CN114539402A

CN114539402A - Antibodies targeting Claudin18.2 and uses thereof

Info

Publication number: CN114539402A
Application number: CN202011354138.3A
Authority: CN
Inventors: 李国坤; 浦容容; 任江涛; 贺小宏; 王延宾; 韩露
Original assignee: Nanjing Bioheng Biotech Co Ltd
Current assignee: Nanjing Bioheng Biotech Co Ltd
Priority date: 2020-11-27
Filing date: 2020-11-27
Publication date: 2022-05-27
Also published as: WO2022111405A1

Abstract

The invention provides an antibody targeting Claudin18.2, as well as a multispecific antibody, a chimeric antigen receptor, an antibody conjugate, a pharmaceutical composition and a kit comprising the same, and applications of the antibody, the antibody conjugate, the pharmaceutical composition and the kit in diagnosis/treatment/prevention of diseases related to Claudin18.2 expression.

Description

Antibodies targeting Claudin18.2 and uses thereof

Technical Field

The present invention is in the field of immunotherapy. More specifically, the invention relates to antibodies targeting claudin18.2 and their use in the prevention and/or treatment and/or diagnosis of disease.

Background

Tight Junction (TJ) complexes exist among epithelial cells of a normal body, have the effects of cell adhesion, maintaining cell polarity and permeability, assisting in transmitting and regulating cell proliferation and differentiation signals and the like, and are very important for maintaining the normal structure and function of the epithelial cells. The Claudin protein is a main component of TJ, participates in maintaining various functions of the TJ, and is closely related to various tumors due to abnormal expression. Among them, Claudin18 protein has two splice variants, Claudin18.1 (also known as CLDN18.1) and Claudin18.2 (also known as CLDN18.2), the former being selectively expressed in normal epithelial cells of lung, while the latter being expressed only on differentiated epithelial cells on gastric mucosa in normal tissues and highly expressed in gastric cancer and pancreatic ductal cancer in tumor tissues. These characteristics make claudin18.2 a clinically significant target in the treatment of gastric cancer and other claudin18.2 positive tumors.

Therefore, the method has important value and significance for drug development and antibody development aiming at the Claudin18.2 target, and research of a new treatment scheme and a combined treatment scheme. The invention aims to provide an antibody targeting Claudin18.2 and application thereof in disease prevention and/or treatment and/or diagnosis.

Disclosure of Invention

In a first aspect, the invention provides an antibody or antigen-binding fragment thereof targeting claudin18.2, comprising an amino acid sequence as set forth in SEQ ID NO: 1, CDR-L1 as set forth in SEQ ID NO: 2, as shown in SEQ ID NO:3, as shown in SEQ ID NO:4, CDR-H1 as shown in SEQ ID NO: 5, and a CDR-H2 as set forth in SEQ ID NO: 6, CDR-H3.

In one embodiment, the antibody or antigen binding fragment thereof of the present invention comprises a heavy chain variable region that is identical to a light chain variable region selected from the group consisting of SEQ ID NOs: 8. 11, 14, 17, 20 and 23, or at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 8. 11, 14, 17, 20 and 23 have one or more amino acid modifications (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) compared to the amino acid sequence; the light chain variable region is identical to a light chain variable region selected from the group consisting of SEQ ID NO: 7. 10, 13, 16, 19 and 22, or an amino acid sequence selected from SEQ ID NOs: 7 has one or more amino acid modifications compared to the amino acid sequence of (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). 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 invention comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 8. 11, 14, 17, 20 and 23 and a heavy 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 invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

(a) as shown in SEQ ID NO: 8 and the heavy chain variable region as set forth in SEQ ID NO: 7;

(b) as shown in SEQ ID NO: 11 and the heavy chain variable region as set forth in SEQ ID NO: 10;

(c) as shown in SEQ ID NO: 14 and the heavy chain variable region as set forth in SEQ ID NO: 13, a light chain variable region;

(d) as shown in SEQ ID NO: 17 and the heavy chain variable region as set forth in SEQ ID NO: 16;

(e) as shown in SEQ ID NO: 20 and the heavy chain variable region as set forth in SEQ ID NO: 19, the light chain variable region shown in seq id no;

(f) as shown in SEQ ID NO: 23 and the heavy chain variable region as set forth in SEQ ID NO: 22;

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 groups (a) - (f);

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

In one embodiment, the antibody or antigen binding fragment thereof of the invention binds to a polypeptide selected from the group consisting of SEQ ID NO: 9. 12, 15, 18, 21 and 24, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to an amino acid sequence selected from SEQ ID NOs: 9. 12, 15, 18, 21 and 24 have one or several amino acid (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) modifications as compared to the amino acid sequence. 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 invention is selected from the group consisting of SEQ ID NO: 9. 12, 15, 18, 21 and 24.

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

The invention also provides nucleic acid molecules encoding the above antibodies or antigen-binding fragments thereof. Thus, in one embodiment, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof binds to a polypeptide selected from the group consisting of SEQ ID NOs: 25-30 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity and encodes an antibody or antigen-binding fragment thereof that is capable of specifically binding to the claudin18.2 antigen. Preferably, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 25-30.

In another aspect, the invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising an anti-claudin 18.2 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.

In one embodiment, the second antibody or antigen-binding portion thereof can be in the form of any antibody or antibody fragment, e.g., full length antibody, Fab ', (Fab')₂Fv, scFv-scFv, minibody, diabody or sdAb.

The invention also provides a vector comprising a nucleic acid molecule encoding the anti-claudin 18.2 antibody or antigen-binding fragment thereof or multispecific antibody described above, and a host cell expressing the anti-claudin 18.2 antibody or antigen-binding fragment thereof or multispecific antibody.

In another aspect, the invention also provides a chimeric antigen receptor comprising an anti-claudin 18.2 antibody, or antigen-binding fragment thereof, described herein, 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 an anti-claudin18.2 antibody, or antigen-binding fragment or multispecific antibody thereof, as provided herein, a CD8 a or CD28 transmembrane region, a CD28 or 4-1BB co-stimulatory domain, and a CD3 ζ intracellular signaling domain.

The invention also provides a nucleic acid molecule encoding a chimeric antigen receptor targeting claudin18.2 as defined above, as well as a vector comprising said nucleic acid molecule.

The invention also provides a cell, preferably an immune cell, e.g. a T cell, NK cell, NKT cell, macrophage, dendritic cell, comprising a chimeric antigen receptor targeting claudin18.2 as defined above. In a preferred embodiment, the engineered immune cell further comprises a second chimeric antigen receptor that targets other tumor antigens.

In another aspect, the present invention also provides an antibody conjugate comprising an anti-claudin 18.2 antibody or antigen-binding fragment thereof as defined herein and a second functional structure, wherein said second functional structure is selected from the group consisting of an Fc, a radioisotope, a half-life extending moiety, a detectable label and a drug.

