204) as the secondary antibody to detect the expression level of the anti-Claudin18.2 single-chain antibody on hCAR-T cells by flow cytometry. The results are as Figure 8 shown.

[0155] It can be seen that the hCAR-T cells prepared with the humanized anti-claudin18.2 single-chain antibody can all effectively express the claudin18.2 single-chain antibody.

[0156] The killing effects of CAR-T cells on target cells NUGC4-18.2-luci and non-target cells NUGC4-luci and 293T-18.1-luci were detected according to the method described in 3.2 of Example 3, and the degranulation of CAR-T cells was detected according to the method described in 3.3 of Example 3. The results are respectively as Figure 9 and Figure 10 shown. It can be seen that the BH232_V1, BH232_V2, BH232_V3, BH232_V4 and BH232_V5 hCAR T cells containing the humanized anti-claudin18.2 single-chain antibody can all produce significant specific killing and degranulation effects on the target cell NUGC4-18.2-luci, and the killing effect is comparable to that of the BH232-CAR T cells constructed with the murine anti-claudin18.2 single-chain antibody, while there is no obvious killing effect on the non-target cells NUGC4-luci and 293T-18.1-luci.

[0157] Example 6. Verification of the tumor suppression effect of CAR-T cells

[0158] Twenty-four healthy female NCG mice at about 7 weeks of age were divided into 4 groups: NT group (negative control), BH232 group, BH232_V1 group and BH232_V3 group. On day 0 (D0), 5×10 6 NUGC4-18.2 cells were subcutaneously injected into each mouse. Ten days later (D10), 2x10 6 NT cells or the corresponding CAR-T cells were injected into the tail vein of each mouse according to the grouping. The changes in the tumor burden of the mice were evaluated twice a week, and the results are as Figure 11 shown.

[0159] It can be seen that the tumor cells in the NT group mice not treated with CAR-T cells continued to grow, while the CAR-T cells constructed with the anti-claudin18.2 antibodies BH232, BH232_V1 and BH232_V3 of the present invention significantly inhibited the growth of tumor cells starting from D25, and maintained the tumor size at a very low level until the end of the experiment. This indicates that the CAR-T cells of the present invention show a strong killing effect on tumor cells in in vivo experiments.

