CN113754780A

CN113754780A - Chimeric antigen receptor targeting CLDN18.2, compositions and uses thereof

Info

Publication number: CN113754780A
Application number: CN202110576654.9A
Authority: CN
Inventors: 常建辉; 朱雁林; 王江漫; 肖亮; 薛彤彤; 王晶翼
Original assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Current assignee: Chengdu Kelun Precision Biotechnology Co.,Ltd.
Priority date: 2020-06-04
Filing date: 2021-05-26
Publication date: 2021-12-07

Abstract

The present invention relates to the field of biomedicine, in particular, the invention relates to single chain antibodies specifically binding to CLDN18.2 and Chimeric Antigen Receptors (CARs) comprising the same. The invention also relates to engineered immune cells expressing the CAR, or co-expressing the CAR and an additional biologically active molecule (e.g., PD-1 antibody or mIL-15), nucleic acid molecules encoding such CARs or co-expression molecules, and methods of making the engineered immune cells. The invention also relates to the use of the CAR and the immune cell for preventing and/or treating cancers such as gastric cancer, gastric adenocarcinoma and pancreatic cancer, and a method for preventing and/or treating cancers such as gastric cancer, gastric adenocarcinoma and pancreatic cancer.

Description

Chimeric antigen receptor targeting CLDN18.2, compositions and uses thereof

Technical Field

Background

Chimeric Antigen Receptor (CAR) -T cells are an emerging therapeutic approach with significant therapeutic efficacy in tumor therapy, and have become a milestone achievement in the history of tumor therapy. In 2017, the FDA approved two CAR-T cell therapies against CD-19, both of which had significant efficacy on B-lymphomas. It uses the method of in vitro culture of immune cell collected from patient body, transduction of specific exogenous gene, in vitro amplification and back-infusion into patient body to excite and enhance the autoimmune function of body so as to attain the goal of curing tumor. However, 90% of cancers are solid tumors, and a greater variety of solid tumors and a greater number of tumor surface-specific target antigens require further confirmation. One of the difficulties in the CAR-T treatment of solid tumors is tumor specific antigen, otherwise a series of problems such as 'off-target' easily occur, so that the search for a specific tumor antigen is very important.

The Claudin protein family was discovered in 1998 by Shorichiro Tsukita et al to be a component of cell tight junctions, they established a cellular barrier that controls the flow of molecules between cells, proteins that mediate cell-to-cell tight junctions, different subtypes of Claudin proteins are expressed in different tissues and are associated with different types of cancers, Claudin1 is highly expressed in colon cancer, and Claudin7 is associated with recurrence of liver cancer. The Claudin protein is a surface protein, suggesting that it may be a useful target for various therapeutic strategies. The expression of Claudin18.2 is highly specific, the expression in normal tissues is limited to differentiated epithelial gastric mucosal cells, but is not present in a gastric stem cell region, is not expressed in other normal tissues, but is highly expressed in several cancer types, such as tumors of stomach, esophagus, pancreas and lung and human cancer cell lines, such as the expression of Claudin18.2 in primary gastric adenocarcinoma and metastatic tumors thereof, which is up to 70 percent, and the biological characteristics show that Claudin18.2 can be used as an ideal tumor treatment target.

Gastric cancer and pancreatic cancer are both recognized cancers with poor prognosis and high mortality. In the treatment mode of the gastric cancer, chemotherapy is a main treatment means of the advanced gastric cancer, and the median survival time of patients with the advanced gastric cancer is only about 11 months by adopting the cytotoxic drug combination scheme for chemotherapy. At present, no accepted standard chemotherapy scheme for advanced gastric cancer exists internationally. In the aspect of biological macromolecular medicaments, except that positive results are obtained in phase III clinical experiments of Trastuzumab (Trastuzumab) and Ramucirumab (Ramucirumab), most of other gastric cancer targeted treatment medicaments have unsatisfactory results or are still in the early stage of clinical research. The core deficiency and unmet clinical needs of the existing drugs make treatment options for advanced or recurrent gastric cancer very limited, with very poor prognosis and high mortality. Pancreatic cancer is the fourth highest in global tumor mortality, with a 5-year survival rate of less than 5%. More than 350,000 new cases of pancreatic cancer worldwide each year; more than 340,000 deaths were reported. Domestic and foreign studies have shown that about 60% of pancreatic cancer subjects have distant metastasis at the time of definitive diagnosis, and 25% of subjects are locally advanced and cannot undergo radical resection, with median survival of only 6-9 months. Therefore, the current medicine for gastric cancer and pancreatic cancer is in great demand.

Based on the expression specificity of claudin18.2, CAR-T cell therapy against claudin18.2 holds the promise of being one of the ways to overcome the above-mentioned refractory cancers. Therefore, the development of CAR-T therapies targeting Claudin18.2 with high specificity and good clinical efficacy is urgent and essential.

Disclosure of Invention

In the present application, the inventors first developed an antibody with excellent properties capable of specifically recognizing/binding to claudin18.2 humanization, and on the basis of this, they paid a lot of creative efforts to further construct a Chimeric Antigen Receptor (CAR) capable of specifically binding to claudin 18.2. The CARs of the invention are able to direct immune effector cell specificity and reactivity in a non-MHC restricted manner towards cells expressing claudin18.2 so that they are cleared and have superior functional properties compared to known CARs targeting claudin 18.2. Therefore, the CAR has the potential of being used for preventing and/or treating Claudin18.2-related tumors such as gastric cancer, gastric adenocarcinoma and pancreatic cancer, and has great clinical value.

Single chain antibodies and conjugates

The present invention relates to a single chain antibody having the ability of specifically binding to CLDN18.2, comprising a heavy chain variable region (VH) and a light chain variable region (VL) of any one of the following groups, said VH and VL being linked by a linker:

(1) a VH having a sequence shown as SEQ ID NO. 13 or a variant thereof and/or a VL having a sequence shown as SEQ ID NO. 14 or a variant thereof; or

(2) A VH having a sequence shown as SEQ ID NO. 16 or a variant thereof and/or a VL having a sequence shown as SEQ ID NO. 17 or a variant thereof;

wherein the variant of (1) - (2) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5, 6, 7,8, 9, or 10 amino acids) to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

In certain embodiments, the linker comprises a linker such as (G)_mS)_nWherein m is an integer from 1 to 6, preferably m is 3, 4, or 5, and n is an integer from 1 to 10, preferably n is 2, 3, 4, 5, or 6. In certain embodiments, the linker comprises the sequence shown as SEQ ID NO: 44.

In certain embodiments, the single chain antibody is an scFv, di-scFv or (scFv)₂。

In certain embodiments, the single chain antibody comprises an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in any one of SEQ ID NOs:19, 23; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:19, 23.

In certain embodiments, the single chain antibody comprises an amino acid sequence selected from the group consisting of: (1) 65, 66 or an amino acid sequence shown in any one of SEQ ID NOs; (2) 65, 66, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs.

Preparation of Single chain antibodies

The single-chain antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombination techniques. For example, a DNA molecule encoding a single-chain antibody of the present invention is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions and the single-chain antibody of the present invention is expressed. The DNA molecule may be a nucleotide sequence optimized for amino acid sequence. The selection and preparation of vectors and host cells can be accomplished according to methods known in the art.

Conjugates

The single chain antibodies of the invention may be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to CLDN18.2 (particularly human CLDN 18.2). Thus, the single chain antibodies of the invention are also intended to include such derivatized forms. For example, a single chain antibody of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular moieties, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and/or a protein or polypeptide (e.g., avidin or a polyhistidine tag) capable of mediating binding of the single chain antibody to another molecule.

Accordingly, the present invention provides a conjugate comprising a single chain antibody of the invention and a modifying moiety linked to the single chain antibody.

In certain embodiments, the modifying moiety is a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. In certain embodiments, the conjugates comprise a single chain antibody of the invention and a variable domain linked to the single chain antibodyThe label of the detection. The detectable label of the present invention may be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g.,³H、¹²⁵I、³⁵S、¹⁴c or³²P), fluorescent dyes (e.g., Fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), Phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridine ester compounds), magnetic beads (e.g.,

) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above. Patents that teach the use of such markers include, but are not limited to, U.S. Pat. nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). Detectable labels as described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation calculators, and fluorescent labels can be detected using photodetectors to detect the emitted light. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label. In certain embodiments, such labels can be suitable for use in immunological assays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, and the like). In certain embodiments, detectable labels as described above can be attached to the single chain antibodies of the invention via linkers of different lengths to reduce potential steric hindrance.

Detection method and kit

The aforementioned single chain antibody or conjugate thereof that specifically binds to CLDN18.2 may be used to detect the presence or level of CLDN18.2 in a sample. The aforementioned single chain antibody or conjugate thereof specifically binding to CLDN18.2 may be used to prepare a kit for detecting the presence or level of CLDN18.2 in a sample. The preparation method and detection method of the kit can refer to the method described in PCT/CN2019/126495 application by the inventor of the application.

Chimeric Antigen Receptor (CAR)

The present invention relates to CLDN 18.2-targeted CARs (anti-CLDN 18.2-CARs) characterized by comprising a non-MHC-restricted CLDN18.2 recognition ability that confers to immune cells (e.g., T cells, NK cells, monocytes, macrophages or dendritic cells) expressing the CAR the ability to recognize cells expressing CLDN18.2 independent of antigen processing and presentation.

Accordingly, in a first aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising an extracellular ligand (or antigen) binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain.

I. Extracellular antigen binding domains

The extracellular antigen-binding domain comprised in the chimeric antigen receptor of the present invention comprises an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2 (e.g., human CLDN 18.2).

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

(1a) the three heavy chain CDRs as follows: CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 13; and/or, the three light chain CDRs as follows: CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 14;

or the like, or, alternatively,

(1b) the three heavy chain CDRs as follows: (1a) the CDR-H1 or a variant thereof containing an amino acid mutation as described in (1a), the CDR-H2 or a variant thereof containing an amino acid mutation as described in (1a), the CDR-H3 or a variant thereof containing an amino acid mutation as described in (1 a); and/or, the three light chain CDRs as follows: (1a) the CDR-L1 or a variant thereof containing an amino acid mutation as described in (1a), the CDR-L2 or a variant thereof containing an amino acid mutation as described in (1a), the CDR-L3 or a variant thereof containing an amino acid mutation as described in (1 a); wherein at least one of the three heavy chain CDRs and/or the three light chain CDRs of (1b) comprises an amino acid mutation compared to the corresponding CDR of (1a), said amino acid mutation being a substitution, deletion or addition of one or several amino acids (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitutions are conservative substitutions.

In certain embodiments, the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering system.

In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system:

the VH comprises: has the sequence shown in SEQ ID NO:1 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; has the sequence shown in SEQ ID NO: 2 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and a polypeptide having the sequence of SEQ ID NO:3 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto;

the VL includes: has the sequence shown in SEQ ID NO:4 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; has the sequence shown in SEQ ID NO:5 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; and a polypeptide having the sequence of SEQ ID NO:6 or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

In certain embodiments, at least one CDR in the heavy chain variable region (VH) and/or the light chain variable region (VL) comprises an amino acid mutation, which is a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof).

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:

the VH comprises: has the sequence shown in SEQ ID NO: 15 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; has the sequence shown in SEQ ID NO: 20 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; and a polypeptide having the sequence of SEQ ID NO: 21 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto;

the VL includes: has the sequence shown in SEQ ID NO: 26 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; has the sequence shown in SEQ ID NO: 27 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; and a polypeptide having the sequence of SEQ ID NO: 30 or a sequence having one or more amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

In certain embodiments, at least one CDR in the heavy chain variable region (VH) and/or light chain variable region (VL) comprises an amino acid mutation as described above, said amino acid mutation being a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof).

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof further comprises Framework Regions (FRs) from a human immunoglobulin.

In certain embodiments, the extracellular antigen-binding domain comprises: a VH having a sequence shown as SEQ ID NO. 13 or a variant thereof and/or a VL having a sequence shown as SEQ ID NO. 14 or a variant thereof; wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids) to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

(2a) the three heavy chain CDRs as follows: CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 16; and/or, the three light chain CDRs as follows: CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 17;

or the like, or, alternatively,

(2b) the three heavy chain CDRs as follows: (2a) the CDR-H1 or a variant thereof containing an amino acid mutation as described in (1), (2a) the CDR-H2 or a variant thereof containing an amino acid mutation as described in (2a), (2a) the CDR-H3 or a variant thereof containing an amino acid mutation as described in (1); and/or, the three light chain CDRs as follows: (2a) the CDR-L1 or a variant thereof containing an amino acid mutation as described in (1), (2a) the CDR-L2 or a variant thereof containing an amino acid mutation as described in (2a), (2a) the CDR-L3 or a variant thereof containing an amino acid mutation as described in (1); wherein at least one of the three heavy chain CDRs and/or the three light chain CDRs of (2b) comprises an amino acid mutation compared to the corresponding CDR of (2a), said amino acid mutation being a substitution, deletion or addition of one or several amino acids (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitutions are conservative substitutions.

the VH comprises: has the sequence shown in SEQ ID NO:7 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; has the sequence shown in SEQ ID NO: 8 or 53 or a sequence having substitution, deletion or addition of one or several amino acids (e.g. substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; and a polypeptide having the sequence of SEQ ID NO: 9 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto;

the VL includes: has the sequence shown in SEQ ID NO: 10 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; has the sequence shown in SEQ ID NO: 11 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; and a polypeptide having the sequence of SEQ ID NO: 12 or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

the VH comprises: has the sequence shown in SEQ ID NO: 31 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; has the sequence shown in SEQ ID NO: 32 or 37 or a sequence having substitution, deletion or addition of one or several amino acids (e.g. substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; and a polypeptide having the sequence of SEQ ID NO: 33 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto;

the VL includes: has the sequence shown in SEQ ID NO: 34 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; has the sequence shown in SEQ ID NO: 35 or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; and a polypeptide having the sequence of SEQ ID NO: 36 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the extracellular antigen-binding domain comprises: a VH having a sequence shown as SEQ ID NO. 16 or a variant thereof and/or a VL having a sequence shown as SEQ ID NO. 17 or a variant thereof; wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids) to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof includes, but is not limited to, camel Ig, IgNAR, Fab fragments, Fab 'fragments, f (ab)'₂Fragment, F (ab)'₃Fragments, Fv, single-chain antibodies (e.g. scFv, di-scFv, (scFv)₂) Minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv) and single domain antibody (sdAb, nanobody).