In one embodiment, the half-life extending moiety is selected from the group consisting of: the half-life extending moiety is selected from the group consisting of a binding structure for albumin, a binding structure for transferrin, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and a white polypeptide (including an antibody) that binds human serum albumin. In one embodiment, the detectable marker is selected from the group consisting of a fluorophore, a chemiluminescent compound, a bioluminescent compound, an enzyme, an antibiotic resistance gene, and a contrast agent. In one embodiment, the drug is selected from the group consisting of cytotoxins and immunomodulators.

In another aspect, the invention also provides a detection kit comprising an anti-claudin 18.2 antibody or antigen-binding fragment thereof, a multispecific antibody, antibody conjugate, or chimeric antigen receptor of the invention.

In another aspect, the invention also provides a pharmaceutical composition comprising an anti-claudin 18.2 antibody or antigen-binding fragment thereof, a chimeric antigen receptor, a multispecific antibody, an engineered immune cell, or an antibody conjugate of the invention, and one or more pharmaceutically acceptable excipients.

In another aspect, the invention also provides a method of treating and/or preventing and/or diagnosing a disease associated with claudin18.2 expression comprising administering to a subject an anti-claudin 18.2 antibody or antigen-binding fragment thereof, chimeric antigen receptor, multispecific antibody, antibody conjugate, engineered immune cell, or pharmaceutical composition as described above.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

anti-Claudin18.2 antibodies or antigen-binding fragments thereof

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

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 to 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 of 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), heavy chain variable region (VH) or light chain variable region (VL) of an antibody, linear antibody, "diabody" having two antigen binding sites, single domain antibody, nanobody, natural ligand of the antigen or a functional fragment thereof, and the like. Thus, unless the context clearly indicates otherwise, an "antibody" of the invention encompasses an antibody fragment or an antigen-binding fragment as defined above.

Typically, an intact antibody comprises two heavy chains and two light chains linked together by disulfide bonds, each light chain being linked to a respective heavy chain by a disulfide bond, in a "Y" shaped configuration. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region, wherein the heavy chain variable region comprises three Complementarity Determining Regions (CDRs): CDR-H1, CDR-H2 and CDR-H3, the heavy chain constant region comprising three constant domains: CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (VL) and a light chain constant region, wherein the light chain variable region comprises three CDRs: CDR-L1, CDR-L2 and CDR-L3, the light chain constant region comprises a constant domain CL. In the heavy/light chain variable region, the CDRs are separated by more conserved Framework Regions (FRs). The variable regions of the heavy/light chains are responsible for recognition and binding to antigens, while the constant regions may mediate binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using a number of numbering schemes well known in the art, including: kabat et al (1991), "Sequences of proteins of Immunological Interest," published Health Service, national institutes of Health, Besseda, 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 mapping," J.mol.biol.262,732-745 "(" Contact "numbering scheme); lefranc MP et al, "IMGTunique number for immunologublins and T cell receptor variable domains and perfect V-like domains," Dev Comp Immunol, month 1 2003; 27(1) 55-77 ("IMGT" numbering scheme); honegger A and Pl ü ckthun A, "Yeast antenna number scheme for immunoglobulin variable domains, an automatic modeling and analysis tool," Jmol Biol, 6.8.2001; 309(3) 657-70 ("Aho" numbering scheme); and Martin et al, "Modeling antibody hypervariable loops: a combined algorithms," PNAS,1989,86(23): 9268-.

The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat approach is based on structural alignment, while the Chothia approach is based on structural information. The numbering of both the Kabat and Chothia schemes is based on the most common antibody region sequence length, with insertions provided by insertion letters (e.g., "30 a") and deletions occurring in some antibodies. These two schemes place certain insertions and deletions (indels) at different locations, resulting in different numbers. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM protocol is a compromise between Kabat and Chothia definitions, which is based on the protocol used by Oxford Molecular's AbM antibody modeling software.

Thus, unless otherwise specified, it is understood that "CDRs" of a given antibody or region thereof (e.g., a variable region thereof) encompass CDRs defined by any of the above-described schemes or other known schemes. For example, where a particular CDR (e.g., CDR3) is designated to contain a given amino acid sequence, it is understood that such CDR can also have the sequence of the corresponding CDR (e.g., CDR3) as defined by any of the above schemes or other known schemes. Likewise, unless otherwise specified, it is understood that the FR of a given antibody or region thereof (e.g., the variable region thereof) encompasses the FR defined by any of the above-described schemes or other known schemes. Unless otherwise indicated, the numbering scheme used herein to define the boundaries of the CDRs and FRs employs the Kabat scheme.

"Single chain antibody" and "scFv" are used interchangeably herein and refer to an antibody comprising an antibody heavy chain variable region (VH) and a light chain variable region (VL) joined by a linker. The optimal length and/or amino acid composition of the linker may be selected. The length of the linker will significantly affect the variable region folding and interaction profiles of the scFv. In fact, if shorter linkers are used (e.g., between 5-10 amino acids), intra-strand folding may be prevented. For the choice of linker size and composition, see, e.g., Hollinger et al, 1993Proc Natl Acad. Sci. U.S.A.90: 6444-; 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 entirety. The scFv may comprise a VH and a VL connected in any order, for example a VH-linker-VL or a VL-linker-VH.

As used herein, the term "chimeric antibody" refers to an antibody in which a portion of each of the heavy and light chain amino acid sequences 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 in another species or belonging to another class. Typically, the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one species, while the constant regions are homologous to antibody sequences from another species. One clear advantage of such chimeric forms is that the variable regions can be conveniently generated from currently known sources using readily available B cells or hybridomas from non-human hosts, in combination with constant regions from, for example, human cells. The variable region has the advantage of being easy to prepare and the specificity is not affected by the source, whereas since the constant region is from a human, the antibody will have a lower probability of eliciting a human immune response upon injection than if the constant region is from a non-human source.

As used herein, a "humanized" antibody refers to an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. "humanized forms" of a non-human antibody refer to variants of the non-human antibody that have undergone humanization to generally reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are well known to those skilled in the art, see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008). Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using a "best fit" approach; framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions; human mature (somatic mutation) framework regions or human germline framework regions; and screening the FR library for the resulting framework region.

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

In one embodiment, the invention provides an antibody or antigen binding fragment thereof targeting claudin18.2 comprising an amino acid sequence as set forth in SEQ ID NO: 1, CDR-L1 as set forth in SEQ ID NO: 2, as shown in SEQ ID NO:3, as shown in SEQ ID NO:4, CDR-H1 as shown in SEQ ID NO: 5, and a CDR-H2 as set forth in SEQ ID NO: 6, CDR-H3.