[0160] It should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Those skilled in the art understand that any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Sequence Listing <110> Nanjing Beiheng Biotechnology Co., Ltd. <120> Antibodies Targeting Claudin18.2 and Their Uses <130> BHCN32 <160> 52 <170> SIPOSequenceListing 1.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR-L1 <400> 1 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr 1 5 10 <210> 2 <211> 3 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR-L2 <400> 2 Trp Ala Ser 1 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR-L3 <400> 3 Gln Asn Asp Tyr Ser Tyr Pro Leu Thr 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR-H1 <400> 4 Gly Tyr Ser Phe Thr Gly Tyr 1 5 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR-H2 <400> 5 Asn Pro Tyr Asn Gly Gly 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR-H3 <400> 6 Met Asn Tyr Gly Asn Ala Met Asp Tyr 1 5 <210> 7 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232 VL <400> 7 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 8 <211> 118 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232 VH <400> 8 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 9 <211> 249 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232 scFv <400> 9 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 130 135 140 Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu 165 170 175 Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn 180 185 190 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 195 200 205 Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val Ser Ser 245 <210> 10 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V1 VL <400> 10 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 11 <211> 118 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V1 VH <400> 11 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Pro Ser 115 <210> 12 <211> 249 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V1 scFv <400> 12 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys 130 135 140 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 165 170 175 Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Ala 180 185 190 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser 195 200 205 Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly 225 230 235 240 Ala Gly Thr Thr Val Thr Val Pro Ser 245 <210> 13 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V2 VL <400> 13 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 14 <211> 118 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V2 VH <400> 14 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Pro Ser 115 <210> 15 <211> 249 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V2 scFv <400> 15 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys 130 135 140 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 165 170 175 Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn 180 185 190 Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 195 200 205 Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Ser Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly 225 230 235 240 Ala Gly Thr Thr Val Thr Val Pro Ser 245 <210> 16 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V3 VL <400> 16 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 17 <211> 118 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V3 VH <400> 17 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Pro Ser 115 <210> 18 <211> 249 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V3 scFv <400> 18 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys 130 135 140 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 165 170 175 Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn 180 185 190 Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 195 200 205 Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Ser Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly 225 230 235 240 Ala Gly Thr Thr Val Thr Val Pro Ser 245 <210> 19 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V4 VL <400> 19 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 20 <211> 118 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V4 VH <400> 20 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Pro Ser 115 <210> 21 <211> 249 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V4 scFv <400> 21 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys 130 135 140 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu 165 170 175 Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Ala 180 185 190 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Lys Ser Ser Ser 195 200 205 Thr Ala Tyr Met Glu Leu Leu Ser Leu Arg Ser Asp Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly 225 230 235 240 Ala Gly Thr Thr Val Thr Val Pro Ser 245 <210> 22 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V5 VL <400> 22 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 23 <211> 118 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V5 VH <400> 23 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 24 <211> 249 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V5 scFv <400> 24 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys 130 135 140 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 165 170 175 Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Ala 180 185 190 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser 195 200 205 Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Met Asn Tyr Gly Asn Ala Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val Ser Ser 245 <210> 25 <211> 747 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232 