In certain embodiments, the antibody or antigen-binding fragment thereof is a single chain antibody. In certain embodiments, the single chain antibody is an scFv, di-scFv or (scFv)₂。

In certain embodiments, the VH and VL comprised by the antibody or antigen-binding fragment thereof are linked by a linker. In certain embodiments, the linker comprises a linker such as (G)_mS)_nThe sequence shown, wherein m is selected from an integer of 1 to 6. In certain preferred embodiments, m is 3, 4, or 5; n is an integer from 1 to 10. In certain preferred embodiments, n is 2, 3, 4, 5, or 6. In certain more preferred embodiments, the linker has the sequence shown as SEQ ID NO 44.

In certain embodiments, the extracellular antigen-binding domain is a single chain antibody.

In certain embodiments, the extracellular antigen-binding domain comprises:

wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids) to the sequence from which it is derived; in certain preferred embodiments, the substitutions are conservative substitutions.

In certain embodiments, the extracellular antigen-binding domain comprises a single chain antibody comprising an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in any one of SEQ ID NOs:19, 23, 65, 66; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:19, 23, 65, 66; or (3) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions) as compared to the amino acid sequence set forth in any of SEQ ID NOs:19, 23, 65, 66; preferably, the substitutions are conservative substitutions.

In certain embodiments, the extracellular antigen-binding domain has the characteristic of specifically binding to more than one antigen or receptor, such as to bind to a molecule, such as CD19, CD20, CD22, CD33, CD123, or CD138, in addition to binding to CLDN 18.2. In certain embodiments, the extracellular antigen-binding domain is the bispecific antibody or multispecific antibody. In certain embodiments, the bispecific or multispecific antibody is capable of specifically binding to CLDN18.2 and has specificity for a target selected from the group consisting of: CD19, CD20, CD22, CD33, CD123, or CD 138.

Transmembrane domain

The transmembrane domain comprised by the chimeric antigen receptor of the invention may be any protein structure known in the art, as long as it is thermodynamically stable in a cell membrane, in particular a eukaryotic cell membrane. The transmembrane domain of a CAR suitable for use in the present invention may be derived from a natural source. In such embodiments, the transmembrane domain may be derived from any membrane-bound or transmembrane protein. Alternatively, the transmembrane domain may be a synthetic non-naturally occurring protein segment, for example a protein segment which comprises predominantly hydrophobic residues such as leucine and valine.

In certain embodiments, the transmembrane domain is a transmembrane region of a protein selected from the group consisting of: an α, β, or zeta chain of a T cell receptor, CD3 epsilon, CD3zeta, CD4, CD5, CD8 alpha, CD137, CD152, CD154, and PD-1, and any combination thereof. In certain preferred embodiments, the transmembrane domain is a transmembrane region of a protein selected from the group consisting of: CD8 α, CD4, PD-1, CD152 and CD 154. In certain preferred embodiments, the transmembrane domain comprises the transmembrane region of CD8 a. In certain exemplary embodiments, the transmembrane domain comprises the amino acid sequence shown as SEQ ID NO 46.

Spacer Domain

The chimeric antigen receptors of the present invention comprise a spacer domain located between the extracellular antigen-binding domain and the transmembrane domain.

In certain embodiments, the spacer domain comprises CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG 4). In such embodiments, without being bound by a particular theory, it is believed that CH2 and CH3 extend the antigen binding domain of the CAR away from the cell membrane of the CAR-expressing cell, and can more accurately mimic the size and domain structure of a native TCR.

In certain embodiments, the spacer domain comprises a hinge domain. The hinge domain may be a segment of amino acids typically found between two domains of a protein, which may allow for flexibility of the protein and movement of one or both domains relative to each other. Thus, the hinge domain may be any amino acid sequence that is capable of providing such flexibility of the extracellular antigen-binding domain and such mobility thereof relative to the transmembrane domain.

In certain embodiments, the hinge domain is a hinge region or portion thereof of a naturally occurring protein. In certain embodiments, the hinge domain comprises a hinge region of CD8 a or a portion thereof, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge region of CD8 a. In certain exemplary embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO 45.

In certain embodiments, the spacer domain comprises a hinge domain comprising a hinge region of PD-1, CD152, or CD 154. In certain embodiments, the spacer domain comprises at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge region of PD-1, CD152, or CD 154.

Signal peptide

In certain embodiments, the CAR of the invention may further comprise a signal peptide at its N-terminus. Typically, a signal peptide is a polypeptide sequence that targets a sequence to which it is linked to a desired site. In certain embodiments, the signal peptide can target the CAR attached thereto to the secretory pathway of the cell and allow the CAR to further integrate and anchor into the lipid bilayer. Signal peptides useful for CARs are known to those skilled in the art. In certain embodiments, the signal peptide comprises a heavy chain signal peptide (e.g., the heavy chain signal peptide of IgG 1), a granulocyte-macrophage colony stimulating factor receptor 2(GM-CSFR2) signal peptide, or a CD8 a signal peptide. In certain preferred embodiments, the signal peptide is selected from the group consisting of CD8 a signal peptide. In certain exemplary embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO 42.

In certain embodiments, the CAR of the invention is co-expressed with an additional biologically active molecule. The additional biologically active molecule may have its own signal peptide, which is distinguished from the signal peptide of the previous paragraph, and is designated signal peptide-2. Signal peptide-2 directs the transport of additional bioactive molecules to specific sites within the cell or outside the cell membrane. The signal peptide-2 may be the same as or different from the signal peptide described in the preceding paragraph. Preferably, the signal peptide-2 may be different from the signal peptide described in the preceding paragraph.

V. intracellular signaling domain

The intracellular signaling domain comprised in the CAR of the invention is involved in signaling the interior of an immune effector cell an effective antigen receptor binding (binding of the CAR of the invention to CLDN18.2), activating at least one normal effector function of the immune effector cell expressing the CAR, or enhancing secretion of at least one cytokine (e.g., IL-2, IFN- γ) by the immune effector cell expressing the CAR.

In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain.

In certain embodiments, the primary signaling domain may be any intracellular signaling domain comprising an Immunoreceptor Tyrosine Activation Motif (ITAM). In certain embodiments, the primary signaling domain comprises an Immunoreceptor Tyrosine Activation Motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of: CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CDs, CD22, CD79a, CD79b, or CD66 d. In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of CD3 ζ.

In certain embodiments, the co-stimulatory signaling domain may be an intracellular signaling domain from a co-stimulatory molecule. In certain embodiments, the co-stimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD270(HVEM), CD278(ICOS), or DAP 10.

In certain embodiments, the co-stimulatory signaling domain is selected from the intracellular signaling domain of CD28, or the intracellular signaling domain of CD137(4-1BB), or a combination of fragments of both.

In certain embodiments, the intracellular signaling domain comprises one co-stimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains. In such embodiments, the two or more co-stimulatory signaling domains may be the same or different.

In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain. The primary signaling domain and the at least one costimulatory signaling domain may be linked in series, in any order, to the carboxy-terminus of the transmembrane domain.

In certain embodiments, the intracellular signaling domain may comprise an intracellular signaling domain of CD3 ζ and an intracellular signaling domain of CD 137. In certain exemplary embodiments, the intracellular signaling domain of CD3 ζ comprises the amino acid sequence set forth in SEQ ID No. 48. In certain exemplary embodiments, the intracellular signaling domain of CD137 comprises the amino acid sequence set forth in SEQ ID NO 47.

In certain exemplary embodiments, the intracellular signaling domain of the chimeric antigen receptor has the amino acid sequence of SEQ ID NO: 49.

Full Length CAR

The present invention provides a chimeric antigen receptor capable of specifically binding to CLDN18.2, comprising, in order from its N-terminus to its C-terminus, an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain. In certain preferred embodiments, wherein the intracellular signaling domain is, from N-terminus to C-terminus, a costimulatory signaling domain and a primary signaling domain.

In certain embodiments, the spacer domain comprises a hinge region of CD8 (e.g., CD8 a) having the amino acid sequence of SEQ ID NO:45, and (b) is shown in the specification. In certain embodiments, the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8 a) having the amino acid sequence of SEQ ID NO:46, or a sequence shown in the figure.

In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3 ζ having the amino acid sequence of SEQ ID NO:48, or a variant thereof. The costimulatory signaling domain comprises the intracellular signaling domain of CD137 having the amino acid sequence of SEQ ID NO: 47. In certain preferred embodiments, the intracellular signaling domain of the chimeric antigen receptor has the amino acid sequence of SEQ ID NO: 49.

In certain preferred embodiments, the chimeric antigen receptor comprises, in order from its N-terminus to its C-terminus, the signal peptide, an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain (a costimulatory signaling domain and a primary signaling domain from the N-terminus to the C-terminus).

In certain exemplary embodiments, the signal peptide comprises the heavy chain signal peptide of IgG1 or the CD8 a signal peptide. In certain exemplary embodiments, the signal peptide comprises a CD8 a signal peptide having the amino acid sequence of SEQ ID NO:42, or a sequence shown in figure 42.

In certain exemplary embodiments, the CAR has an amino acid sequence selected from the group consisting of: (1) an amino acid sequence set forth in any one of SEQ ID NOs:25, 29, (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to an amino acid sequence set forth in any one of SEQ ID NOs:25, 29 and which sequence substantially retains at least one biological activity of the amino acid sequence from which it is derived (e.g., the ability to direct the specificity and reactivity of immune effector cells to cells expressing CLDN18.2 in a non-MHC-restricted manner); or (3) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions) as compared to the amino acid sequence set forth in any of SEQ ID NOs:25, 29, and in certain preferred embodiments, the substitutions are conservative substitutions.

VII-Co-expressed CAR and additional bioactive molecule

The invention provides a co-expressed CAR and a further biologically active molecule comprising a chimeric antigen receptor of the first aspect capable of specifically binding to CLDN18.2 and a further biologically active molecule. Self-cleaving peptides prevent the formation of covalent bonds from amino acids during translation and maintain translation, such that the translation product is "self-cleaved" to separate chimeric antigen receptors that specifically bind to CLDN18.2 from other biologically active molecules. A chimeric antigen receptor capable of specifically binding to CLDN18.2 becomes an independent CAR having an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain. The additional biologically active molecule is either secreted extracellularly or expresses a chimeric polypeptide or protein in the form of a membrane. With the expansion and enrichment of immune cells expressing a CLDN18.2-CAR that specifically binds to a CLDN in a tumor microenvironment, additional bioactive molecules are enriched in the tumor microenvironment, exerting an anti-tumor effect in synergy with the anti-CLDN 18.2-CAR.

In certain embodiments, the additional bioactive molecule is selected from one or more of the following components: an antibody or antigen-binding fragment thereof that specifically binds an immune checkpoint (e.g., an anti-PD-1, PD-L1, CTLA-4, or LAG-3 antibody or antigen-binding fragment thereof), a cytokine (e.g., IL-15, IL-7, IL-12, IL-18, IL-21), or a membrane-chimeric polypeptide (e.g., mIL-15, mIL-7, mIL-12, mIL-18, mIL-21);

in certain embodiments, the nucleic acid sequence encoding an anti-CLDN 18.2-CAR is linked to the nucleic acid sequence of the additional biologically active molecule by a nucleic acid sequence of a self-cleaving peptide. The nucleic acid sequence encoding an anti-CLDN 18.2-CAR may be at the 5 'end or 3' end of the nucleic acid sequence encoding the additional biologically active molecule. In certain exemplary embodiments, the nucleic acid sequence encoding an anti-CLDN 18.2-CAR is 5' to the nucleic acid sequence encoding the additional biologically active molecule. Any self-cleaving peptide that can cause cleavage of the fusion protein into two separate proteins can be used in the present invention. In certain exemplary embodiments, the self-cleaving peptide is P2A having the sequence shown in SEQ ID NO 50. The nucleotide sequence can be optimized according to the requirements of gene recombination.

In certain embodiments, a fusion protein comprising a CAR and an additional biologically active molecule has the structure:

n '-signal peptide-specifically binds to the extracellular antigen-binding domain of human CLDN 18.2-spacer domain-transmembrane domain-intracellular signaling domain-self-cleaving peptide- (signal peptide-2) p-further biologically active molecule-C'. Wherein the signal peptide-2 is the same as or different from the signal peptide of N'. P is 0 or 1.

In certain embodiments, the additional biologically active molecule has a signal peptide immediately upstream thereof that is different from the N-terminus of the CLDN 18.2-CAR. In certain exemplary embodiments, the signal peptide-2 is human IL2 signal peptide having the amino acid sequence set forth in SEQ ID NO: 43, or a sequence as shown in figure 43.

In certain embodiments, the additional biologically active molecule is an anti-PD-1 linear antibody, preferably, the linear antibody has the amino acid sequence as set forth in SEQ ID NO:51, or a sequence shown in seq id no.

In certain embodiments, the fusion protein has the amino acid sequence as set forth in SEQ ID NO: 39 or a variant sequence thereof. In certain embodiments, the variant sequence is identical to SEQ ID NO: 39 having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1, 2, 3, 4, or 5 amino acids or any combination thereof), which variant sequence retains the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the variant sequence has a sequence identical to SEQ ID NO: 39, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 39.

In certain embodiments, the additional bioactive molecule is membrane-chimeric IL-15, preferably, the membrane-chimeric IL-15 has the amino acid sequence as set forth in SEQ ID NO:52, respectively, in sequence as shown in fig. 52.

In certain embodiments, the fusion protein consists of SEQ ID NO:38 or a variant thereof. The variant has a sequence identical to SEQ ID NO:38, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. The variant substantially retains the amino acid sequence of SEQ ID NO:38 (e.g., capable of encoding an expression vector having the ability to direct the specificity and reactivity of immune effector cells to cells expressing CLDN18.2 in a non-MHC-restricted manner, and encoding an expressed bioactive molecule that retains the essential activity of a pre-expressed bioactive molecule).