In one embodiment, the antibody or antigen binding fragment thereof of the present invention comprises a heavy chain variable region that is identical to a light chain variable region selected from the group consisting of SEQ ID NOs: 8. 11, 14, 17, 20 and 23, or 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% identical to an amino acid sequence selected from SEQ ID NOs: 8. 11, 14, 17, 20 and 23 with one or several (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications as compared to the amino acid sequence; the light chain variable region is identical to a light chain variable region selected from the group consisting of SEQ ID NO: 7. 10, 13, 16, 19 and 22, or 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% identical to an amino acid sequence selected from SEQ ID NOs: 7. 10, 13, 16, 19 and 22 has one or more amino acid modifications (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) as compared to the amino acid sequence. 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 invention comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 8. 11, 14, 17, 20 and 23 and a heavy 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 invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

(g) as shown in SEQ ID NO: 8 and the heavy chain variable region as set forth in SEQ ID NO: 7;

(h) as shown in SEQ ID NO: 11 and the heavy chain variable region as set forth in SEQ ID NO: 10;

(i) as shown in SEQ ID NO: 14 and the heavy chain variable region as set forth in SEQ ID NO: 13, a light chain variable region;

(j) as shown in SEQ ID NO: 17 and the heavy chain variable region as set forth in SEQ ID NO: 16;

(k) as shown in SEQ ID NO: 20 and the heavy chain variable region as set forth in SEQ ID NO: 19, the light chain variable region shown in seq id no;

(l) As shown in SEQ ID NO: 23 and the heavy chain variable region as set forth in SEQ ID NO: 22;

optionally, the heavy chain variable region and the light chain variable region are 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% identical to the heavy chain variable region and the light chain variable region of any one of groups (a) - (f);

In one embodiment, the antibody or antigen binding fragment thereof of the invention binds to a polypeptide selected from the group consisting of SEQ ID NO: 9. 12, 15, 18, 21 and 24 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to an amino acid sequence selected from SEQ ID NOs: 9. 12, 15, 18, 21 and 24 have one or several amino acid modifications compared to the amino acid sequence, e.g., 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. Preferably, the amino acid sequence of the antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of SEQ ID NO: 9. 12, 15, 18, 21 and 24.

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. Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including 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 may be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

As used herein, the term sequence "identity" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is often expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% identity. Those skilled in the art know 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.

In one aspect, the invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising an anti-claudin 18.2 antibody or antigen-binding fragment thereof as described above, further comprising one or more secondary antibodies that specifically bind to other antigens.

As used herein, the term "multispecific" refers to an antigen binding protein having polyepitopic specificity (i.e., capable of specifically binding to two, three, or more different epitopes on one biomolecule or capable of specifically binding to epitopes on two, three, or more different biomolecules). As used herein, the term "bispecific" means that the antigen binding protein has two different antigen binding specificities.

In one embodiment, the second antibody may have any antibody or antibodiesAntibody fragment forms, e.g. full length antibody, Fab ', (Fab')₂Fv, scFv-scFv, minibody, diabody or sdAb.

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, CD126, CD138, B54, MUC-1, Ia, HM1.24, HLA-DR, tenascin, angiogenic factor, VEGF, PIGF, ED-B fibronectin, oncogene product, CD66 54-d, necrotic antigen, Ii, IL-2, T101, TAC, IL-6, R54, DR 54, tEGFR, Her 54, L4-CAM, mesothelin, CEA, hepatitis B surface antigen, anti-CD receptor, CD54, EGFR 54, HERB 54, EGFR-54, EGCG 54, EGFR-2, EGFR-54, EGFR-2, EGCG 54, EGFR-2, EGFR-54, EGFR-D54, EGFR-2, EGFR-D54, EGFR-2, and EGFR-D54, EGFR-D54, EGFR, and ErbB-54, and B-D54, kdr, kappa 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, bi-antigen, antigen associated with a universal TAG, cancer-testis antigen, MUC D, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, VEGF-R D, CEA, PSM D, PSM-A D, prostate specific antigen, Met-D, Met-AG D, Met-CD D, Met-D D, Met-K D, and monoclonal antibody, CCL-1, hTERT, MDM2, CYP1B, WT1, activin, AFP, p53, cyclin (D1), CS-1, BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecules, cyclins (such as cyclin A1(CCNA1)) and/or pathogen-specific antigens, biotinylated molecules, molecules expressed by HIV, HCV, HBV, and/or other pathogens; and/or a neoepitope or neoantigen.

Nucleic acids, vectors, host cells

In another aspect, the invention relates to a nucleic acid molecule encoding the anti-claudin18.2 antibody or multispecific antibody of the invention. The nucleic acid of the present invention may be RNA, DNA or cDNA. According to one embodiment of the invention, the nucleic acid of the invention is a substantially isolated nucleic acid.

In one embodiment, the nucleic acid molecule encoding said anti-claudin18.2 antibody hybridizes with a sequence selected from the group consisting of SEQ ID NO: 25-30 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity and the anti-claudin 18.2 antibody that it encodes is capable of specifically binding to claudin18.2 (i.e., binds little to non-target antigens such as claudin 18.1). Preferably, the nucleic acid molecule encoding said anti-claudin18.2 antibody is as set forth in SEQ ID NO: 25-30.

The nucleic acid of the invention may also be in the form of a vector, may be present in and/or may be part of a vector, such as a plasmid, cosmid or YAC. The vector may especially be an expression vector, i.e.a vector providing for 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 typically comprises at least one nucleic acid molecule of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, and the like). The selection of such regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory and other elements useful or necessary for expression of the Claudin18.2 antibody of the invention include, but are not limited to, promoters, enhancers, terminators, integration factors, selectable markers, leaders, and reporters.

In another aspect, the invention also provides host cells expressing the claudin18.2 antibody, multispecific antibody and/or containing the nucleic acid or vector of the invention. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

Suitable bacterial cells include cells of gram-negative bacterial strains, such as Escherichia coli, Proteus and Pseudomonas strains, and gram-positive bacterial strains, such as Bacillus (Bacillus), Streptomyces, Staphylococcus and Lactococcus strains.

Suitable fungal cells include cells of species of the genera Trichoderma (Trichoderma), Neurospora (Neurospora) and Aspergillus (Aspergillus); or cells of species including Saccharomyces (Saccharomyces) such as Saccharomyces cerevisiae, Schizosaccharomyces (Schizosaccharomyces pombe), Pichia (Pichia) such as Pichia pastoris and Pichia methanolica, and Hansenula.

Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins may also be used in the present invention.

Chimeric antigen receptors

In another aspect, the invention also provides a recombinant receptor, such as a recombinant TCR receptor or a chimeric antigen receptor, comprising an anti-claudin18.2 antibody as described above. Preferably, the invention also provides a chimeric antigen receptor comprising an anti-claudin18.2 antibody as described above.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide generally comprising a ligand binding domain (e.g., an antigen-binding portion of an antibody), a transmembrane domain, an optional costimulatory domain, and an intracellular signaling domain, each linked by a linker. CARs are able to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner using the antigen-binding properties of antibodies.

In one embodiment, the present invention provides a chimeric antigen receptor comprising an anti-claudin 18.2 antibody or antigen-binding fragment thereof as described above or a multispecific antibody, transmembrane domain and intracellular signaling domain comprising said anti-claudin 18.2 antibody.