scFv <400> 25 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120 tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaag gcagcaccag cggcagcggc 360 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaag gcagcaccag cggcagcggc 360 aaaccgggca gcggcgaagg cagcaccaaa ggcgaggtcc agctgcaaca gtctggacct 420 aaaccgggca gcggcgaagg cagcaccaaa ggcgaggtcc agctgcaaca gtctggacct 420 gagctggtga agcctggagc ttcaatgaag atatcctgca aggcttctgg ttactcattc 480 gagctggtga agcctggagc ttcaatgaag atatcctgca aggcttctgg ttactcattc 480 actggctaca ccatgaactg ggtgaagcag agccatggaa agaaccttga gtggattgga 540 actggctaca ccatgaactg ggtgaagcag agccatggaa agaaccttga gtggattgga 540 cttattaatc cttacaatgg tggtactagc tacaaccaga agttcaaggg caaggccaca 600 cttattaatc cttacaatgg tggtactagc tacaaccaga agttcaaggg caaggccaca 600 ttaactgtag acaagtcatc cagcacagcc tacatggagc tcctcagtct gacatctgag 660 ttaactgtag acaagtcatc cagcacagcc tacatggagc tcctcagtct gacatctgag 660 gactctgcag tctattactg tgcaagaatg aactatggta atgctatgga ctactggggt 720 gactctgcag tctattactg tgcaagaatg aactatggta atgctatgga ctactggggt 720 caaggaacct cagtcaccgt ctcctca 747 caaggaacct cagtcaccgt ctcctca 747 <210> 26<210> 26 <211> 747<211> 747 <212> DNA<212> DNA <213> Artificial Sequence(Artificial Sequence)<213> Artificial Sequence(Artificial Sequence) <220><220> <223> BH232_V1 <223> BH232_V1 <400> 26 <400> 26 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120 tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 180 tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaag gcagcaccag cggcagcggc 360 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaag gcagcaccag cggcagcggc 360 aaaccgggca gcggcgaagg cagcaccaaa ggcgaggtcc agctgcaaca gtctggacct 420 aaaccgggca gcggcgaagg cagcaccaaa ggcgaggtcc agctgcaaca gtctggacct 420 gagctggtga agcctggagc ttcaatgaag atatcctgca aggcttctgg ttactcattc 480 gagctggtga agcctggagc ttcaatgaag atatcctgca aggcttctgg ttactcattc 480 actggctaca ccatgaactg ggtgaagcag agccatggaa agaaccttga gtggattgga 540 actggctaca ccatgaactg ggtgaagcag agccatggaa agaaccttga gtggattgga 540 cttattaatc cttacaatgg tggtactagc tacaaccaga agttcaaggg caaggccaca 600 cttattaatc cttacaatgg tggtactagc tacaaccaga agttcaaggg caaggccaca 600 ttaactgtag acaagtcatc cagcacagcc tacatggagc tcctcagtct gacatctgag 660 ttaactgtag acaagtcatc cagcacagcc tacatggagc tcctcagtct gacatctgag 660 gactctgcag tctattactg tgcaagaatg aactatggta atgctatgga ctactggggt 720 gactctgcag tctattactg tgcaagaatg aactatggta atgctatgga ctactggggt 720 caaggaacct cagtcaccgt ctcctca 747 <210> 27 <211> 747 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V2 <400> 27 gatatagtaa tgacacagtc accagacagc ctcgccgtca gcctcggaga gcgagcgaca 60 attaattgta agtcatcaca atcattgttg aacagcggaa atcaaaagaa ttatcttact 120 tggtaccagc aaaaaccagg acagccgcca aaactcctca tttattgggc gagtactcgc 180 gaaagtggtg tcccggaccg cttctccggc tctggaagcg gaactgactt taccctcacc 240 attagcagtc ttcaggcaga agatgtcgca gtgtattact gccaaaatga ctattcatac 300 ccgcttacct ttggcgccgg aaccaaattg gaacttaaag gcagcaccag cggcagcggc 360 aaaccgggca gcggcgaagg cagcaccaaa ggcgaagtcc aactgcagca aagcggtgcg 420 gaggttaaaa aaccaggagc ttctgtaaag gtttcttgca aggcaagtgg ttactctttc 480 actggataca cgatgaattg ggttaagcaa gcgccgggcc agggtctgga atggatcggt 540 ctgataaacc cttacaacgg tggtacatcc tacaaccaaa agttccaagg caaagcaacg 600 cttacggtgg ataagtcatc ttcaactgct tacatggagc tttcccgact ccgatctgaa 660 gactccgccg tgtattactg cgcaaggatg aactatggga acgcgatgga ttactggggg 720 gcaggcacaa cggtgacggt accgagc 747 <210> 28 <211> 747 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V3 <400> 28 gatattgtga tgacgcaaag ccctgacagc ctggccgtgt cacttgggga gagagcgaca 60 atgtcttgca agagtagtca gtccctgctg aactctggaa accagaaaaa ttatttgaca 120 tggtatcagc agaaaccagg ccaaccgccc aaattgctga tctattgggc ctcaacccga 180 gagtcaggcg tgccggatcg attctctggc agtggctctg ggactgattt cacccttacc 240 ataagctccg tgcaagcaga ggacgtggcc gtttattatt gtcaaaacga ctactcttat 300 ccacttacat ttggcgctgg cactaaactt gaactgaaag gcagcaccag cggcagcggc 360 aaaccgggca gcggcgaagg cagcaccaaa ggcgaggttc aactccaaca atctggcgcc 420 gaggttaaga agcctggggc gagtgtaaaa gtttcttgta aggcaagtgg ttactctttt 480 accggataca cgatgaattg ggtgaaacaa gcaccgggtc agggtttgga gtggataggg 540 ctcattaatc cgtacaatgg agggacatct tacaatcaga agtttcaagg gaaagcaact 600 ctgactgtcg ataagtcatc tagcaccgcg tacatggagt tgtcacgctt gaggagtgaa 660 gacagtgcgg tgtattattg tgcgcggatg aactatggaa acgcaatgga ctattgggga 720 gctggaacaa cagtgacagt gcctagc 747 <210> 29 <211> 747 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V4 <400> 29 gatattgtca tgactcaatc cccagactcc ctcgccgtga gtcttgggga gcgagctacg 60 attaactgta aatcatcaca gagtcttctc aacagtggta atcaaaagaa ttaccttacc 120 tggtatcaac agaagcctgg tcagcctcct aaactcctga tatattgggc atctacgcga 180 gagtctgggg tgccggatag gtttagcgga agtggtagtg gaacagattt cacactcaca 240 ataagctctt tgcaagcgga agatgtagcc gtatactact gccaaaacga ctattcctat 300 cccctgacct ttggagcggg aacgaaactt gagctgaagg gcagcaccag cggcagcggc 360 aaaccgggca gcggcgaagg cagcaccaaa ggcgaagtgc aactgcaaca gtcaggcgcg 420 gaagtaaaga aaccgggggc ctctgtgaaa gtcagctgca aggcctcagg atacagcttt 480 acagggtaca caatgaactg ggtcaaacag agccacggta aaaaccttga gtggataggc 540 ttgataaatc catataatgg aggtacatct tacgcgcaaa agtttcaggg gcgagtaact 600 atgactagag ataaaagctc ctcaacggct tatatggagc tgctgtccct tcgctctgac 660 gataccgccg tatattactg tgctagaatg aactacggta acgctatgga ttattggggg 720 gcagggacga ccgtaactgt tcctagc 747 <210> 30 <211> 747 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> BH232_V5 <400> 30 gatattgtta tgacacaaag tcctgattca ctggcggtat ctctcggcga aagggcgaca 60 atcaattgca agtcaagtca aagcctcctt aactcaggaa atcaaaaaaa