In certain embodiments, the fusion protein has the amino acid sequence as set forth in SEQ ID NO: 41 or a variant sequence thereof. In certain embodiments, the variant sequence is identical to SEQ ID NO: 41 (e.g., substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids, or any combination thereof; e.g., substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids, or any combination thereof) that retains the amino acid sequence of SEQ ID NO: 41. In certain embodiments, the variant sequence has a sequence identical to SEQ ID NO: 41, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 41.

In certain embodiments, the fusion protein consists of SEQ ID NO:40 or a variant thereof. The variant has a sequence identical to SEQ ID NO:40, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. The variant substantially retains the amino acid sequence of SEQ ID NO:40 (e.g., capable of encoding an expression vector having the ability to direct the specificity and reactivity of immune effector cells to cells expressing CLDN18.2 in a non-MHC-restricted manner, and encoding an expressed bioactive molecule that retains the essential activity of a pre-expressed bioactive molecule).

Preparation of chimeric antigen receptors

Methods of generating chimeric antigen receptors and immune effector cells (e.g., T cells) comprising the chimeric antigen receptors are known in the art and can include transfecting a cell with at least one polynucleotide encoding a CAR and expressing the polynucleotide in the cell. For example, a nucleic acid molecule encoding a CAR of the invention can be included in an expression vector (e.g., a lentiviral vector) capable of being expressed in a host cell, such as a T cell, to produce the CAR.

Accordingly, in a second aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of the first aspect.

It is understood by those skilled in the art that due to the degeneracy of the genetic code, a nucleotide sequence encoding one chimeric antigen receptor of the present invention may have a variety of different sequences. Thus, unless otherwise indicated, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

In certain exemplary embodiments, the nucleotide sequence encoding the chimeric antigen receptor of the first aspect is selected from the group consisting of: (1) 24, 28 or a degenerate variant thereof; (2) a sequence that is substantially identical to a sequence described in (1), e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence described in (1), or a sequence having one or more nucleotide substitutions to a sequence described in (1); and which sequence substantially retains at least one biological activity of the nucleotide sequence from which it is derived (e.g., is capable of encoding a polypeptide having the ability to direct the specificity and reactivity of immune effector cells to cells expressing CLDN18.2 in a non-MHC-restricted manner).

In certain instances, a first nucleotide sequence encoding a CAR according to the first aspect may be linked to a second nucleotide sequence encoding an additional biologically active molecule by a nucleotide sequence encoding a self-cleaving peptide. It is known to those skilled in the art that two proteins linked by a self-cleaving peptide cannot form a normal peptide chain link during translation, so that a proteolytic enzyme-like action is formed to "cleave" the two proteins in cis, and therefore, when the nucleic acid molecule is expressed in a cell, the CAR is cleaved at the self-cleaving peptide from another bioactive molecule, so that the bioactive molecule is secreted out of the CAR-T cell or is expressed in a chimeric manner on the CAR-T cell membrane, thereby exerting an antitumor effect synergistically.

Accordingly, in a third aspect the present invention provides a nucleic acid construct comprising a first nucleotide sequence encoding a chimeric antigen receptor according to the first aspect and further comprising a second nucleotide sequence encoding a further biologically active molecule.

In certain embodiments, the first and second nucleotide sequences are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A). In certain embodiments, the self-cleaving peptide is P2A (e.g., P2A of the sequence shown as SEQ ID NO: 50).

In certain embodiments, the additional biologically active molecule has anti-tumor activity.

In certain embodiments, the additional bioactive molecule is selected from one or more of the following components: an immune checkpoint inhibitor (e.g., an anti-PD-1, PD-L1, CTLA-4, or LAG-3 antibody or antigen-binding fragment), a cytokine (e.g., IL-15, IL-7, IL-12, IL-18, or IL-21), or a membrane-chimeric polypeptide (e.g., mIL-15, mIL-7, mIL-12, mIL-18, or mIL-21).

In certain embodiments, the additional biologically active molecule is selected from the group consisting of an anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof. In certain embodiments, the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof is an scFv.

In certain exemplary embodiments, the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region of any one of the following groups: (1) a heavy chain variable region and/or a light chain variable region of Nivolumab or a variant thereof, (2) a heavy chain variable region and/or a light chain variable region of Pembrolizumab or a variant thereof, (3) a heavy chain variable region and/or a light chain variable region of Atezolizumab or a variant thereof, (4) a heavy chain variable region and/or a light chain variable region of Durvalumab or a variant thereof, (5) a heavy chain variable region and/or a light chain variable region of Avelumab or a variant thereof, (6) a VH having a sequence shown in SEQ ID NO:67 or a variant thereof and/or a VL having a sequence shown in SEQ ID NO:68 or a variant thereof, (7) a VH having a sequence shown in SEQ ID NO:63 or a variant thereof and/or a VL having a sequence shown in SEQ ID NO:64 or a variant thereof.

In certain embodiments, the PD-1 or PD-L1 antibody or antigen-binding fragment thereof includes, but is not limited to, camel Ig, IgNAR, Fab fragment, Fab 'fragment, F (ab)'₂Fragment, F (ab)'₃Fragments, Fv, single-chain antibodies (e.g. scFv, di-scFv, (scFv)₂) Minibodies, diabodies, triabodies, tetradiabodies, disulfide stabilized Fv proteins ("dsFv"), and single domain antibodies (sdabs, nanobodies).

In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a single chain antibody. In some embodiments of the present invention, the substrate is,the single-chain antibody is scFv, di-scFv or (scFv)₂。

In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof has the heavy chain variable region of SEQ ID NO:63 or a variant sequence thereof and/or the light chain variable region of SEQ ID NO:64 or a variant sequence thereof. The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions, or additions) to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

In certain embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof. In certain embodiments, the PD-L1 antibody or antigen-binding fragment thereof comprises: (1) 67, the heavy chain variable region shown in SEQ ID NO; and/or the light chain variable region of SEQ ID NO. 68; or (2) a heavy chain variable region (VH) and/or a light chain variable region (VL) wherein at least one of the sequences has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (1), or at least one of the sequences has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids) to the sequence set forth in (1). Preferably, the substitutions are conservative substitutions.

In certain preferred embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is an scFv and the single chain antibody has a heavy chain variable region of the sequence shown in SEQ ID NO:63 and/or a light chain variable region of the sequence shown in SEQ ID NO: 64.

In certain preferred embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is an scFv and the single chain antibody has the heavy chain variable region of the sequence shown in SEQ ID NO:67 and/or the light chain variable region of the sequence shown in SEQ ID NO: 68.

In certain preferred embodiments, the VH and VL are connected by a linker. In certain preferred embodiments, the linker comprises a linker such as (G)_mS)_nThe sequence shown, wherein m is selected from an integer of 1 to 6. In certain preferred embodiments, m is 3, 4, or 5; n is an integer from 1 to 10. In certain preferred embodiments, n is 2, 3, 4, 5, or 6. In certain preferred embodiments, the linker has the amino acid sequence of SEQ ID NO:44, or a sequence of seq id no.

In certain exemplary embodiments, the anti-PD-1 single chain antibody comprises an amino acid sequence selected from the group consisting of: (1) 51, SEQ ID NO; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO 51; (3) has one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:51 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

In certain exemplary embodiments, the nucleic acid construct encoding a specific CLDN18.2 chimeric antigen receptor and anti-PD-1 single chain antibody comprises a nucleotide sequence selected from the group consisting of: (1) 38 or a degenerate variant thereof; (2) a sequence that is substantially identical to a nucleotide sequence described in (1), e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence described in (1), or a sequence having one or more nucleotide substitutions to a sequence described in (1).

In certain embodiments, the additional biologically active molecule is selected from the group consisting of a membrane-chimeric polypeptide which is mIL-15. In certain embodiments, the membrane-chimeric polypeptide mIL-15 sequence comprises an amino acid sequence selected from the group consisting of seq id nos: (1) SEQ ID NO: 52; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO 52; (3) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 52. In certain preferred embodiments, the substitutions are conservative substitutions.

In certain exemplary embodiments, the nucleic acid construct encoding a specific binding to a CLDN18.2 chimeric antigen receptor and a membrane-chimeric polypeptide mIL-15 comprises a nucleotide sequence selected from the group consisting of: (1) 40 or a degenerate variant thereof; (2) a sequence that is substantially identical to a nucleotide sequence described in (1), e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence described in (1), or a sequence having one or more nucleotide substitutions to a sequence described in (1).

In certain exemplary embodiments, the additional biologically active molecule is SEQ ID NO:51, or a sequence shown in seq id no. The additional bioactive molecule comprises a signal peptide-2 at the N-terminus. In certain embodiments, the signal peptide-2 linked to the additional biologically active molecule is different from the signal peptide contained in the chimeric antigen receptor. In certain embodiments, the signal peptide-2 linked to the additional bioactive molecule is an IL2 signal peptide, an IL2 signal peptide refers to a signal peptide sequence contained in the sequence of the native gene of IL2, preferably, the native gene of IL2 is the native gene of human IL2 and the signal peptide of IL2 is the signal peptide of human IL 2. In certain exemplary embodiments, the IL2 signal peptide comprises the amino acid sequence set forth as SEQ ID NO: 43, or a sequence as shown in figure 43.

In certain embodiments, the first nucleotide sequence is upstream of the second nucleotide sequence. In certain exemplary embodiments, the sequence encoding the self-cleaving peptide is linked to the 3 'end of the first nucleotide sequence and linked to the 5' end of the second nucleotide sequence.

In a fourth aspect, the invention provides a vector comprising the isolated nucleic acid molecule of the second aspect or the nucleic acid construct of the third aspect.

In certain embodiments, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, a viral vector.

In certain embodiments, the vector is an expression vector.

In certain embodiments, the vector is an episomal vector.

In certain embodiments, the vector is a viral vector.

In certain exemplary embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.

In certain embodiments, the vector is an episomal or non-integrating viral vector, such as an integration defective retrovirus or lentivirus.

In a fifth aspect, the invention provides a host cell comprising an isolated nucleic acid molecule as described in the second aspect above, a nucleic acid construct as described in the third aspect or a vector as described in the fourth aspect. The vectors described above can be introduced into the host cell by a variety of suitable means, such as calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, electroporation, TALEN methods, ZFN methods, non-viral vector mediated transfection (e.g., liposomes) or viral vector mediated transfection (e.g., lentiviral infection, retroviral infection, adenoviral infection), as well as other physical, chemical or biological means for transfer into a host cell, such as transposon technology, CRISPR-Cas9, and the like.

In certain embodiments, the host cell comprising the isolated nucleic acid molecule of the second aspect or a vector comprising the nucleic acid molecule expresses the chimeric antigen receptor of the invention.

In certain embodiments, the host cell comprising the nucleic acid construct of the third aspect or a vector comprising the nucleic acid construct expresses the chimeric antigen receptor of the invention and an additional biologically active molecule.

In certain embodiments, the host cell is selected from mammalian (e.g., human) immune cells. In certain embodiments, the immune cell is derived from a patient or a healthy donor. In certain embodiments, the immune cell is selected from a T lymphocyte, a Natural Killer (NK) cell, a monocyte, a macrophage, or a dendritic cell, and any combination thereof.

In a sixth aspect, the present invention provides a method of making a cell expressing a chimeric antigen receptor of the present invention, comprising: (1) providing a host cell; (2) obtaining a host cell capable of expressing the chimeric antigen receptor; wherein the step (2) comprises: introducing the isolated nucleic acid molecule of the second aspect or a vector comprising said nucleic acid molecule into said host cell. In another aspect, the invention provides a method of making a cell co-expressing a chimeric antigen receptor of the invention and an additional biologically active molecule, comprising: (1) providing a host cell; (2) obtaining a host cell capable of co-expressing the chimeric antigen receptor and an additional biologically active molecule; wherein the step (2) comprises: introducing the nucleic acid construct of the third aspect or a vector comprising the nucleic acid construct into the host cell.

In certain embodiments, the host cell is selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages, and any combination thereof. In certain embodiments, the immune cell is selected from a T lymphocyte, an NK cell, a monocyte, a macrophage, or a dendritic cell, and any combination of these cells.

In certain embodiments, in step (1), the immune cells are pretreated; the pretreatment comprises sorting, activation and/or proliferation of immune cells; in certain embodiments, the pretreatment comprises contacting the immune cells with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.

In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host cell by viral infection. In certain embodiments, the nucleic acid molecule or vector is introduced into the host cell in step (2) by means of transfection with a non-viral vector, such as by means of a vector system of transposons, CRISPR/Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.

In certain embodiments, after step (2), the method further comprises: amplifying the host cell obtained in step (2).

Engineered immune cells

Immune cells derived from a patient or a healthy donor can be engineered by the above described production methods provided by the present invention into immune cells expressing a CAR that specifically binds to CLDN18.2 and optionally additional biologically active molecules.

Accordingly, the seventh aspect of the invention also provides an engineered immune cell expressing a CAR of the invention that specifically binds to CLDN 18.2.

In certain embodiments, the engineered immune cell comprises the isolated nucleic acid molecule of the second aspect or a vector comprising the nucleic acid molecule.

In certain embodiments, the engineered immune cell also expresses an additional biologically active molecule. In certain embodiments, the engineered immune cell comprises the nucleic acid construct of the third aspect or a vector comprising the nucleic acid construct.

In certain embodiments, the additional biologically active molecule is selected from an immune checkpoint antibody or antigen binding fragment thereof (e.g., an anti-PD-1, PD-L1, CTLA-4, or LAG-3 antibody or antigen binding fragment thereof), a cytokine (e.g., IL-15, IL-7, IL-12, IL-18, IL-21), or a membrane-chimeric polypeptide (e.g., mIL-15, mIL-7, mIL-12, mIL-18, mIL-21).