As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables a chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and to direct the cellular response of the immune cell against a target cell. The transmembrane domain may be natural or synthetic, and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric antigen receptor binds to a target antigen. Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, the TCR α chain, the TCR β chain, the TCR γ chain, the TCR δ chain, the CD3 ζ subunit, the CD3 ε subunit, the CD3 γ subunit, the CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. Alternatively, the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the CD8 a chain or CD28, 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. 31 or 33, or whose 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.

As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs a cell to perform a specified function. In one embodiment, the intracellular signaling domains comprised by the chimeric antigen receptors of the present invention may be the intracellular domain sequences of T cell receptors and co-receptors which work together to trigger signaling upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar function. The intracellular signaling domain may contain a number of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). Non-limiting examples of intracellular signaling domains of the invention include, but are not limited to, intracellular regions of FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, claudin18.29a, claudin18.29b, and CD66d, among others. In preferred embodiments, the signalling domain of a CAR of the invention may comprise the intracellular region of CD3 ζ having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 39 or 41, or a coding sequence thereof having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleic acid molecule set forth in SEQ ID No. 38 or 40.

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 connect a transmembrane domain to an antibody. In particular, 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 derived in whole or in part from a naturally occurring molecule, such as the extracellular region of CD8, CD4, or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a portion of the hinge region of CD8 a, CD28, Fc γ RIII a receptor, IgG4 or IgG1, more preferably a CD8 a, 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 whose 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.

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. "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of co-stimulatory domains of the invention include, but are not limited to, co-stimulatory signaling domains derived from: 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, ghlit, TRIM, and ZAP 70. Preferably, the co-stimulatory domain of the CAR of the invention is from 4-1BB, CD28 or 4-1BB + CD 28. 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 to the amino acid sequence depicted 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 to the nucleic acid molecule depicted 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 depicted 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 depicted in SEQ ID NO. 36.

In one embodiment, the CAR of the invention may further comprise a signal peptide such that when it is expressed in a cell, for example 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 alpha-helix. At the end of the signal peptide there is usually a stretch of amino acids which is recognized and cleaved by the signal peptidase. The signal peptidase may cleave during translocation or after completion 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 those derived from B2M, CD8 α, IgG1, GM-CSFR α, and the like. In one embodiment, the signal peptide useful in the present invention is derived from B2M or CD8 α having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO 43 or 45, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleic acid molecule shown in SEQ ID NO 44 or 46.

In one embodiment, the CAR comprises an anti-claudin 18.2 antibody or antigen-binding fragment thereof as provided herein or a multispecific antibody comprising the anti-claudin 18.2 antibody, a CD8 a or CD28 transmembrane region, a CD28 or 4-1BB co-stimulatory domain, and a CD3 ζ intracellular signaling domain. In this embodiment, the CAR may further comprise a signal peptide from B2M, CD8 a, IgG1, or GM-CSFR a.

As used herein, the term "vector" is a vector nucleic acid molecule used as a vehicle for transferring (foreign) genetic material into a host cell where it can, for example, be replicated and/or expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering an isolated nucleic acid to the interior of a cell, for example, by homologous recombination or by using a hybrid recombinase that targets sequences at a site specifically. 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 invention) in a suitable host cell and the translation of their mRNA. Suitable carriers for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, rous sarcoma viruses (RSV, polyoma viruses and adeno-associated viruses (AAV), etc.), bacteriophages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). the vectors themselves are typically nucleic acid molecules, typically DNA sequences comprising an insert (transgene) and a larger sequence that serves as a "backbone" for the vector. The vector is an in vitro transcription vector.

Engineered immune cells

In one aspect, the invention also provides an engineered immune cell expressing a CAR of the invention.

As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune cell may be a T cell, macrophage, dendritic cell, monocyte, NK cell, and/or NKT cell. In one embodiment, the immune cell is derived from a stem cell, such as an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell, and the like. Preferably, the immune cell is a T cell. The T cell may be any T cell, such as an in vitro cultured T cell, e.g., a primary T cell, or a T cell from an in vitro cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. 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 the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells may also be concentrated or purified. The T cells may be at any developmental stage, 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, γ δ -T cells, α β -T cells, and the like. In a preferred embodiment, the immune cell is a human T cell. T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled in the art, such as Ficoll isolation.

The nucleic acid sequence encoding the chimeric antigen receptor can be introduced into an immune cell using conventional methods known in the art (e.g., by transduction, transfection, transformation, etc.). "transfection" is the process of introducing a nucleic acid molecule or polynucleotide (including vectors) into a target cell. One example is RNA transfection, the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into a host cell. The term is used primarily for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe viral-mediated transfer of a nucleic acid molecule or polynucleotide. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of the material. Transfection may be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to create liposomes that fuse with the cell membrane and deposit their cargo into the interior. Exemplary techniques for transfecting 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 the non-viral transfer of a nucleic acid molecule or polynucleotide (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells). Thus, transformation is a genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by direct uptake of the cell membrane from its surroundings and subsequent incorporation of foreign genetic material (nucleic acid molecules). The transformation may be achieved by artificial means. In order for transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques may include heat shock mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation. After introducing the nucleic acid or vector into the immune cells, the resulting immune cells can be expanded and activated by one of ordinary skill in the art by conventional techniques.

In one embodiment, to reduce the risk of graft versus host disease, the engineered immune cell further comprises at least one gene whose expression is suppressed or silenced selected from the group consisting of: CD, GR, dCK, TCR/CD genes (e.g., TRAC, TRBC, CD γ, CD δ, CD ε, CD ζ), MHC associated genes (HLA- - -2, HLA-DPA, HLA-DQ, HLA-DRA, TAP, LMP, RFX, RFXAP, RFXANK, CIITA) and immune checkpoint genes such as PD, LAG, TIM, CTLA, PPP2, PTPN, PDCD, HAVCR, BTLA, CD160, TIGIT, CD, CRTAM, TNFRSF10, CASP, CASDD, FAS, TGFBRII, FRTGBRI, SMAD, SKAD, SKI, SKIL, TGIF, IL10, HMIL 6, IL6, EIF2AK, CSK, BAT, PAG, PRCY, GUCY1, GUDM 1, GUA, GUB 1, GU, and GU. Preferably, the engineered immune cell further comprises at least one gene whose expression is inhibited or silenced selected from the group consisting of: 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.

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, Transdominant Negative Protein (TNP), chimeric/antibody conjugates, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFv), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors can be used to inhibit gene expression. In addition, DNA fragmentation can also be mediated by, for example, meganucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in CRISPR systems to silence the gene.