ctatttgact 120 tggtatcagc agaagccagg acaaccaccg aaactcctta tttattgggc ctccacaagg 180 gagtcaggtg taccagacag gttctcagga agtggctcag gaaccgattt tacccttacc 240 atttcatccc tccaggcgga agacgtggca gtatattact gccagaatga ttactcatat 300 cccctcacct tcggcgcagg gactaagctg gagcttaagg gcagcaccag cggcagcggc 360 aaaccgggca gcggcgaagg cagcaccaaa ggcgaagtcc aacttcagca aagcggagct 420 gaggtcaaaa aacctggcgc aagtgttaaa gtgagctgca aggcaagtgg atatagtttc 480 accggatata cgatgaattg ggtcaaacag gcgcctggac agggactgga atggatagga 540 ctcataaatc cgtacaatgg cggaacaagt tatgcacaga agttccaagg gcgcgtaact 600 atgaccaggg acacctcaat tagcactgct tacatggagc tctcaaggtt gcgatcagac 660 gataccgcgg tttactactg cgccagaatg aattacggca acgcgatgga ctactggggg 720 cagggtacga gcgttatactgt ttcaagc 747 <210> 31 <211> 25 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α transmembrane domain <400> 31 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Lys 20 25 <210> 32 <211> 75 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α transmembrane domain <400> 32 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gcaaa 75 <210> 33 <211> 27 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 transmembrane domain <400> 33 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 34 <211> 81 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 transmembrane domain <400> 34 ttttgggtcc tcgtcgtagt tggaggggta cttgcctgtt atagcctcct ggttaccgta 60 gcatttatta tattctgggt g 81 <210> 35 <211> 41 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 costimulatory domain <400> 35 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 36 <211> 123 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 costimulatory domain <400> 36 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <210> 37 <211> 40 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> 4-1BB costimulatory domain <400> 37 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 1 5 10 15 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 20 25 30 Glu Glu Glu Glu Gly Gly Cys Glu 35 40 <210> 38 <211> 120 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> 4-1BB costimulatory domain <400> 38 cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 60 actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 120 <210> 39 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD3ζ signaling domain <400> 39 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 1 5 10 15 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 20 25 30 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 35 40 45 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 100 105 110 Arg <210> 40 <211> 339 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD3ζ signaling domain <400> 40 ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 60 ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 120 ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 180 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300 acctacgacg cccttcacat gcaggccctg ccccctcgc 339 <210> 41 <211> 114 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD3ζ signaling domain <400> 41 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 1 5 10 15 Gly Gln Asn Gln Leu Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 20 25 30 Phe Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 35 40 45 Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 50 55 60 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 65 70 75 80 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser 85 90 95 Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu Pro 100 105 110 Pro Arg <210> 42 <211> 342 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD3ζ signaling domain <400> 42 ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 60 ctctttaacg agctcaatct aggacgaaga gaggagttcg atgttttgga caagagacgt 120 ggccgggacc ctgagatggg gggaaagccg cagagaagga agaaccctca ggaaggcctg 180 tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 240 gagcgccgga ggggcaaggg gcacgatggc cttttccagg gtctcagtac agccaccaag 300 gacacctttg acgcccttca catgcaggcc ctgccccctc gc 342 <210> 43 <211> 20 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> B2M signal peptide <400> 43 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala 20 <210> 44 <211> 60 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> B2M signal peptide <400> 44 atgtcccgct ctgttgcttt ggctgtgctg gcccttttgt cccttagcgg actggaggcc 60 <210> 45 <211> 21 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α signal peptide <400> 45 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 46 <211> 63 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α signal peptide <400> 46 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 47 <211> 45 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α hinge region <400> 47 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 48 <211> 135 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α hinge region <400> 48 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 49 <211> 39 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 hinge region <400> 49 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro 35 <210> 50 <211> 117 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 hinge region <400> 50 attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60 catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagccc 117 <210> 51 <211> 12 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> IgG4 hinge region <400> 51 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 52 <211> 36 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> IgG4 hinge region <400> 52 gaaagcaaat acgggccgcc gtgtccaccc tgtccg 36