In certain embodiments, the immune checkpoint antibody or antigen binding fragment thereof is selected from an anti-PD-1 antibody or antigen binding fragment thereof (e.g., scFv). In certain embodiments, the PD-1 antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:51, or a sequence shown in seq id no.

In certain embodiments, the immune checkpoint antibody or antigen binding fragment thereof is selected from an anti-PD-1 or anti-PD-L1 antibody or antigen binding fragment thereof (e.g., scFv). In certain embodiments, the immune checkpoint antibody or antigen binding fragment thereof is selected from an anti-PD-1 or anti-PD-L1 antibody or antigen binding fragment thereof is selected from Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, or Avelumab. In certain embodiments, the anti-PD-1 or anti-PD-L1 antibody or antigen-binding fragment thereof is selected from the group consisting of PD-L1 antibodies in PCT/CN 2019/106599. In certain embodiments, the anti-PD-1 or anti-PD-L1 antibody or antigen-binding fragment thereof is selected from the group consisting of PD-1 antibodies in CN201910939944.8 or its cognate patent application.

In certain embodiments, the anti-PD-1 or anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region selected from the group consisting of:

(1) a heavy chain variable region and/or a light chain variable region of Nivolumab or a variant thereof;

(2) a heavy chain variable region and/or a light chain variable region of Pembrolizumab or a variant thereof;

(3) (ii) the heavy chain variable region and/or the light chain variable region of Atezolizumab or a variant thereof;

(4) a heavy chain variable region and/or a light chain variable region of Durvalumab or a variant thereof;

(5) the heavy chain variable region and/or the light chain variable region of Avelumab or a variant thereof;

(6) a VH having the sequence shown by SEQ ID NO. 67 or a variant thereof and/or a VL having the sequence shown by SEQ ID NO. 68 or a variant thereof; or the like, or, alternatively,

(7) a VH having the sequence shown in SEQ ID NO. 63 or a variant thereof and/or a VL having the sequence shown in SEQ ID NO. 64 or a variant thereof;

wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids) to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In certain embodiments, the membrane-chimeric polypeptide is mIL-15. In certain embodiments, the membrane-chimeric polypeptide mIL-15 has the sequence set forth in SEQ ID NO: shown at 52.

In certain embodiments, the engineered immune cell comprises the isolated nucleic acid molecule of any of the second aspect, the nucleic acid construct of any of the third aspect, or the vector of any of the fourth aspect, or a nucleic acid molecule, nucleic acid construct, or vector having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or having a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids).

In certain embodiments, wherein the immune cell is derived from a T lymphocyte, NK cell, monocyte, macrophage or dendritic cell of the patient or healthy donor and any combination thereof. The immune cells are obtained from a patient or a healthy donor. These immune cells are prepared as engineered immune cells by introducing the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, or the vector of the fourth aspect by the methods provided above.

In certain embodiments, the engineered immune cell further has a binding specificity other than a CLDN18.2 target, such as the engineered immune cell further binds CD19, CD20, CD22, CD33, CD123, or CD 138.

In certain embodiments, the engineered immune cell further comprises a knockout of one or more endogenous genes, wherein the endogenous gene is selected from the group consisting of: genes encoding TCR α, TCR β, CD52, Glucocorticoid Receptor (GR), deoxycytidine kinase (dCK), or immune checkpoint proteins, such as programmed death-1 (PD-1).

In an eighth aspect, the present invention provides a method of preparing the aforementioned engineered immune cell, comprising: (1) providing immune cells from a patient or a healthy donor; (2) obtaining an immune cell capable of expressing a CLDN 18.2-specific binding chimeric antigen receptor or an immune cell co-expressing a CLDN 18.2-specific binding chimeric antigen receptor and a biologically active molecule; wherein step (2) comprises introducing the isolated nucleic acid molecule of the second aspect or the nucleic acid construct of the third aspect or the vector of the fourth aspect into the immune cell of step (1).

In certain embodiments, the immune cell is selected from a T lymphocyte, an NK cell, a monocyte, a macrophage, or a dendritic cell, and any combination of these cells.

In certain embodiments, in step (2), the nucleic acid molecule, nucleic acid construct or vector is introduced into the immune cell by viral infection. In certain embodiments, the nucleic acid molecule, nucleic acid construct or vector is introduced into the immune cell in step (2) by means of transfection with a non-viral vector, such as by means of vector systems of transposons, CRISPR/Cas9 vectors, TALEN methods, ZFN methods, electroporation methods, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection, and the like.

In certain embodiments, after step (2), the method further comprises: amplifying the immune cells obtained in the step (2).

Immune cell composition

In a ninth aspect, the present invention also provides an immune cell composition comprising the aforementioned engineered immune cells, and optionally unmodified and/or unsuccessfully engineered immune cells that do not express a CAR specific for CLDN 18.2. Not all immune cells have been engineered to express a CAR specific for CLDN18.2, limited to the current state of the art and for some unknown reasons. And the immune cells not expressing the CAR also have certain biological activity, so the immune cell composition can contain the immune cells expressing and not expressing the CAR specific to CLDN18.2, and the immune cell composition can still meet the requirement of clinical application. In certain embodiments the engineered immune cells expressing a CAR specific for CLDN18.2 comprise between about 10% and 100%, preferably between 40% and 80% of the total number of cells of the immune cell composition.

In certain embodiments, the immune cell composition is cultured as an immune cell line, and thus, in another aspect, the invention also provides an immune cell line comprising the immune cell composition.

In a tenth aspect, the present invention provides a kit for the preparation of a chimeric antigen receptor that specifically binds to CLDN18.2, or for the preparation of cells expressing said chimeric antigen receptor or immune cells co-expressing said chimeric antigen receptor and a further biologically active molecule. In certain embodiments, the kit comprises the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect or the vector of the fourth aspect, or the host cell of the fifth aspect, and an essential solvent, such as sterile water, physiological saline, or a cell culture broth, such as an LB medium, such as an EliteCell primary T lymphocyte culture system (product number: PriMed-EliteCell-024), and optionally, instructions for use.

In another aspect, the invention provides the use of the aforementioned kit for the preparation of a chimeric antigen receptor capable of specifically binding to CLDN18.2, or a cell expressing said chimeric antigen receptor, or an immune cell co-expressing said chimeric antigen receptor and a further biologically active molecule.

Pharmaceutical composition

In an eleventh aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor of the first aspect of the present invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, or the immune cell composition of the ninth aspect, and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug having anti-tumor activity.

In certain embodiments, the additional pharmaceutically active agent is an interferon, interleukin-2, or a chemotherapeutic drug.

In certain embodiments, the additional pharmaceutically active agent is selected from: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, leucovorin, paclitaxel and albumin-bound paclitaxel.

In certain embodiments, in the pharmaceutical composition, the chimeric antigen receptor of the first aspect of the invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, or the immune cell composition of the ninth aspect of the invention and the additional pharmaceutically active agent may be provided as separate components or as a mixed component. Thus, the chimeric antigen receptor of the first aspect of the invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect or the immune cell composition of the ninth aspect and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In certain embodiments, the pharmaceutical compositions of the present invention comprise: the chimeric antigen receptor of the first aspect, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, or the immune cell composition of the ninth aspect.

The chimeric antigen receptor of the first aspect of the invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, or the immune cell composition of the ninth aspect of the invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier, e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof, prior to use.

The chimeric antigen receptor of the first aspect of the invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, or the immune cell composition of the ninth aspect of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the chimeric antigen receptor of the first aspect, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, or the immune cell composition of the ninth aspect of the invention is administered by intravenous injection or bolus injection.

The pharmaceutical composition of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the chimeric antigen receptor of the first aspect of the invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, or the immune cell composition of the ninth aspect. A "prophylactically effective amount" is an amount sufficient to prevent, or delay the onset of disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the chimeric antigen receptor of the first aspect of the invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, or the immune cell composition of the ninth aspect of the invention may vary according to: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g. age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, etc.

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

Methods of treatment and uses

In another aspect, the present invention provides a method for preventing and/or treating a tumor, and/or delaying tumor progression, and/or reducing or inhibiting tumor recurrence in a subject, the method comprising administering to a subject in need thereof an effective amount of the chimeric antigen receptor of the first aspect, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, the immune cell composition of the ninth aspect, or the pharmaceutical composition of the eleventh aspect of the invention.

In certain embodiments, the method comprises administering to the subject an effective amount of a single chain antibody of the invention.

In certain embodiments, the method comprises administering to the subject an effective amount of the chimeric antigen receptor of the first aspect, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, the immune cell composition of the ninth aspect, or the pharmaceutical composition of the eleventh aspect. In certain embodiments, the host cell is an immune cell (e.g., a human immune cell).

In certain embodiments, the method comprises the steps of: (1) providing an immune cell (e.g., a T lymphocyte, an NK cell, a monocyte, a macrophage, a dendritic cell, or any combination of these) in need of the subject; (2) introducing into the immune cell of step (1) a vector comprising the isolated nucleic acid molecule of the second aspect of the invention, the nucleic acid construct of the third aspect or the vector of the fourth aspect of the invention, to obtain a cell expressing the chimeric antigen receptor or an immune cell co-expressing the chimeric antigen receptor and an additional biologically active molecule; (3) administering the immune cells obtained in step (2) to the subject for treatment.

In certain embodiments, the method comprises administering to the subject a partial dose by dose fractionation, e.g., once, twice, three times, or more divided administrations, e.g., a first percentage of the total dose on a first day of treatment, a second percentage of the total dose on a subsequent (e.g., second, third, fourth, fifth, sixth, or seventh or later) day of treatment, e.g., a third percentage (e.g., remaining percentage) of the total dose on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or later) day of treatment.

In certain embodiments, 10% of the total dose of cells is administered on the first day of treatment, 30% of the total dose of cells is administered on the second day, and the remaining 60% of the total dose of cells is administered on the third day.

In certain embodiments, 50% of the total dose of cells is administered on the first day of treatment and 50% of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh or later) day of treatment. In certain embodiments, the total dose of 1/3 cells is administered on the first day of treatment, the total dose of 1/3 cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth, or seventh or later) day of treatment, and the total dose of 1/3 cells is administered on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or later) day of treatment.

In certain embodiments, the total cell dose comprises 1 × 10⁷To 10X 10⁸Individual CAR-positive immune cells, e.g. comprising (1-5). times.10⁷To (5-10). times.10⁸Individual CAR-positive immune cells.

In certain embodiments, the physician can adjust the dosage or treatment regimen according to the patient's condition, the size and stage of the tumor, or the clinical circumstances of the drug combination.

In certain embodiments, the tumor is selected from the group consisting of a solid tumor, a hematologic tumor, and a metastatic, refractory or recurrent lesion of the cancer.

In certain embodiments, the tumor or cancer is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, 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, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia.

In certain embodiments, the tumor is selected from the group consisting of gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer).

In certain embodiments, the tumor is selected from gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma, such as locally advanced unresectable or metastatic gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma.

In certain embodiments, the tumor is CLDN18.2 positive. In certain embodiments, the tumor is HER2 negative. In certain embodiments, the tumor is CLDN18.2 positive and HER2 negative. HER2 negative means that the cell surface is free of significant amounts of HER2 protein, including IHC 1+, or IHC 2+/FISH negative, and also including the range of IHC 0 to 1+ and IHC 1+ to 2 +.

In certain embodiments, the chimeric antigen receptor of the first aspect, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, the immune cell composition of the ninth aspect, or the pharmaceutical composition of the eleventh aspect of the invention is administered in combination with an additional agent. In certain embodiments, the additional agent comprises (i) an agent that increases the efficacy of a cell comprising a CAR nucleic acid or CAR polypeptide (e.g., an immune cell expressing a CAR of the invention, an engineered immune cell of the invention, or an immune cell composition); (ii) an agent that ameliorates one or more side effects associated with administration of a cell comprising a CAR nucleic acid or CAR polypeptide (e.g., an immune cell expressing a CAR of the invention, an engineered immune cell or immune cell composition of the invention); (iii) additional pharmaceutically active agents having anti-tumor activity. In certain preferred embodiments, the additional pharmaceutically active agent is selected from: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, leucovorin, paclitaxel and albumin-bound paclitaxel. These agents may be administered prior to, concurrently with or subsequent to the administration of the chimeric antigen receptor of the first aspect of the invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, the immune cell composition of the ninth aspect or the pharmaceutical composition of the eleventh aspect.

In certain embodiments, the methods described above further comprise administering to the subject a second therapy, which can be any therapy known for tumors, such as surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

In certain embodiments, the second therapy may be applied separately, in combination, simultaneously, or sequentially with the methods described above.

In certain preferred embodiments, the chemotherapy is selected from the group consisting of: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, leucovorin, paclitaxel and albumin-bound paclitaxel.

In certain embodiments, the subject may be a mammal, such as a human.

In another aspect, there is provided a use of the chimeric antigen receptor of the first aspect of the invention, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the engineered immune cell of the seventh aspect, the immune cell composition of the ninth aspect or the pharmaceutical composition of the eleventh aspect in the manufacture of a medicament for the prevention and/or treatment and/or co-treatment of a tumor, and/or delay of tumor progression, and/or reduction or inhibition of tumor recurrence in a subject. The dosage, dosage form, administration route, indications, combination therapy and the like of the aforementioned treatment methods can be applied to the application of the medicament.

Acronyms

CLDN18.2 Claudin 18.2

CAR chimeric antigen receptor

Complementarity determining regions in CDR immunoglobulin variable regions

CDR-H1 complementarity determining region 1 in the immunoglobulin heavy chain variable region

CDR-H2 complementarity determining region 2 in the immunoglobulin heavy chain variable region

CDR-H2-1 immunoglobulin heavy chain variable region complementarity determining region 2 first sequence

CDR-H2-2 immunoglobulin heavy chain variable region complementarity determining region 2 second sequence

CDR-H3 complementarity determining region 3 in the immunoglobulin heavy chain variable region

CDR-L1 variable region of immunoglobulin light chain complementarity determining region 1

CDR-L2 complementarity determining region 2 in the immunoglobulin light chain variable region

CDR-L3 CDR3 in the immunoglobulin light chain variable region

FR antibody framework regions: amino acid residues other than CDR residues in antibody variable regions

VH antibody heavy chain variable region

VL antibody light chain variable region

The AbM AbM CDR definition format was derived from Martin's related studies (Martin ACR, Cheetham JC, Rees AR (1989) modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-.