In one embodiment, the engineered immune cell further comprises a second chimeric antigen receptor that targets other tumor antigens. The additional tumor antigen targeted by the second chimeric antigen receptor may 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, CD 685126, CD138, B54, MUC-1, HM1.24, angiogenic factors, VEGF, PIGF, ED-B fibronectin, CD66 54-d, IL-2, T101, TAC, IL-6, ROR 54, DR 54, TEGFR, Her 54, L54-mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD54, EGP 6852, EGP 54, EGFP 54, HAFCRL 54, ErbR 54, ErbB-D2, EGFR, ErbB-D2-54, EGFR, ErbB-2-D54, EGFR, ErbB-D2, EGFR, ErbB 54, ErbB-D2, and ErbB-D2, and E2, and its derivatives, and their use, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A6, PRAME, survivin, EGP 6, TAG 6, B6-H6, IL-13Ra 6, CA 6, CD171, G250/CAIX, HLA-A6, NY-ESO-1, PSCA, folate receptor-a, CD44v 6/8, avb6 integrin, 8H 6, NCAM, VEGF receptor, 5T 6, fetal AchR, MUC 6, NY-ESO-1, MART-1, gp100, oncofetal embryonic antigen, VEGF-R6, CEA, prostate specific antigen, PSMA, Herma 6/neu, estrogen receptor, progesterone receptor, heparin B6, acetylated CD123, Met-A6, Met-6, GD-6, GG-6, CTD 6, CTG 6, CTD 6, CTG-6, and CTD 6, BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecules, MAGE-A1, MAGEA3, CCNA1 and/or pathogen-specific antigens, biotinylated molecules, molecules expressed by HIV, HCV, HBV, and/or other pathogens.

In one embodiment, a plurality of immune cells are provided, each immune cell 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 and/or targets claudin18.2 (e.g., a CAR comprising an anti-claudin 18.2 antibody described herein), and another cell is engineered to express a chimeric antigen receptor that binds and/or targets another antigen. In one embodiment, the immune cells may also express a multispecific chimeric antigen receptor that targets one or more antigens including claudin 18.2. For example, such a multispecific chimeric antigen receptor may comprise a multispecific antibody targeting claudin18.2, or both an anti-claudin 18.2 antibody as described herein and an antibody targeting another antigen. In such embodiments, the plurality of engineered immune cells may be administered together or separately. In one embodiment, the plurality of immune cells can be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of the present application.

Antibody conjugates

In one aspect, the present invention provides an antibody conjugate comprising an anti-claudin 18.2 antibody as defined herein and a second functional structure, wherein said second functional structure is selected from the group consisting of an Fc, a radioisotope, a half-life extending moiety, a detectable label and a drug.

In one embodiment, the invention provides an antibody conjugate comprising an anti-claudin 18.2 antibody as defined herein 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 and variant Fc. "native Fc" refers to a molecule or sequence comprising a non-antigen-binding fragment, whether monomeric or multimeric, produced by digestion of an intact antibody. The immunoglobulin source that produces native Fc is preferably derived from human. Native Fc fragments are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (e.g., disulfide) and non-covalent linkages. Natural Fc molecule monomer subunits have 1-4 intermolecular disulfide bonds between them, depending on the class (e.g., IgG, IgA, IgE, IgD, IgM) or subtype (e.g., IgG1, IgG2, IgG3, IgA1, IgGA 2). An example of a native Fc is a 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 differs from the amino acid sequence of a "native" or "wild-type" Fc due to at least one "amino acid modification" as defined herein, also referred to as an "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 is capable of folding naturally into a functional dimeric Fc region. In one embodiment, the Fc is preferably that of a human immunoglobulin, more preferably human IgG 1.

In one embodiment, the invention provides an antibody conjugate comprising an anti-claudin 18.2 antibody as defined herein and a radioisotope. Examples of radioisotopes useful in the present invention include, but are not limited to, At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²、^99mTc、¹²³I、¹⁸F and⁶⁸Ga。

in one embodiment, the invention provides an antibody conjugate comprising an anti-claudin 18.2 antibody as defined herein and a half-life extending moiety selected from the group consisting of a binding structure for albumin, a binding structure for transferrin, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin and a white polypeptide (including an antibody) that binds human serum albumin.

In one embodiment, the invention provides an antibody conjugate comprising an anti-claudin 18.2 antibody as defined herein and a detectable label. The term "detectable label" means herein a compound that produces a detectable signal. For example, the detectable label may 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, beta-galactosidase), antibiotic (e.g., kanamycin, ampicillin, chloramphenicol, tetracycline, etc.) resistance genes, and contrast agents (such as nanoparticles or gadolinium). One skilled in the art can select an appropriate detectable label depending on the detection system used.

In one embodiment, the invention provides an antibody conjugate comprising an anti-claudin 18.2 antibody as defined herein and a drug, such as a cytotoxin or an immunomodulator (i.e. an antibody drug conjugate), conjugated to said anti-claudin 18.2 antibody. Typically, the drug is covalently linked to the antibody, and usually 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, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, mechlorethamine, busulfan, dibromomannitol, streptozocin, mitomycin, cis-dichlorodiamine platinum (II) (DDP), cisplatin, carboplatin, zorubicin, doxorubicin, ditobicin, carminomycin, idarubicin, epirubicin, mitoxantrone, actinomycin D, bleomycin, calicheamicin, mithramycin, Atramycin (AMC), vincristine, vinblastine, taxol, ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, cytochalasin, fluxan, flutriafol, flutolysin, flutamide, flutamsultamide, and other drugs, Gramicidin D, ethidium bromide, emidine, etoposide, teniposide, colchicine, dihydroxyanthracenedione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O, P' - (DDD)), interferon, and combinations thereof. Examples of immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate, methotrexate, glucocorticoids and analogs thereof, cytokines, stem cell growth factor, lymphotoxins, 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 factors designated "S1 factor", erythropoietin and thrombopoietin, or combinations thereof.

Kit and pharmaceutical composition

In another aspect, the invention also provides a detection kit comprising a humanized antibody, a multispecific antibody, an antibody conjugate or a chimeric antigen receptor described herein.

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

As used herein, the term "pharmaceutically acceptable excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient (i.e., capable of eliciting a desired therapeutic effect without causing any undesirable local or systemic effects), which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by genomic AR,19th ed. pennsylvania: mach Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adherents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of suitable excipients to prepare the desired pharmaceutical compositions of the present invention is known to those skilled in the art. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the choice of suitable excipients depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

The pharmaceutical composition according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally. Methods of parenteral delivery include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or extracts of fluid extracts, or in a form which is particularly suitable for the desired method of administration. Processes known in the art for the manufacture of medicaments may comprise, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions comprising immune cells such as described herein are typically provided in solution and preferably comprise a pharmaceutically acceptable buffer.

The pharmaceutical compositions according to the invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the diseases to be treated. Preferred examples of the pharmaceutical agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rebeccin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium ii (sorfimer Sodiumtofrin ii), temozolomide, topotecan, glucuronide (trimetrenate glucoside), oritavastin e (auristatin E), vincristine, and adriamycin; peptide cytotoxins such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme, 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, and the like; antibodies or fragments thereof, such as anti-CD 3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral/bacterial protein domains, and viral/bacterial peptides. In addition, the pharmaceutical compositions of the present invention may also be used in combination with one or more other therapeutic methods, such as chemotherapy, radiation therapy.