Claims

1. An antibody targeting Claudin18.2, which comprises CDR-L1 shown in SEQ ID NO: 1, CDR-L2 shown in SEQ ID NO: 2, CDR-L3 shown in SEQ ID NO: 3, CDR-H1 shown in SEQ ID NO: 4, CDR-H2 shown in SEQ ID NO: 5, and CDR-H3 shown in SEQ ID NO:

6.

2. The antibody according to claim 1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NOs: 8, 11, 14, 17, 20, and 23, or has conservative modifications of one or several amino acids compared with the amino acid sequences selected from SEQ ID NOs: 8, 11, 14, 17, 20, and 23; the light chain variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NOs: 7, 10, 13, 16, 19, and 22, or has conservative modifications of one or several amino acids compared with the amino acid sequence selected from SEQ ID NO:

7.

3. The antibody according to claim 1, wherein the amino acid sequence of the antibody has at least 90% identity with an amino acid sequence selected from SEQ ID NOs: 9, 12, 15, 18, 21, and 24, or has conservative modifications of one or several amino acids compared with the amino acid sequences selected from SEQ ID NOs: 9, 12, 15, 18, 21, and 24.

4. The antibody according to any one of claims 1-3, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

5. A nucleic acid molecule encoding the antibody according to any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity with a nucleotide sequence selected from SEQ ID NOs: 25-30, and the antibody encoded by it can specifically bind to Claudin18.

2.

7. A multispecific antibody, which comprises the antibody according to any one of claims 1-4 and one or more second antibodies or antigen-binding portions thereof that specifically bind to other antigens.

8. The multispecific antibody according to claim 7, wherein the second antibody or its antigen-binding portion is selected from a full-length antibody, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv, minibody, diabody or sdAb.

9. A vector, which comprises a nucleic acid molecule encoding the antibody according to any one of claims 1-4 or the multispecific antibody according to claim 7 or 8.

10. A host cell, which expresses the antibody according to any one of claims 1-4 or the multispecific antibody according to claim 7 or 8.

11. A chimeric antigen receptor, which comprises the antibody according to any one of claims 1-4 or the multispecific antibody according to claim 7 or 8, a transmembrane domain, and an intracellular signaling domain.

12. The chimeric antigen receptor according to claim 11, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

13. The chimeric antigen receptor according to claim 11, wherein the intracellular signaling domain is selected from the intracellular regions of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

14. The chimeric antigen receptor according to claim 11, wherein the chimeric antigen receptor further comprises one or more co-stimulatory domains, which are selected from the co-stimulatory signaling domains of the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CLAUDIN18.2, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278(ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70.

15. An engineered immune cell comprising the chimeric antigen receptor according to any one of claims 11-14.

16. The engineered immune cell according to claim 15, which is selected from T cells, NK cells, NKT cells, macrophages, dendritic cells.

17. The engineered immune cell according to claim 15, which further comprises a second chimeric antigen receptor targeting other tumor antigens.

18. The engineered immune cell according to any one of claims 15-17, further comprising the expression of at least one gene selected from the following being inhibited or silenced: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3 and CTLA4.

19. An antibody conjugate comprising the antibody according to any one of claims 1-4 or the multispecific antibody according to claim 7 or 8, and a second functional structure, wherein the second functional structure is selected from Fc, radioisotope, a structure moiety for extending the half-life, a detectable label and a drug.

20. The antibody conjugate according to claim 19, wherein the structure portion for extending the half-life is selected from: the binding structure of albumin, the binding structure of transferrin, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and white polypeptides that bind to human serum albumin; the detectable label is selected from fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents; and the drug is selected from cytotoxins and immunomodulators.

21. A detection kit, which comprises the antibody according to any one of claims 1-4, the multispecific antibody according to claim 7 or 8, the chimeric antigen receptor according to any one of claims 11-14, or the antibody conjugate according to claim 19 or 20.

22. A pharmaceutical composition comprising the antibody according to any one of claims 1-4, the multispecific antibody according to claim 7 or 8, the chimeric antigen receptor according to any one of claims 11-14, the engineered immune cell according to any one of claims 15-18, or the antibody conjugate according to claim 19 or 20, and one or more pharmaceutically acceptable excipients.

23. Use of the antibody according to any one of claims 1-4, the multispecific antibody according to claim 7 or 8, the chimeric antigen receptor according to any one of claims 11-14, the engineered immune cell according to any one of claims 15-18, or the antibody conjugate according to claim 19 or 20, or the pharmaceutical composition according to claim 22, in the preparation of a medicament for the treatment and / or prevention and / or diagnosis of gastric cancer.

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