The immunoglobulin alignment and numbering system proposed by Kabat of Elvin a (see, e.g., Kabat et al, Sequences of Proteins of Immunological Interest,5th ed. public Health Service, National Institutes of Health, Bethesda, Md., 1991).

Chothia by Chothia et al proposed immunoglobulin numbering system, which is based on the structural loop region position identification CDR region boundary classic rules (see, for example, Chothia & Lesk (1987) J.mol.biol.196:901 and 917; Chothia et al (1989) Nature 342:878 and 883).

IMGT is based on the International ImmunoGeneTiCs information System (International ImmunoGeneTiCs information) initiated by Lefranc et al

(IMGT)) see Lefranc et al, dev.company.immunol.27: 55-77,2003.

IL-2 Interleukin 2

IFN interferon

PCR polymerase chain reaction

FACS flow cytofluorescent sorting

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of molecular biology, microbiology, cell biology, biochemistry, immunology, etc. used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "antibody" refers to a polypeptide capable of recognizing a variable region of an immunoglobulin molecule by virtue of at least one antigen recognition site located in the variable region of the immunoglobulin moleculeImmunoglobulin molecules that specifically bind a target (e.g., carbohydrates, polynucleotides, lipids, polypeptides, etc.). As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab ', F (ab')2, Fv), single chains (e.g., scFv, di-scFv, (scFv)₂) And domain antibodies (including, for example, shark and camelid antibodies), as well as fusion proteins including antibodies, and immunoglobulin molecules including any other modified configuration of the antigen recognition site. The antibodies of the invention are not limited by any particular method of producing the antibodies. Antibodies include any type of antibody, such as IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of any particular type. Depending on the amino acid sequence of the constant region of the heavy chain of an antibody, immunoglobulins can be assigned to different classes. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant regions corresponding to the different types of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known. The heavy chain constant region consists of 4 domains (CH1, hinge region, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The VH and VL regions of an antibody can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each V_HAnd V_LBy the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antigen-binding sites, respectively. Division of amino acids into regions or domainsThe formulations may follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia&Lesk (1987) J.mol.biol.196: 901-917; chothia et al (1989) Nature 342: 878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs contained in the variable regions of the heavy and light chains, respectively, designated CDR1, CDR2, and CDR 3. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md.,1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol.biol.196: 901-917; Chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, Dev.Complex.Immunol.27: 55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al, Dev. company. Immunol.27:55-77,2003).

In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia, or IMGT numbering system. In certain embodiments, the CDRs contained by the antibodies or antigen-binding fragments thereof of the present invention are determined by the IMGT numbering system. In the present invention, the definitions of the numbering systems for the CDRs are defined by the IMGT numbering system, except for the specific labels.

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

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide that is a fragment of an antibody, e.g., a polypeptide that is a fragment of a full-length antibody,which retain the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or compete with the full-length antibody for specific binding to the antigen, also referred to as "antigen-binding portions". See generally, Fundamental Immunology, Ch.7(Paul, W., ed., 2nd edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes.₂Fragment, F (ab)'₃Fragment, Fd, Fv, scFv, di-scFv, (scFv)₂A minibody, a diabody, a triabody, a tetrafunctional antibody, a disulfide stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al, 2005; nat Biotechnol,23: 1126-.

As used herein, the term "camel Ig" or "camel VHH" refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte et al, FASEB J.,21:3490-3498 (2007)). "heavy chain antibody" or "camelid antibody" refers to an antibody containing two VH domains and no light chain (Riechmann L. et al, J.Immunol. methods)231:25-38 (1999); WO 94/04678; WO 94/25591; U.S. Pat. No. 6,005,079).

As used herein, the term "IgNAR" or "immunoglobulin neoantigen receptor" refers to a class of antibodies from the shark immune repertoire consisting of homodimers of one variable neoantigen receptor (VNAR) domain and five constant neoantigen receptor (CNAR) domains.

As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al, Nature 341: 544546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F (ab')₂Fragment "means a fragment obtained byAn antibody fragment of two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reductively linked F (ab')₂The fragment obtained after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete Fd fragment of the light and heavy chains, consisting of the VH and CH1 domains.

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

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

As used herein, The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein The VL and VH are linked by a linker (linker) (see, e.g., Bird et al, Science 242: 423-. Such scFv molecules can have the general structure: NH (NH)₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS)₄But variants thereof can also be used (Holliger et al (1993), Proc. Natl. Acad. Sci. USA 90: 6444-. Other linkers useful in the present invention are represented by Alfthan et al (1995), Protein Eng.8:725-731, Choi et al (2001), Eur.J.Immunol.31:94-106, Hu et al (1996), Cancer Res.56:3055-3061, Kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), Cancer immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, containing NH₂-VH-VH-COOH、NH_2-scFv of VL-VL-COOH. The scFv may form any engineering possible structure, single chain antibodies (scFv), tandem antibodies (tandem di-scFvs), bifunctional, trifunctional, tetrafunctional, disulfide stabilized Fv proteins, camel Ig, IgNAR, etc. In certain embodiments of the invention, the scFv may form a di-scFv, which refers to two or more individual scFv connected in tandem to form an antibody. In certain embodiments of the invention, the scFv may form a (scFv)₂It refers to two or more individual scfvs connected in parallel to form an antibody.

As used herein, the term "bifunctional antibody" refers to an antibody fragment having two antigen binding sites, said fragment comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with the complementary domains of the other strand and two antigen binding sites are created. The bifunctional antibody may be bivalent or bispecific. Bifunctional antibodies are described more fully in, for example, EP 404,097; WO 1993/01161; hudson et al, Nature medicine (nat. Med.)9: 129-; and Hollinger et al, PNAS USA 90: 6444-. Tri-and tetra-functional antibodies are also described in Hudson et al, Nature medicine 9: 129-.

As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen to which a full-length antibody binds (Holt, l. et al, Trends in Biotechnology, 21(11):484-490, 2003). Single domain antibodies are also known as nanobodies (nanobodies).

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

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

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

As used herein, the expression "specific binding" or "specific targeting" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction may be the equilibrium dissociation constant (K) of the interaction_D) And (4) showing. In the present invention, the term "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, Nature,1993,361: 186-187). The ratio of kdis/kon is equal to the dissociation constant K_D(see Davies et al, Annual Rev Biochem, 1990; 59: 439-. K can be measured by any effective method_DKon and kdis values. In certain embodiments, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, dissociation constants can be measured using bioluminescence interferometry or Kinexa.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.appl biosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48: 444-.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl Acad. set USA94:412-417(1997), which are incorporated herein by reference).

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

As used herein, the terms "nucleic acid molecule," "nucleotide sequence," "polynucleotide" refer to messenger RNA (mrna), RNA, genomic RNA (grna), positive strand RNA (+), negative strand RNA (-), genomic DNA (gdna)), complementary DNA (cdna), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide may be inserted. The vector may comprise a sequence which is autonomously replicating directly in the cell, or may comprise a sequence sufficient to allow integration into the host cell DNA. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage and viral vector. Non-limiting examples of viral vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyomavirus vacuoles (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "episomal vector" episomal refers to a vector that is capable of replicating without integrating into the chromosomal DNA of a host and without being gradually lost by dividing host cells, and also means that the vector replicates extrachromosomally or episomally.

As used herein, the term "viral vector" is used broadly to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer. In addition to nucleic acids, viral particles will typically include various viral components and sometimes host cell components.

The term "viral vector" may refer to a virus or viral particle capable of transferring a nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements derived primarily from viruses.

As used herein, the term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or parts thereof derived primarily from a retrovirus.

As used herein, the term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from lentiviruses, or portions thereof (including LTRs). In certain embodiments, the terms "lentiviral vector", "lentiviral expression vector" may be used to refer to a lentiviral transfer plasmid and/or an infectious lentiviral particle. Where reference is made herein to elements (e.g., cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.), it is understood that the sequences of these elements are present in the lentiviral particles of the present invention in the form of RNA and are present in the DNA plasmids of the present invention in the form of DNA.

As used herein, an "integration-defective" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that is unable to integrate the viral genome into the genome of a host cell. In certain embodiments, the integrase protein is mutated to specifically reduce its integrase activity. Integration-deficient lentiviral vectors can be obtained by modifying the pol gene encoding the integrase protein to generate a mutant pol gene encoding an integration-deficient integrase. Such integration-defective viral vectors have been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as E.coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells, immune cells (e.g., T lymphocytes, NK cells, monocytes, macrophages or dendritic cells, etc.). The host cell may comprise a single cell or a population of cells.

In certain embodiments, the host cell may comprise a cell electroporated, transfected, infected or transduced in vivo, ex vivo or in vitro with the isolated nucleic acid molecule of the second aspect of the invention, the nucleic acid construct of the third aspect or a vector comprising the nucleic acid molecule or nucleic acid construct. In such embodiments, the host cell is preferably an immune cell.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, a spacer domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that combines antibody-based specificity for an antigen of interest (e.g., CLDN18.2) with an immune effector cell activating intracellular domain to exhibit specific immune activity against a cell expressing the antigen of interest (e.g., CLDN 18.2). In the present invention, the expression "immune effector cell expressing a CAR" refers to an immune effector cell expressing a CAR and having antigen specificity determined by the targeting domain of the CAR. Methods of making CARs (e.g., for cancer therapy) are known in the art, and can be found, for example, in Park et al, Trends biotechnol., 29:550-557, 2011; grupp et al, N Engl J Med.,368: 1509-; han et al, j.hematol.oncol.,6:47,2013; PCT patent publications WO2012/079000, WO 2013/059593; and U.S. patent publication 2012/0213783, which is incorporated herein by reference in its entirety.

As used herein, the terms "extracellular antigen-binding domain" and "extracellular ligand-binding domain" are used interchangeably to refer to a polypeptide capable of specifically binding an antigen or receptor of interest. This domain will be able to interact with cell surface molecules. For example, extracellular antigen-binding domains can be selected to recognize antigens that are cell surface markers of target cells associated with a particular disease state.

As used herein, the term "intracellular signaling domain" refers to a protein moiety that conducts signals of effector signaling functions and directs a cell to perform a specialized function. Thus, the intracellular signaling domain has the ability to activate at least one normal effector function of the CAR-expressing immune effector cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.

As used herein, the term "primary signaling domain" refers to a protein moiety that is capable of modulating primary activation of a TCR complex in a stimulatory manner or in an inhibitory manner. The primary signaling domain, which acts in a stimulatory manner, typically contains a signaling motif known as an immunoreceptor tyrosine-based activation motif (I TAM). Non-limiting examples of ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, C D3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66 d.

As used herein, the term "co-stimulatory signaling domain" refers to the intracellular signaling domain of a co-stimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide the second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Non-limiting examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD270(HVEM), CD278(ICOS), DAP 10.

As used herein, the term "self-cleaving peptide" refers to a class of peptides that can induce cleavage of recombinant proteins in cells, such as 2A self-cleaving peptides, which are a family of peptides 18-22 aa long. Four members of the 2A peptide family are often used for life science research. They are P2A, E2A, F2A and T2A. F2A was from foot and mouth disease virus 18. The sequence of T2A can be found, for example, in SEQ ID NO:56, the sequence of P2A can be found, for example, in SEQ ID NO:50, the sequence of E2A can be found, for example, in SEQ ID NO:57, and the sequence of F2A can be found, for example, in SEQ ID NO: 58.

In this context, a membrane-chimeric polypeptide refers to a polypeptide or protein that, after expression, does not completely detach from the cell membrane of the cell in which it is expressed, which polypeptide or protein may have a membrane-penetrating structure, or may be attached to the cell membrane but free from the cell.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19th ed. pennsylvania: mach Publishing Company,1995), and include, but are not limited to: sterile water, physiological saline, a pH regulator, a surfactant, an adjuvant, an ionic strength enhancer, a diluent, an agent for maintaining osmotic pressure, an agent for delaying absorption, and a preservative. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in a medicament, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solution, and any combination thereof.

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

As used herein, the term "subject" refers to a mammal, e.g., a primate mammal, e.g., a human. In certain embodiments, the term "subject" is meant to include living organisms in which an immune response can be elicited. In certain embodiments, the subject (e.g., human) has a tumor (e.g., a tumor associated with CLDN18.2), or is at risk for having a disease as described above.

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

As used herein, the term "immune cell" refers to a cell involved in an immune response, e.g., involved in promoting immune effector function. Examples of immune cells include T cells (e.g., α/β T cells and γ/δ T cells), B cells, Natural Killer (NK) cells, natural killer T (nkt) cells, mast cells, and bone marrow-derived macrophages.

The immune cells of the invention may be autologous/autologous ("self") or non-self ("non-self", e.g., allogeneic, syngeneic or xenogeneic). As used herein, "autologous" refers to cells from the same subject; "allogeneic" refers to cells of the same species that are genetically different from the comparative cells; "syngeneic" refers to cells from a different subject that are genetically identical to the comparative cells; "allogeneic" refers to cells from a different species than the comparative cells. In a preferred embodiment, the cells of the invention are allogeneic.

Exemplary immune cells that can be used for the CARs described herein include T lymphocytes. The term "T cell" or "T lymphocyte" is well known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell may be a T helper (Th) cell, such as a T helper 1(Th1) or a T helper 2(Th2) cell. The T cells may be helper T cells (HTL; CD4T cells), CD4T cells, cytotoxic T cells (CTL; CD8T cells), CD4CD8T cells, CD4CD8T cells, or any other subset of T cells. In certain embodiments, T cells may include naive T cells and memory T cells.

One skilled in the art will understand that other cells can also be used as immune cells with the CARs as described herein. Specifically, immune cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils and macrophages. The immune cells also include progenitor cells of the immune cells, wherein the progenitor cells can be induced to differentiate into immune cells in vivo or in vitro. Thus, in certain embodiments, the immune cells include progenitor cells of immune cells, such as Hematopoietic Stem Cells (HSCs) contained within a CD34+ cell population derived from cord blood, bone marrow, or flowing peripheral blood, which differentiate into mature immune cells upon administration in a subject, or which can be induced to differentiate into mature immune cells in vitro.