Therapeutic/prophylactic/diagnostic uses

In another aspect, the invention also provides a method of treating and/or preventing and/or diagnosing a disease associated with claudin18.2 expression comprising administering to a subject a humanized antibody, a chimeric antigen receptor, a multispecific antibody, an antibody conjugate, an engineered immune cell, or a pharmaceutical composition as described above.

In one embodiment, diseases associated with Claudin18.2 expression include, but are not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., 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, thymus cancer, cholangiocarcinoma, gallbladder carcinoma, melanoma, mesothelioma, lymphoma, myeloma (e.g., 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, otorhinolaryngological (ENT) cancer, and metastatic, recurrent or refractory lesions of these cancers.

In a preferred embodiment, the disease associated with claudin18.2 expression is selected from the group consisting of gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer.

The invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.

Drawings

FIG. 1: the expression levels of Claudin18.1 or Claudin18.2 in various constructed cells overexpressing Claudin18.1(A) or Claudin18.2(B) are shown.

FIG. 2: the GFP expression levels in the constructed various cells containing luciferase-GFP are shown.

FIG. 3: the binding of clone BH232 to Claudin18.1(A) and Claudin18.2(B) is shown.

FIG. 4: the levels of scFv expression in BH232-CAR T cells (a) and NT cells (B) are shown.

FIG. 5: the killing effect of BH232-CAR T cells (a) and NT cells (B) on different target and non-target cells at various effective target ratios is shown.

FIG. 6: shows degranulation of BH232-CAR T cells and NT cells after co-culture with various target and non-target cells.

FIG. 7: the levels of IL2(a) and IFN- γ (B) release after co-culture of BH232-CAR T and NT cells with various target and non-target cells are shown.

FIG. 8: the expression level of Claudin18.2scFv in hCR-T cells constructed with humanized antibody is shown.

FIG. 9: shows the killing effect of hCR-T cells on target cells NUGC4-18.2-luci (A) and non-target cells NUGC4-luci (B), 293T-18.1-luci (C) at various effective target ratios.

FIG. 10: shows the degranulation of hCR-T cells after co-culture with target cells NUGC4-18.2-luci and non-target cells NUGC4-luci, 293T-18.1-luci.

FIG. 11: the inhibitory effect of CAR-T cells on mouse tumors is shown.

Detailed Description

Example 1 construction of cell lines overexpressing Claudin18.1 or Claudin18.2

The complete coding sequence for claudin18.1 (GeBank: NM-016369) and claudin18.2 (GeBank: NM-001002026) were synthesized separately and cloned into vector pGEM-T Easy (Promega, cat # A1360) to give pLV-claudin18.1 and pLV-claudin18.2 plasmids.

293T cells and CHO cells were transfected with pLV-claudin18.1 plasmid using a lipofectamine (Roche, cat # 06366546001), and the monoclonal was isolated by limiting dilution 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 were isolated by limiting dilution to obtain 293T-18.2, NUGC4-18.2 and CHO-18.2 monoclonal cell lines. Then, expression of Claudin18.1 or Claudin18.2 in the above monoclonal cell line was detected by flow cytometry using the antibodies CLDN18.2 antibody (Sanyo organism, cat # SY11-362) and CLDN18 antibody (biorbyt Co., cat # orb39924), and the results are shown in FIG. 1.

It can be seen that 293T-18.1 and CHO-18.1 cells can bind to anti-CLDN 18 antibody but hardly to anti-CLD 18.2 antibody, indicating that they specifically express CLDN18.1 (FIG. 1A). CHO-18.2, 293T-18.2 and NUGC4-18.2 cells strongly bound to anti-CLDN 18.2 antibodies (FIG. 1B).

pLVX-acGFP-N1-Fluc plasmid (PPL company, Cat. No. PPL00157-4a) containing nucleic acid encoding luciferase-GFP fusion protein was packaged as lentivirus, and 293T cells, 293T-18.1 cells, 293T-18.2 cells, NUGC4 cells and NUGC4-18.2 cells were infected with the lentivirus, respectively, to obtain 293T-luci, 293T-18.1-luci, 293T-18.2-luci, NUGC4-luci and NUGC4-18.2-luci cell strains. The GFP signal was detected in the cell lines by flow cytometry, and the results are shown in FIG. 2. As can be seen, the prepared 5 cell lines can efficiently express the luciferase-GFP fusion protein and can be used for subsequent experiments.

Example 2 screening of anti-Claudin18.2 antibodies

The pLV-Claudin18.2 plasmid was administered intramuscularly to Balb/c mice of the appropriate age, followed by repeated immunizations every 2-3 weeks for a total of 4 times. Then, mouse spleen lymphocytes were taken, mixed with SP2/0 myeloma cells and cell fusion was mediated by the addition of PEG to prepare hybridoma cells. Hybridoma clones which bound to Claudin18.2 were screened by ELISA or flow cytometry using a Claudin18.2 positive cell line (293T-18.2 cells, NUGC4-18.2 cells), while hybridoma clones which did not bind to Claudin18.1 were screened by ELISA or flow cytometry using a Claudin18.1 positive cell line (293T-18.1 cells). After multiple rounds of screening, 1 antibody clone which can specifically bind to Claudin18.2 but not to Claudin18.1 was obtained and named BH232 clone.

To detect binding of BH232 to Claudin18.1 and Claudin18.2, CHO-18.1 cells or CHO-18.2 cells were stained with supernatant from BH232 clones and detected by flow cytometry, as shown in FIG. 3. It can be seen that the binding to claudin18.1 is very weak (fig. 3A, less than 10%) while the binding to claudin18.2 is as high as 98.5% (fig. 3B), indicating that the BH232 clones screened were able to bind specifically to claudin 18.2.

Total RNA from BH232 clones was extracted and reverse transcribed using cDNA synthesis kit (Novozam, cat # R211-01) to obtain cDNA. PCR was performed using cDNA as a template and degenerate primers (Zhou H et al, Nucleic Acids Research 22: 888-394 (1994); Chardes T et al, FEBS Letters452:386-394 (1999)). The PCR product was recovered and cloned into pMD18-T vector (Takara, cat # 6011) followed by transformation of competent strain JM109 (general organism, cat # CS 03020). The next day, clones were selected for sequencing and the amino acid sequence of the resulting BH232 clone is shown in table 1 below.

TABLE 1 amino acid sequence of BH232 clone

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

Example 3 preparation of CAR-T cells targeting Claudin18.2 and validation of their function

3.1 preparation of CAR-T cells

Sequences encoding the following proteins were synthesized and cloned into a pLVX vector (Public Protein/Plasmid Library (PPL), cat # PPL00157-4 a): CD8 alpha signal peptide (SEQ ID No:45), anti-claudin 18.2 single chain antibody (SEQ ID No:9), CD8 alpha hinge region (SEQ ID No:47), CD8 alpha transmembrane region (SEQ ID No:31), 4-1BB intracellular region (SEQ ID No:37) and CD3 zeta intracellular region (SEQ ID No:39), and correct insertion of the target sequence was confirmed by sequencing.