As used herein, the term "engineered immune cell" refers to an immune cell that expresses any of the CARs described herein, or into which any of the isolated nucleic acids or vectors described herein has been introduced. The CAR polypeptide can also be synthesized in situ in the cell after introduction of the polynucleotide encoding the CAR polypeptide into the cell by a variety of methods. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, the polynucleotide construct may be integrated into the genome of the cell using stable transformation methods. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, virus-mediated methods may be used. The polynucleotide may be introduced into the cell by any suitable method, such as recombinant viral vectors (e.g., retrovirus, adenovirus), liposomes, and the like. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or microprojectile bombardment. The polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.

As used herein, the term "immune effector function" refers to a function or response of an immune effector cell that enhances or promotes immune attack (e.g., killing, or inhibits growth or proliferation) of a target cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.

The terms "cancer," "tumor," and "tumors" are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in vivo. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may migrate to remote sites in the body through the lymphatic system or the bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

The term "hematological malignancy" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B cell lymphomas, including, for example, hodgkin lymphoma. T cell lymphomas, including, for example, cutaneous T cell lymphomas. Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphocytic leukemia. Hematological malignancies also include myelomas (e.g., multiple myeloma) and other hematological and/or B-cell or T-cell related cancers.

In this context, combination therapy includes the use of a single chain antibody of the invention, a host cell of the ninth aspect, an engineered immune cell or immune cell composition of the invention in combination with one or more additional active therapeutic agents of a second therapy (e.g., chemotherapeutic agents) or other prophylactic or therapeutic modalities (e.g., radiation therapy).

Exemplary anticancer agents for the second therapy can include chemotherapeutic agents (e.g., mitotic inhibitors), alkylating agents (e.g., Nitrogen Mustard nitrosgen Mustard), antimetabolites (e.g., folic acid analogs), natural products (e.g., Vinca alkaloids Vinca alkoids), various agents (e.g., platinum coordination complexes), hormones and antagonists (e.g., adrenal corticosteroids), immunomodulators (e.g., bripirimimine, Upjohn), and the like. Other anti-cancer therapies include other antibodies that specifically target cancer cells.

In such combination therapies, the various active agents often have different complementary mechanisms of action, which may result in synergistic effects. Combination therapy includes therapeutic agents that affect an immune response (e.g., enhance or activate a response) and therapeutic agents that affect (e.g., inhibit or kill) tumor/cancer cells. Combination therapy can reduce the likelihood of drug resistant cancer cells. Combination therapy may allow for a reduction in the dosage of one or more of the agents to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder or condition.

As used herein, "combination" includes therapies that can be administered separately, e.g., formulated separately for separate administration (e.g., as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., "co-formulation"). In certain embodiments, the single chain antibody of the invention, the host cell of the ninth aspect, the engineered immune cell of the invention, or the immune cell composition may be administered sequentially with the second therapy. In other embodiments, the single chain antibody of the invention, the host cell of the ninth aspect, the engineered immune cell of the invention, or the immune cell composition can be administered concurrently with the second therapy. The single chain antibody of the invention, the host cell of the ninth aspect, the engineered immune cell or the immune cell composition of the invention may be used in any combination with at least one other (active) agent.

Herein, Claudin18.2 positive, obtained by immunohistochemistry and staining intensity evaluation by a professional clinician. HER2 negative, including IHC 1+, or IHC 2+/FISH negative, further including ranges of IHC 0 to 1+ and IHC 1+ to 2 +.

Advantageous effects of the invention

The invention provides a chimeric antigen receptor targeting Claudin18.2, immune effector cells expressing the chimeric antigen receptor only generate specific killing on Claudin18.2 target tumor cells, have no killing effect on Claudin18.1 and tumor cells not expressing Claudin18.2, and have good specificity. Meanwhile, in vivo experiments show that the immune effector cells expressing the chimeric antigen receptor can obviously inhibit the growth of tumors under low dose, and the animal tolerance is good, which indicates that the chimeric antigen receptor of the invention can bring potential benefits for solid tumor patients. Furthermore, the invention provides an immune cell co-expressing the chimeric antigen receptor of the invention and an additional biologically active molecule comprising a nucleic acid expressing the chimeric antigen receptor targeting claudin18.2 and an additional biologically active molecule nucleic acid linked downstream of the nucleic acid encoding the chimeric antigen receptor targeting claudin18.2 via a lytic peptide nucleic acid. The biologically active molecule is expressed and then cleaved at the cleavage peptide, secreted outside the immune cell, or expressed chimerically in the immune cell membrane.

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

Drawings

FIGS. 1-4 show the results of the killing activity of CAR-T cells (1E9.2-T, 2C6.9-T, blank T) against target cells.

FIGS. 5-6 show the results of measurements of IL-2 secretion levels following activation of CAR-T cells (1E9.2-T, 2C6.9-T, blank T).

FIGS. 7-8 show the results of measurements of IFN- γ secretion levels following activation of CAR-T cells (1E9.2-T, 2C6.9-T, blank T).

FIG. 9 shows the results of the killing activity of CAR-T cells (2C6.9-T, 2C6.9-T-PD-1, 2C6.9-T-mIL-15) against target cells.

FIG. 10 shows the results of measuring the amount of PD-1 secreted from 2C6.9-T cells and 2C6.9-T-PD-1 cells after stimulation of target cells.

FIG. 11 shows the tumor volume change curves of tumor-bearing B-NDG mice treated with 2C6.9-T cells and targeted T cells.

FIG. 12 shows the body weight change curves of tumor-bearing B-NDG mice treated with 2C6.9-T cells and target T cells.

FIG. 13 shows the tumor volume change curves of tumor-bearing NOD/SCID mice treated with 2C6.9-T cells, naive T cells.

FIG. 14 shows the body weight change curves of tumor-bearing NOD/SCID mice treated with 2C6.9-T cells, naive T cells.

FIG. 15 shows tissue sections of target organs of tumor-bearing NOD/SCID mice treated with 2C6.9-T cells, naive T cells.

Sequence information

The sequence information to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

Detailed Description

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

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2nd edition, cold spring harbor laboratory press, 1989, and f.m. ausubel et al, eds. molecular biology laboratory guidelines, 3 rd edition, John Wiley & Sons, inc., 1995. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Example 1: preparation of human Claudin18.2 scFv antibody targeting

2 murine anti-CLDN 18.2 monoclonal antibodies 1E9.2 and 2C6.9 are obtained by screening through a hybridoma technology. The above murine monoclonal antibody was analyzed to obtain the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VH, VL (see sequence information Table). Then humanizing the above method to finally obtain a humanized antibody 1E9.2-HZ11 (whose VH and VL are shown in SEQ ID NOS: 13 and 14, respectively); 2C6.9-HZ21 (VH and VL are shown in SEQ ID NO:16 and 17, respectively). The sequence of CDR-H2 of 2C6.9-HZ21 was further engineered into the sequence shown in SEQ ID NOs:37 or 53.

The VH and VL of the above humanized antibody were linked by linker (SEQ ID NO:44) to obtain scFv, and the sequence information of each scFv is shown in the following table.

Table 2: structure of scFv

Example 2: construction and preparation of Chimeric Antigen Receptor (CAR) lentiviral expression vector

1) Construction of lentiviral plasmids:

based on the scFv sequences in the above examples and the scFv sequence of a target T (the target T is derived from the literature J Natl Cancer Inst.2019Apr 1; 111(4): 409) and 418, which are hu8E5 in the literature), a CAR lentiviral expression vector was further constructed. The intracellular domain of CD137 and ITAM region of CD3Zeta were used as activation signals, and fused with the above scFv, together with a signal peptide, CD8 hinge region, CD8 transmembrane region, and a chimeric antigen receptor expression vector was constructed using primer F (SEQ ID NO:54) and primer R (SEQ ID NO:55), and the constructed chimeric antigen receptor structure was as shown in Table 3 below.

Table 3: structure of chimeric antigen receptor

2) And (3) packaging the virus:

the CAR lentiviral plasmid and transfection reagent mixture constructed above was added drop-wise to 293t (atcc) cells and mixed well. Placing the culture dish at 37 ℃ and 5% CO₂After the incubator continuously cultures for 48 hours, culture medium supernatant containing viruses in the culture dish is collected, filtered, centrifuged, and the viruses are resuspended in PBS buffer and stored at-80 ℃.

Example 3: construction and preparation of co-expression CAR lentivirus expression vector

On the basis of the CAR structure of example 2, sequences encoding other bioactive molecules (e.g., PD-1scFv whose nucleotide sequence is shown in SEQ ID NO: 72 and upstream P2A self-cleaving peptide nucleotide sequence is shown in SEQ ID NO: 69; or mIL-15 whose nucleotide sequence is shown in SEQ ID NO:73 and upstream P2A self-cleaving peptide nucleotide sequence is shown in SEQ ID NO: 70) were linked by a sequence encoding P2A self-cleaving peptide (amino acid sequence is SEQ ID NO:50) to obtain a co-expressed CAR. The lentiviral expression vector co-expressing the CAR was prepared by the same method as in example 2. When the co-expressed CAR is expressed in cells, the co-expressed CAR is cleaved from PGP in the P2A sequence, so that the linked bioactive molecule is secreted out of the CAR-T cell or is expressed on the CAR-T cell membrane in a chimeric way, and the anti-tumor effect is exerted synergistically. For example, since CLDN18.2 CAR-T specifically binds to tumor cells, 2C6.9-T-PD-1CAR-T reduces or eliminates immunosuppression by secreting anti-PD-1 antibodies after activation, increasing the anti-tumor effect. 2C6.9-T-mIL-15 after CAR-T activation, CAR-T cells express membrane-chimeric IL-15, which increases and makes more durable the anti-tumor effect by stimulating secretion of some cytokines. The structure of the co-expressed CAR and the encoding nucleic acid sequence are shown in the table below.

Table 4: structure of co-expressed CAR

Example 4: preparation of CAR-T cells

1) Primary T cell separation: human PBMC cells were incubated with Dynabeads (thermo) at room temperature, magnetic polarity, T cells resuspended in X-vivo 15 medium and supplemented with 10% FBS, 300U/mL IL-2,5ng/mL IL-15 and 10ng/mL IL-7, (IL-2, IL-15, IL-7 from near-shore protein technologies, Inc.) at 37 ℃, 5% CO₂Is stored in the incubator.

2) Activation of T cells:

adjusting cell density to 1X 10⁶cells/mL, six-well plates were added with cytokine and antibody complexes (300U/mL IL-2, 10ng/mL IL-7, 5ng/mL IL-15, 500ng/mL Anti-CD3(OKT3), 2. mu.g/mL Anti-CD28) and incubated continuously for 48 hours.

3) Viral infection:

(1) the required amount of virus was calculated according to MOI of 20. The calculation formula is as follows: required amount of virus (mL) ═ number of cells (MOI)/viral titer.

(2) The virus was quickly rewarmed to 37 ℃. The amount of the virus obtained by the above calculation was added to a six-well plate, polybrene was added thereto to a final concentration of 6. mu.g/mL, and the mixture was thoroughly mixed and centrifuged.

(3) After centrifugation was complete, 5% CO at 37 deg.C₂The culture is continued in the incubator of (1) for standby.

CAR-T cells expressing CARs described in example 2 (1E9.2-T, 2C6.9-T) and CAR-T cells expressing co-expressed CARs described in example 3 were obtained by the above method, respectively.

Experimental example 5: positive rate detection of CAR-T cells

In the lentivirus expression plasmid, the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the truncated EGFR are expressed under the drive of the same promoter, and since the primary T cell membrane does not express the EGFR on the surface, the lentivirus transfected T cells can be labeled with an antibody recognizing the EGFR and assayed by flow-based assay, thereby reflecting the expression level of the CAR on the surface of the T cells. The CAR-positive rate of CAR-T cells obtained in example 4 was determined by the above method, and FACS detection results are shown in the following table. The results show that the CAR positive rate of all CAR-T cells is more than 50%, and the CAR is successfully expressed after the effector cells are transfected by lentiviruses, and the chimeric antigen receptor T cells expressing Claudin18.2-CAR are successfully constructed.

Table 5: positive rate test result of CAR

Chimeric antigen receptors	Positive rate of CAR
		1E9.2-T	50.31％
2C6.9-T	57.92％

Experimental example 6: evaluation of killing Activity of CAR-T cells against target cells

6.1 construction of human Claudin18.2 and human Claudin18.1 overexpression cell lines

A complete coding sequence of human Claudin18.2 (gene number: NM-001002026.2, synthesized by Nanjing King-Story Biotechnology Co., Ltd.) and a complete coding sequence of human Claudin18.1 (gene number: NM-016369.3, synthesized by Nanjing King-Story Biotechnology Co., Ltd.) were cloned into a lentiviral vector pLVX-IRES-puro, and viruses were prepared by a lentiviral packaging system as described in the literature (Mohammadi Z et., Mol Biotechnol.2015Sep; 57(9):793- & gt 800.), and HEK293T and L929 cells were infected after the viruses were obtained, respectively, and HEK293T-Claudin 18.1 (293T-18.1 for short), HEK293T-Claudin 18.2 (293T-18.2 for short), L929-Claudin 18.2 (L929-Claudin 18.2 for short) and stable cell lines for subsequent experiments were obtained by puromycin screening and monoclonal selection.

6.2 evaluation of the killing Activity of 1E9.2-T and 2C6.9-T cells against target cells

The killing activity of the CAR-T cells is evaluated by measuring the cracking ability of the 1E9.2-T and 2C6.9-T cells to target cells and the ability of the cells to release cytokines, and the specific steps are as follows:

1) cleavage activity: target cells L929-18.2 and L929 in E-Plate, RPMI1640+ 10% FBS, 5% CO₂Culturing in 37 deg.C incubator until cell index (live cell interacts with microelectrode in detection plate hole to generate electrical impedance change, converting these signals into specific parameter-cell index by standard formula, the cell index well measures cell state-growth, diffusion, shape change, death, stress, etc., the cell index is a dimensionless parameter, and the impedance value measured by RTCA is calculated by standard formula) reaches 1 (cell index is 1, cell state is best), taking 1E9.2-T, 2C6.9-T and untransfected CAR control T cell (blank T) as effector cell, and then E/T (effector cell) according to 1:1 and 2.5:1Target cells) ratio, real-time cell analysis, real-time measurement of the lysis results of effector/target cell co-incubation (cell index is generated autonomously by the instrument, in response to changes in cell number and cell adhesion status, increased cell number, and increased cell index. And the change of the cell adherence state can also influence the change of the cell index, such as the cell index is increased after the cell is adhered to the wall, the cell index is decreased after the adherent cell is killed, the cell index is cracked, and the cell index is also decreased).

2) Cytokine release assay: 1E9.2-T, 2C6.9-T, blank T cells not transfected with CAR were used as effector cells, then the cells were mixed according to 10:1, 5:1 and 1: 1E/T (Effector/target cells, results only representative data) ratio to 293T-18.2 and 293T-18.1 in 96-well plates, DMEM + 10% FBS, 5% CO₂Incubated overnight in a 37 ℃ incubator. After the culture was completed, the secretion levels of IL-2 and IFN-. gamma.were measured using ELISA kit, and the OD 450nm value was read using a microplate reader.

The processed data were plotted using GraphPad 6.0.

5) And (4) analyzing results:

the results of the detection of the killing activity of 1E9.2-T and 2C6.9-T are shown in FIGS. 1-4. The results of the IL-2 secretion level measurements are shown in FIGS. 5-6. The results of IFN- γ secretion level measurements are shown in FIGS. 7-8.

The above results show that 1E9.2-T, 2C6.9-T can activate primary T cells and mediate killing of T cells against CLDN18.2 expressing tumor cells with high efficiency (fig. 1, 2) and cause a significant increase in cytokine secretion (fig. 5, 7). In particular, 1E9.2-T and 2C6.9-T did not kill wild type L929 cells (FIGS. 3 and 4) and hardly induced 293T-18.1 tumor cells to release IL-2 or IFN-gamma (FIGS. 6 and 8), and the above results indicate that 1E9.2-T and 2C6.9-T can only specifically lyse Claudin 18.2-expressing tumor cells and cause significant increase in IL-2 and IFN-gamma secretion, cannot specifically lyse Claudin 18.1-expressing tumor cells, and cannot cause increase in IL-2 or IFN-gamma secretion without off-target phenomenon.

6.3 evaluation of the killing Activity of Co-expressed CAR-T on target cells

The killing activity of the CAR-T cells is evaluated by measuring the cracking capacity of the co-expressed CAR-T cells on target cells and the PD-1 release capacity, and the specific steps are as follows:

blank T, 2C6.9-T and 2C6.9-T-mIL-15,2C6.9-T-PD-1 and target cells NUGC4-luciferase (human luciferase-expressing stable cell line NUGC4-luciferase prepared by the method of example 6.1) were added to white 96-well plates at an E/T (effector cells/target cells) ratio of 3:1, 1:1, 0.5:1, 0.1:1, 5% CO₂And (3) co-incubating and culturing for 18-24 h in an incubator at 37 ℃, adding 20 mu l of fluorescence detection reagent after the culture is finished, and detecting the fluorescence intensity by using an enzyme-labeling instrument.

4) ELISA detection of PD-1 secretion: the detection method is similar to conventional ELISA. 5 days after viral transduction, 3.3X 10 cells were harvested⁶Each CAR-T/100 μ l was set to 2 duplicate wells, added to a 96-well plate and cultured for 24 hours, and the supernatant was collected and frozen at-80 ℃. The specific detection method comprises the following steps: 1 mu/ml gPD-1-hFcH (self-made), and coating overnight at 4 ℃; washing BSA PBS, blocking, adding a PD-1scFv (home-made) standard (ng/ml) diluted in a gradient manner, and incubating for 2-6 hours at 37 ℃; washed 3 times with 300 μ l PBST; biotin-labeled Protein L was diluted with PBS (2% BSA), 100. mu.l was added to the corresponding wells, and incubated at 37 ℃ for 1.5 hours; washed 5 times with 300 μ l PBST; add 100 u l HRP labeled streptomycin, 37 degrees C were incubated for 30 minutes, with 300u l PBST cleaning; adding 100 mul of TMB color developing solution for color development; after adding the stop solution, reading by an enzyme-labeling instrument at 450 nm.

The processed data were plotted using GraphPad 6.0.

5) And (4) analyzing results:

as shown in FIG. 9, CAR-T was able to lyse tumor cells efficiently at different E/T ratios. After 2C6.9-T-PD-1 is cultured for 24h, the secretion capacity of PD-1 is obviously higher than that of the traditional 2C6.9-T, as shown in figure 10.

Experimental example 7: CAR-T cell in vivo efficacy evaluation

A stable cell line N87-Claudin 18.2 (abbreviated as N87-18.2) expressing human Claudin18.2 was prepared by the method of example 6.1.

Animal grouping: tumor-bearing N87-18.2 cell (5X 10)⁶/) successful 48B-NDG mice, 6-8 week old female mice, randomized 4 groups, group 1, PBS, group 2, blank T cells (UTD), group 3CAR-T (2C6.9-T) was given, and group 4 was given a targeting T.

And (3) treatment: 24h before administration, 100mg/kg cyclophosphamide is injected into abdominal cavity, CAR-T is infused with day1, and the injection is injected into tail vein at a dose of 5 × 10⁵Mice tumor volume and body weight were observed and measured periodically after dosing.

The tumor diameter was measured with a vernier caliper and the tumor volume was calculated as follows: v is 0.5a × b²Wherein a and b represent the major and minor diameters of the tumor, respectively. Animal mortality was recorded daily.

Tumor growth inhibition ratio TGI (%) for CAR-T tumor inhibition efficacy was calculated using the following formula:

TGI(％)＝[1-(V_Tend-V_Tbeginning)/(V_Cend-V_CBeginning)]*100％

Wherein V_TAnd (3) finally: mean tumor volume at the end of treatment group experiment

V_TFirstly: mean tumor volume at the beginning of treatment group dosing

V_CAnd (3) finally: mean tumor volume at the end of the negative control experiment

V_CFirstly: mean tumor volume at the beginning of negative control group administration

And (4) conclusion: as shown in figure 11, CAR-T showed inhibition of growth of N87-18.2 in mice from day 14 onwards, with the inhibition continuing until the termination of observation and being dose dependent, and CAR-T significantly inhibited growth of N87-18.2 in mice from day 20, with TGI for 2C6.9-T and for target T being 108.16% and 93.12%, respectively (23% for the blank T group). Animals in the 2C6.9-T group were well tolerated for treatment (fig. 12).

Experimental example 8: evaluation of in vivo efficacy and safety of CAR-T cells

Animal grouping: tumor-bearing N87-18.2 cell (5X 10)⁶/one) successful 16 NOD/SCID mice, 6-8 week old female mice, until the tumor grows to 80-100 mm³When the mice with too small or too large tumor volume are removed, the mice are randomly divided into 2 groups, and the group 1Blank T cells (UTD) were administered, and CAR-T (2C6.9-T) was administered in group 2.

And (3) treatment: 24h before administration, 100mg/kg cyclophosphamide is injected into abdominal cavity, CAR-T is infused with day0, and the injection is injected into tail vein at a dose of 1 × 10⁷Mice tumor volume and body weight were observed and measured periodically after dosing. After the test, gross anatomical observation, organ weight analysis and histopathological examination were performed.

TGI(％)＝[1-(V_Tend-V_Tbeginning)/(V_Cend-V_CBeginning)]*100％

V_TFirstly: mean tumor volume at the beginning of treatment group dosing

And (4) conclusion: as shown in FIG. 13, CAR-T showed inhibitory effect on the growth of N87-18.2 in mice from day 7 onwards, and animals were dissected on day 24. The TGI of 2C6.9-T was 68% (0% for blank T group). Animals in the 2C6.9-T group were well tolerated for treatment (fig. 14). No changes that could be associated with the 2C6.9-T candidate were detected by analyzing the data from gross anatomical observations, biochemical index measurements of mouse blood, gravimetric organ analysis, and histopathological examination, and thus, as described above, this safety evaluation showed 1X 10 intravenous injections⁷No obvious toxicological target organs were found for 2C6.9-T (FIG. 15).

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

SEQUENCE LISTING

<110> Sichuan Kolun Bordete biomedical corporation

<120> chimeric antigen receptor targeting CLDN18.2, composition and use thereof

<130> IDC210085

<150> CN202010500359.0

<151> 2020-06-04

<160> 73

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 1E9.2 CDR-H1

<400> 1

Gly Phe Ser Phe Ser Asn Ser Ala

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 1E9.2 CDR-H2

<400> 2

Ile Ser Ser Gly Asp Ser Tyr Thr

1 5

<210> 3

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 1E9.2 CDR-H3

<400> 3

Ala Arg Gln Gly Tyr Gly Asn Ala Leu Asp Tyr

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 1E9.2 CDR-L1

<400> 4

Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr

1 5 10

<210> 5

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 1E9.2 CDR-L2

<400> 5

Trp Ser Ser

1

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 1E9.2 CDR-L3

<400> 6

Gln Asn Asp Tyr Tyr Tyr Pro Leu Thr

1 5

<210> 7

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 2C6.9 CDR-H1

<400> 7

Gly Phe Ser Leu Thr Arg Tyr Gly

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 2C6.9 CDR-H2-1

<400> 8

Ile Trp Gly Asp Gly Asn Thr

1 5

<210> 9

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 2C6.9 CDR-H3

<400> 9

Ala Arg Val Asn Phe Gly Asn Ala Leu Asp Tyr

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 2C6.9 CDR-L1

<400> 10

Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr

1 5 10

<210> 11

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 2C6.9 CDR-L2

<400> 11

Trp Ala Ser

1

<210> 12

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 2C6.9 CDR-L3

<400> 12

Gln Asn Asp Phe Ile Phe Pro Leu Thr

1 5

<210> 13

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> humanized antibody 1E9.2hz11 heavy chain variable region

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Ser

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Ser Gly Asp Ser Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Tyr Gly Asn Ala Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 14

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> humanized antibody 1E9.2hz11 light chain variable region

<400> 14

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 Ser 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 Phe Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Tyr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 15

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 1E9.2 CDR-H1

<400> 15

Gly Phe Ser Phe Ser Asn Ser Ala Met Ser

1 5 10

<210> 16

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> humanized antibody 2C6.9hz21 heavy chain variable region

<400> 16

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Glu Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Asn Phe Gly Asn Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 17

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> humanized antibody 2C6.9hz21 light chain variable region

<400> 17

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 Asp 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 Phe Ile Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 18

<211> 738

<212> DNA

<213> Artificial sequence

<220>

<223> 1E9.2-HZ11-scfv nucleotides

<400> 18

gacatcgtga tgacccagag ccccgactcc ctggccgtga gcctgggcga gagagccacc 60

atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctgacc 120

tggtatcagc agaagcccgg ccagccccct aagctgctga tctactggag cagcaccaga 180

gagagcggcg tgcctgacag gttcagcggc tccggcagcg gcaccgactt caccctgacc 240

atcagcagcc tgcaggccga ggacttcgcc gtgtactact gccagaacga ctactactac 300

cccctgacct tcggcggcgg caccaaggtg gagatcaagg gcggcggcgg ctccggcggc 360

ggcggaagcg gcggcggcgg cagcgaggtg cagctggtgg agagcggcgg cggcctggtg 420

aagcccggcg gcagcctgcg gctgagctgc gccgccagcg gcttcagctt cagcaacagc 480

gccatgagct gggtgcggca ggcccccggc aagggcctgg agtgggtgag caccatcagc 540

agcggcgact cctacaccta ctacgccgac agcgtgaagg gcagattcac catcagcaga 600

gacaacgcca agaactccct gtacctgcag atgaacagcc tgagggccga ggacaccgcc 660

gtgtactact gcgccagaca gggctacggc aacgccctgg actactgggg ccagggcacc 720

ctggtgaccg tgagcagc 738

<210> 19

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<223> 1E9.2-HZ11-scfv amino acid

<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 Ser 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 Phe Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Tyr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

130 135 140

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Ser

145 150 155 160

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

165 170 175

Ser Thr Ile Ser Ser Gly Asp Ser Tyr Thr Tyr Tyr Ala Asp Ser Val

180 185 190

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

195 200 205

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Gln Gly Tyr Gly Asn Ala Leu Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ser

245

<210> 20

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 1E9.2 CDR-H2

<400> 20

Thr Ile Ser Ser Gly Asp Ser Tyr Thr Tyr

1 5 10

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 1E9.2 CDR-H3

<400> 21

Gln Gly Tyr Gly Asn Ala Leu Asp Tyr

1 5

<210> 22

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> 2C6.9-HZ21-scfv nucleotides

<400> 22

gacatcgtga tgacccagtc ccccgacagc ctggccgtga gcctgggcga gagagccaca 60

atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctgacc 120

tggtatcagc agaagcccgg ccagccccct aagctgctga tctactgggc ctccaccaga 180

gactccggcg tgcccgacag attcagcggc agcggcagcg gcaccgactt caccctgacc 240

atcagcagcc tgcaggccga ggacgtggcc gtgtactact gccagaatga ctttatcttc 300

cccctgacct tcggcggcgg caccaaggtg gagatcaagg gcgggggcgg cagcggcggc 360

ggcggcagcg gcggcggcgg cagccaggtg cagctgcagg agagcggccc cggcctggtg 420

aagcccagcg agaccctgag cctgacctgc accgtgtccg gcttcagcct gaccagatac 480

ggcgtgagct ggatcagaca gcctcccggc aagggcctgg agtggatcgg cgtgatctgg 540

ggcgagggca acaccaacta caaccccagc ctgaagagca gagtgaccat cagcaaggac 600

agcagcaaga gccaggtgtc cctgaagctg agcagcgtga ccgccgccga caccgccgtg 660

tactactgcg ccagagtgaa cttcggcaac gccctggact actggggcca gggcaccctg 720

gtgaccgtga gcagc 735

<210> 23

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> 2C6.9-HZ21-scfv amino acids