After diluting the above plasmid by adding 3ml of Opti-MEM (Gibco, cat # 31985-: the packaging vector psPAX2(Addgene, cat # 12260) and the envelope vector pmd2.g (Addgene, cat # 12259) were added at a ratio of 4:2:1 for the viral envelope vector. Then, 120ul of X-treme GENE HP DNA transfection reagent (Roche, cat # 06366236001) was added, mixed immediately, incubated at room temperature for 15min, and the plasmid/vector/transfection reagent mixture was added dropwise to the 293T cell culture flask. The viruses were collected at 24 hours and 48 hours, and after combining them, concentrated lentiviruses were obtained by ultracentrifugation (25000g, 4 ℃, 2.5 hours).

T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, cat. No. 40203D) and cultured at 37 ℃ and 5% CO2 for 1 day. Then, concentrated lentivirus was added and after 3 days of continuous culture, BH232-CAR T cells targeted to claudin18.2 were obtained. Unmodified wild-type T cells (NTs) were used as controls.

After 11 days of culture at 37 ℃ and 5% CO2, the expression level of Anti-Claudin 18.2 single-chain antibody on CAR-T cells was measured by flow cytometry using Biotin-SP (long spacer) Affinipure Goat Anti-Mouse IgG, F (ab')2Fragment specificity (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat # 115-065-072) as a primary antibody and APC Streptavidin (BD Pharmingen, cat # 554067) as a secondary antibody, as shown in FIG. 4.

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

3.2 testing the killing Effect of CAR-T cells on target cells

At 1x10⁴Concentration of individual cells/well 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 plated in 96-well plates, then NT cells and BH232-CAR T cells were plated in 96-well plates at an effective target ratio (i.e., ratio of effector T cells to target cells) of 16:1, 8:1, 4:1, 2:1, 1:1 for co-culture, and fluorescence was measured 16-18 hours later using a microplate reader. According to the calculation formula: (mean value of fluorescence of target cells-mean value of fluorescence of sample)/mean value of fluorescence of target cells x 100%, and the killing efficiency was calculated, and the results are shown in FIG. 5.

It can be seen that at various effect-to-target ratios, the BH232-CAR T cells of the present invention showed strong killing effect on the two target cells 293T-18.2-luci cells and NUGC4-18.2-luci cells, while the non-target cells 293T-18.1-luci cells, NUGC4-luci cells and 293T-luci cells were less killed, indicating that BH232-CAR T cells exhibited specific killing only on cells expressing claudin 18.2.

3.3 detection of degranulation of CAR-T cells

At 1 × 10⁵Concentration of individual cells/well 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)Cell, CHO-18.1-luci cell) were plated in 96-well plates, BH232-CAR T cells and NT cells (negative control) were added at a ratio of 1:1, 10. mu.L of PE Mouse anti-human CD107a antibody (BD, cat # 555801) was added to each well, and incubated at 37 ℃ under 5% CO2 protected from light. After 1h, 20. mu.L of Golgi Stop (BD, cat # 51-2092K2) was added to each well and incubated at 37 ℃ under 5% CO2 protected from light for 2.5 h. Then 10. mu.L of APC anti-human CD8(BD, cat # 555369) was added to each well and incubated at 37 ℃ under 5% CO2 for 0.5h in the absence of light. Cell samples from each well were examined by flow cytometry and analyzed for the proportion of T cells that were double positive for CD107a and CD8, the results of which are shown in fig. 6.

It can be seen that the BH232-CAR T cells prepared according to the invention showed significantly increased degranulation of specificity compared to NT cells for all three target cells 293T-18.2-luci, NUGC4-18.2-luci and CHO-18.2-luci, whereas no significant increase in degranulation was observed for non-target cells.

3.4 detection of cytokine Release levels from CAR-T cells

At 1x10⁵Concentration of individual cells/well 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 plated in 96-well plates, BH232-CAR T cells and NT cells (negative control) were added in a ratio of 1:1, respectively, and cell co-culture supernatants were collected after 18-24 hours of co-culture.

The contents of IL2 and IFN-. gamma.in the co-culture supernatants were measured using Human IL-2DuoSet ELISA Kit (R & D systems, cat # DY202), Human IFN-gamma DuoSet ELISA Kit (R & D systems, cat # DY285), respectively, according to the manufacturer's recommendations, and the results are shown in FIG. 7.

It can be seen that the level of release of cytokines IL2(a) and IFN- γ (B) was significantly increased after co-culture of the BH232-CAR T cells of the present invention with three target cells compared to NT cells, and that this cytokine release was specific.

Example 4 preparation of humanized anti-Claudin18.2 antibody

Murine anti-claudin 18.2 antibodies were humanized using CDR grafting techniques. Specifically, the CDR regions of the BH232 clone were grafted to the framework regions of the heavy chain subtype III and the light chain kappa subtype I of human antibodies (Carter P, PNAS 89:4285-4289 (1992); Presta LG et al, Cancer Research 57:4593-4599(1997)) and the amino acid residues in the framework regions were point-mutated to enhance the affinity of the antibodies, to finally obtain 5 humanized anti-Claudin 18.2 antibodies whose sequences are shown in Table 2 below.

TABLE 2 amino acid sequence of humanized anti-Claudin18.2 antibody

Cloning	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

Example 5 preparation of hCRAR-T cells comprising humanized anti-Claudin18.2 antibody and validation of their function

Sequences encoding the following proteins were synthesized and cloned into a pLVX vector (Public Protein/Plasmid Library (PPL), cat # PPL00157-4 a): B2M signal peptide (SEQ ID No:43), humanized anti-claudin 18.2 single chain antibody (selected from any one of 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 CD3 zeta intracellular region (SEQ ID No:41), and correct insertion of the target sequence was confirmed by sequencing.

The above plasmids were packaged as lentiviruses according to the method described in 3.1 of example 3 and infected with activated T cells to obtain hCRAR-T cells containing humanized anti-Claudin 18.2 antibody. The expression level of Anti-Claudin 18.2 single-chain antibody on hCR-T cells was measured by flow cytometry using Biotin-SP (long spacer) affinity Goat Anti-Mouse IgG, F (ab')2Fragment specificity (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat # 115-065-072) as a primary antibody and PE Streptavidin (Biolegend, cat # 405204) as a secondary antibody, and the results are shown in FIG. 8.

It can be seen that hCR-T cells prepared with the humanized anti-claudin 18.2 single chain antibody all efficiently expressed the Claudin18.2 single chain antibody.

The killing effect of CAR-T cells on target cells nucc 4-18.2-luci and non-target cells nucc 4-luci, 293T-18.1-luci was tested according to the method described in 3.2 of example 3 and the degranulation of CAR-T cells was tested according to the method described in 3.3 of example 3, with the results shown in fig. 9 and 10, respectively. It can be seen that the BH232_ V1, BH232_ V2, BH232_ V3, BH232_ V4 and BH232_ V5 hCR T cells containing the humanized anti-Claudin 18.2 single-chain antibody can generate obvious specific killing and degranulation effects on target cells NUGC4-18.2-luci, and the killing effects are equivalent to those of BH232-CAR T cells constructed by a murine anti-claudin 18.2 single-chain antibody, while the killing effects on non-target cells NUGC4-luci and 293T-18.1-luci are not obvious.

Example 6 demonstration of the tumor-inhibiting Effect of CAR-T cells

24 healthy female NCG mice, about 7 weeks old, were divided into 4 groups: NT group (negative control), BH232 group, BH232_ V1 group, and BH232_ V3 group. On day 0 (D0), each mouse was injected subcutaneously with 5X 10⁶NUGC4-18.2 cells. After 10 days (D10), 2x10 was injected intravenously into each mouse tail according to the grouping⁶Individual NT cells or corresponding CAR-T cells. Mice were evaluated 2 times per week for changes in tumor burden and the results are shown in figure 11.

It can be seen that tumor cells in NT group mice not treated with CAR-T cells continued to grow, whereas CAR-T cells constructed with the anti-claudin 18.2 antibodies BH232, BH232_ V1 and BH232_ V3 of the present invention significantly inhibited tumor cell growth from D25 and maintained tumor size at a very low level until the end of the experiment. This indicates that the CAR-T cells of the invention show a strong killing effect against tumor cells in vivo experiments.

It should be noted that the above-mentioned embodiments are merely preferred examples of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Sequence listing

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<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

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300

ccgctcacgt tcggtgctgg gaccaagctg gagctgaaag gcagcaccag cggcagcggc 360

aaaccgggca gcggcgaagg cagcaccaaa ggcgaggtcc agctgcaaca gtctggacct 420

gagctggtga agcctggagc ttcaatgaag atatcctgca aggcttctgg ttactcattc 480

actggctaca ccatgaactg ggtgaagcag agccatggaa agaaccttga gtggattgga 540

cttattaatc cttacaatgg tggtactagc tacaaccaga agttcaaggg caaggccaca 600

ttaactgtag acaagtcatc cagcacagcc tacatggagc tcctcagtct gacatctgag 660

gactctgcag tctattactg tgcaagaatg aactatggta atgctatgga ctactggggt 720

caaggaacct cagtcaccgt ctcctca 747

<210> 26

<211> 747

<212> DNA

<213> Artificial Sequence(Artificial Sequence)

<220>

<223> BH232_V1

<400> 26

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

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300

ccgctcacgt tcggtgctgg gaccaagctg gagctgaaag gcagcaccag cggcagcggc 360

aaaccgggca gcggcgaagg cagcaccaaa ggcgaggtcc agctgcaaca gtctggacct 420

gagctggtga agcctggagc ttcaatgaag atatcctgca aggcttctgg ttactcattc 480

actggctaca ccatgaactg ggtgaagcag agccatggaa agaaccttga gtggattgga 540

cttattaatc cttacaatgg tggtactagc tacaaccaga agttcaaggg caaggccaca 600

ttaactgtag acaagtcatc cagcacagcc tacatggagc tcctcagtct gacatctgag 660

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 gcgttactgt ttcaagc 747

<210> 31

<211> 25

<212> PRT

<213> Artificial Sequence(Artificial Sequence)

<220>

<223> CD8 alpha 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> CD8 alpha 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> transmembrane domain of CD28

<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> transmembrane domain of CD28

<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 Co-stimulatory 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 Co-stimulatory 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> CD3 zeta 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> CD3 zeta 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> CD3 zeta 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> CD3 zeta 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> CD8 alpha 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> CD8 alpha 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> CD8 alpha 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> CD8 alpha 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 comprising an amino acid sequence as set forth in SEQ ID NO: 1, CDR-L1 as set forth in SEQ ID NO: 2, as shown in SEQ ID NO:3, as shown in SEQ ID NO:4, CDR-H1 as shown in SEQ ID NO: 5 and a CDR-H2 as set forth in SEQ ID NO: 6, CDR-H3.

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

3. The antibody of claim 1, wherein the amino acid sequence of the antibody is identical to a sequence selected from the group consisting of SEQ ID NO: 9. 12, 15, 18, 21 and 24, or an amino acid sequence selected from SEQ ID NOs: 9. 12, 15, 18, 21 and 24 have one or several amino acid conservative modifications compared to the amino acid sequence.

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

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

6. The nucleic acid molecule of claim 5 which hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 25-30 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity and encodes an antibody capable of specifically binding to claudin 18.2.

7. A multispecific antibody comprising the antibody of 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 method of claim 7A multispecific antibody, wherein the second antibody or antigen-binding portion thereof is selected from the group consisting of a full-length antibody, Fab ', (Fab')₂Fv, scFv-scFv, minibody, diabody or sdAb.

9. A vector comprising a nucleic acid molecule encoding the antibody of any one of claims 1-4 or the multispecific antibody of claim 7 or 8.

10. A host cell expressing the antibody of any one of claims 1-4 or the multispecific antibody of claim 7 or 8.

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

12. The chimeric antigen receptor of claim 11, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154.

13. The chimeric antigen receptor of claim 11, wherein the intracellular signaling domain is selected from the intracellular domains of: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66 d.

14. The chimeric antigen receptor of claim 11, further comprising one or more costimulatory domains selected from the group consisting of the costimulatory 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, nkslp g2C, SLP76, PD-1, LIGHT, TRIM, and ZAP 70.

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

16. The engineered immune cell of claim 15, selected from a T cell, NK cell, NKT cell, macrophage, dendritic cell.

17. The engineered immune cell of claim 15, further comprising a second chimeric antigen receptor that targets other tumor antigens.

18. The engineered immune cell of any one of claims 15-17, further comprising suppression or silencing of expression of at least one gene selected from the group consisting of: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3 and CTLA 4.

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

20. The antibody conjugate of claim 19, wherein the half-life extending moiety is selected from the group consisting of: a binding structure of albumin, a binding structure of transferrin, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and a white polypeptide binding human serum albumin; the detectable marker is selected from the group consisting of a fluorophore, a chemiluminescent compound, a bioluminescent compound, an enzyme, an antibiotic resistance gene, and a contrast agent; the drug is selected from cytotoxins and immunomodulators.

21. A test kit comprising the antibody of any one of claims 1-4, the multispecific antibody of claim 7 or 8, the chimeric antigen receptor of any one of claims 11-14, or the antibody conjugate of claim 19 or 20.

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

23. Use of the antibody of any one of claims 1-4, the multispecific antibody of claim 7 or 8, the chimeric antigen receptor of any one of claims 11-14, the engineered immune cell of any one of claims 15-18, or the antibody conjugate of claim 19 or 20 or the pharmaceutical composition of claim 22 in the manufacture of a medicament for the treatment and/or prevention and/or diagnosis of a disease associated with claudin18.2 expression.