<400> 23

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 Asp 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 Phe Ile Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

130 135 140

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr

145 150 155 160

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

165 170 175

Gly Val Ile Trp Gly Glu Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys

180 185 190

Ser Arg Val Thr Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Ser Leu

195 200 205

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Arg Val Asn Phe Gly Asn Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser

245

<210> 24

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<223> 1E9.2-T nucleotides

<400> 24

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

cccgacatcg tgatgaccca gagccccgac tccctggccg tgagcctggg cgagagagcc 120

accatcaact gcaagagcag ccagagcctg ctgaacagcg gcaaccagaa gaactacctg 180

acctggtatc agcagaagcc cggccagccc cctaagctgc tgatctactg gagcagcacc 240

agagagagcg gcgtgcctga caggttcagc ggctccggca gcggcaccga cttcaccctg 300

accatcagca gcctgcaggc cgaggacttc gccgtgtact actgccagaa cgactactac 360

taccccctga ccttcggcgg cggcaccaag gtggagatca agggcggcgg cggctccggc 420

ggcggcggaa gcggcggcgg cggcagcgag gtgcagctgg tggagagcgg cggcggcctg 480

gtgaagcccg gcggcagcct gcggctgagc tgcgccgcca gcggcttcag cttcagcaac 540

agcgccatga gctgggtgcg gcaggccccc ggcaagggcc tggagtgggt gagcaccatc 600

agcagcggcg actcctacac ctactacgcc gacagcgtga agggcagatt caccatcagc 660

agagacaacg ccaagaactc cctgtacctg cagatgaaca gcctgagggc cgaggacacc 720

gccgtgtact actgcgccag acagggctac ggcaacgccc tggactactg gggccagggc 780

accctggtga ccgtgagcag caccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaagcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatttgggc ccctctggct 960

ggtacttgcg gggtcctgct gctttcactc gtgatcactc tttactgtaa gcgcggtcgg 1020

aagaagctgc tgtacatctt taagcaaccc ttcatgaggc ctgtgcagac tactcaagag 1080

gaggacggct gttcatgccg gttcccagag gaggaggaag gcggctgcga actgcgcgtg 1140

aaattcagcc gcagcgcaga tgctccagcc tacaagcagg ggcagaacca gctctacaac 1200

gaactcaatc ttggtcggag agaggagtac gacgtgctgg acaagcggag aggacgggac 1260

ccagaaatgg gcgggaagcc gcgcagaaag aatccccaag agggcctgta caacgagctc 1320

caaaaggata agatggcaga agcctatagc gagattggta tgaaagggga acgcagaaga 1380

ggcaaaggcc acgacggact gtaccaggga ctcagcaccg ccaccaagga cacctatgac 1440

gctcttcaca tgcaggccct gccgcctcgg 1470

<210> 25

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<223> 1E9.2-T amino acid

<400> 25

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 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu

20 25 30

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln

35 40 45

Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln

50 55 60

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ser Ser Thr

65 70 75 80

Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Phe Ala Val

100 105 110

Tyr Tyr Cys Gln Asn Asp Tyr Tyr Tyr Pro Leu Thr Phe Gly Gly Gly

115 120 125

Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

145 150 155 160

Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

165 170 175

Ser Phe Ser Asn Ser Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

180 185 190

Gly Leu Glu Trp Val Ser Thr Ile Ser Ser Gly Asp Ser Tyr Thr Tyr

195 200 205

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

210 215 220

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg Gln Gly Tyr Gly Asn Ala Leu Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

305 310 315 320

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

325 330 335

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

340 345 350

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

355 360 365

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 26

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 1E9.2 CDR-L1

<400> 26

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr

<210> 27

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 1E9.2 CDR-L2

<400> 27

Trp Ser Ser Thr Arg Glu Ser

1 5

<210> 28

<211> 1467

<212> DNA

<213> Artificial sequence

<220>

<223> 2C6.9-T nucleotides

<400> 28

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

cccgacatcg tgatgaccca gtcccccgac agcctggccg tgagcctggg cgagagagcc 120

acaatcaact gcaagagcag ccagagcctg ctgaacagcg gcaaccagaa gaactacctg 180

acctggtatc agcagaagcc cggccagccc cctaagctgc tgatctactg ggcctccacc 240

agagactccg gcgtgcccga cagattcagc ggcagcggca gcggcaccga cttcaccctg 300

accatcagca gcctgcaggc cgaggacgtg gccgtgtact actgccagaa tgactttatc 360

ttccccctga ccttcggcgg cggcaccaag gtggagatca agggcggggg cggcagcggc 420

ggcggcggca gcggcggcgg cggcagccag gtgcagctgc aggagagcgg ccccggcctg 480

gtgaagccca gcgagaccct gagcctgacc tgcaccgtgt ccggcttcag cctgaccaga 540

tacggcgtga gctggatcag acagcctccc ggcaagggcc tggagtggat cggcgtgatc 600

tggggcgagg gcaacaccaa ctacaacccc agcctgaaga gcagagtgac catcagcaag 660

gacagcagca agagccaggt gtccctgaag ctgagcagcg tgaccgccgc cgacaccgcc 720

gtgtactact gcgccagagt gaacttcggc aacgccctgg actactgggg ccagggcacc 780

ctggtgaccg tgagcagcac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaagcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tttgggcccc tctggctggt 960

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 1020

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1140

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1200

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1260

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1320

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1380

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1440

cttcacatgc aggccctgcc gcctcgg 1467

<210> 29

<211> 489

<212> PRT

<213> Artificial sequence

<220>

<223> 2C6.9-T amino acids

<400> 29

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 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu

20 25 30

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln

35 40 45

Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln

50 55 60

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr

65 70 75 80

Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

100 105 110

Tyr Tyr Cys Gln Asn Asp Phe Ile Phe Pro Leu Thr Phe Gly Gly Gly

115 120 125

Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

145 150 155 160

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe

165 170 175

Ser Leu Thr Arg Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

180 185 190

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Glu Gly Asn Thr Asn Tyr

195 200 205

Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Ser Ser Lys

210 215 220

Ser Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

225 230 235 240

Val Tyr Tyr Cys Ala Arg Val Asn Phe Gly Asn Ala Leu Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro

260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

275 280 285

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

290 295 300

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

305 310 315 320

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

325 330 335

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

340 345 350

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

355 360 365

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg

485

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 1E9.2 CDR-L3

<400> 30

Gln Asn Asp Tyr Tyr Tyr Pro Leu Thr

1 5

<210> 31

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 2C6.9 CDR-H1

<400> 31

Gly Phe Ser Leu Thr Arg Tyr Gly Val Ser

1 5 10

<210> 32

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 2C6.9 CDR-H2-1

<400> 32

Val Ile Trp Gly Asp Gly Asn Thr Asn

1 5

<210> 33

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 2C6.9 CDR-H3

<400> 33

Val Asn Phe Gly Asn Ala Leu Asp Tyr

1 5

<210> 34

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 2C6.9 CDR-L1

<400> 34

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr

<210> 35

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 2C6.9 CDR-L2

<400> 35

Trp Ala Ser Thr Arg Asp Ser

1 5

<210> 36

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 2C6.9 CDR-L3

<400> 36

Gln Asn Asp Phe Ile Phe Pro Leu Thr

1 5

<210> 37

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> AbM 2C6.9 CDR-H2-2

<400> 37

Val Ile Trp Gly Glu Gly Asn Thr Asn

1 5

<210> 38

<211> 2319

<212> DNA

<213> Artificial sequence

<220>

<223> 2C6.9-T-PD-1 nucleotides

<400> 38

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

cccgacatcg tgatgaccca gtcccccgac agcctggccg tgagcctggg cgagagagcc 120

acaatcaact gcaagagcag ccagagcctg ctgaacagcg gcaaccagaa gaactacctg 180

acctggtatc agcagaagcc cggccagccc cctaagctgc tgatctactg ggcctccacc 240

agagactccg gcgtgcccga cagattcagc ggcagcggca gcggcaccga cttcaccctg 300

accatcagca gcctgcaggc cgaggacgtg gccgtgtact actgccagaa tgactttatc 360

ttccccctga ccttcggcgg cggcaccaag gtggagatca agggcggggg cggcagcggc 420

ggcggcggca gcggcggcgg cggcagccag gtgcagctgc aggagagcgg ccccggcctg 480

gtgaagccca gcgagaccct gagcctgacc tgcaccgtgt ccggcttcag cctgaccaga 540

tacggcgtga gctggatcag acagcctccc ggcaagggcc tggagtggat cggcgtgatc 600

tggggcgagg gcaacaccaa ctacaacccc agcctgaaga gcagagtgac catcagcaag 660

gacagcagca agagccaggt gtccctgaag ctgagcagcg tgaccgccgc cgacaccgcc 720

gtgtactact gcgccagagt gaacttcggc aacgccctgg actactgggg ccagggcacc 780

ctggtgaccg tgagcagcac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaagcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tttgggcccc tctggctggt 960

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 1020

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1140

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1200

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1260

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1320

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1380

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1440

cttcacatgc aggccctgcc gcctcggcgg gccaagagag gatctggagc cacaaatttt 1500

agcctgctga aacaggccgg agatgtggag gaaaaccctg gccctatgta taggatgcag 1560

ctgctgagct gtattgccct gagccttgca ctggtgacca atagcgaggt gcagctggtg 1620

cagagcggcg ccgaggtgaa gaagcctggc gccagcgtga aggtgagctg caaggcctcc 1680

ggctacacct tcaccgacta ctacatgaac tgggtgcgag aggcccccgg ccagggcctg 1740

gagtggatgg gcgagatcaa tcccaagtac ggcgatacca cctacaatca gaagttcaag 1800

gaccgggcca ccctgaccgt ggataagtct acatctacag cctatatgga gctgagcagc 1860

ctgagatccg atgataccgc agtgtattat tgtgccaaag gaattagact gtttgactct 1920

tggggccagg gaaccctagt gaccgtaagc agcggaggag gaggcagcgg aggaggcgga 1980

tctggcggcg gcggctccga catccagctg acccagagcc ccagcagcct gagcgccagc 2040

gtgggcgaca gggtgaccat cacctgccgg ccctccggca acgtgcacaa ttacttcgcc 2100

tggtaccagc agaagcccgg caagagccct aagctgctgg tgtataatgc caagacactg 2160

gctgaaggcg tgcctagtag gtttagcggc agcggaagcg gcactgatta tacactgaca 2220

atttctagcc tgcagcctga ggattttgct acatattatt gtcatcatta ctatacagcc 2280

ccttttacat ttggaggcgg aacaaaggtg gagattaag 2319

<210> 39

<211> 773

<212> PRT

<213> Artificial sequence

<220>

<223> 2C6.9-T-PD-1 amino acid

<400> 39

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 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu

20 25 30

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln

35 40 45

Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln

50 55 60

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr

65 70 75 80

Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

100 105 110

Tyr Tyr Cys Gln Asn Asp Phe Ile Phe Pro Leu Thr Phe Gly Gly Gly

115 120 125

Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

145 150 155 160

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe

165 170 175

Ser Leu Thr Arg Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

180 185 190

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Glu Gly Asn Thr Asn Tyr

195 200 205

Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Ser Ser Lys

210 215 220

Ser Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

225 230 235 240

Val Tyr Tyr Cys Ala Arg Val Asn Phe Gly Asn Ala Leu Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro

260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

275 280 285

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

290 295 300

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

305 310 315 320

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

325 330 335

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

340 345 350

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

355 360 365

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala Lys Arg Gly Ser Gly

485 490 495

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

500 505 510

Pro Gly Pro Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser

515 520 525

Leu Ala Leu Val Thr Asn Ser Glu Val Gln Leu Val Gln Ser Gly Ala

530 535 540

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

545 550 555 560

Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Asn Trp Val Arg Glu Ala Pro

565 570 575

Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Asn Pro Lys Tyr Gly Asp

580 585 590

Thr Thr Tyr Asn Gln Lys Phe Lys Asp Arg Ala Thr Leu Thr Val Asp

595 600 605

Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp

610 615 620

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Ile Arg Leu Phe Asp Ser

625 630 635 640

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

645 650 655

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln

660 665 670

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

675 680 685

Cys Arg Pro Ser Gly Asn Val His Asn Tyr Phe Ala Trp Tyr Gln Gln

690 695 700

Lys Pro Gly Lys Ser Pro Lys Leu Leu Val Tyr Asn Ala Lys Thr Leu

705 710 715 720

Ala Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

725 730 735

Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

740 745 750

Tyr Cys His His Tyr Tyr Thr Ala Pro Phe Thr Phe Gly Gly Gly Thr

755 760 765

Lys Val Glu Ile Lys

770

<210> 40

<211> 2730

<212> DNA

<213> Artificial sequence

<220>

<223> 2C6.9-T-mIL-15 nucleotides

<400> 40

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

cccgacatcg tgatgaccca gtcccccgac agcctggccg tgagcctggg cgagagagcc 120

acaatcaact gcaagagcag ccagagcctg ctgaacagcg gcaaccagaa gaactacctg 180

acctggtatc agcagaagcc cggccagccc cctaagctgc tgatctactg ggcctccacc 240

agagactccg gcgtgcccga cagattcagc ggcagcggca gcggcaccga cttcaccctg 300

accatcagca gcctgcaggc cgaggacgtg gccgtgtact actgccagaa tgactttatc 360

ttccccctga ccttcggcgg cggcaccaag gtggagatca agggcggggg cggcagcggc 420

ggcggcggca gcggcggcgg cggcagccag gtgcagctgc aggagagcgg ccccggcctg 480

gtgaagccca gcgagaccct gagcctgacc tgcaccgtgt ccggcttcag cctgaccaga 540

tacggcgtga gctggatcag acagcctccc ggcaagggcc tggagtggat cggcgtgatc 600

tggggcgagg gcaacaccaa ctacaacccc agcctgaaga gcagagtgac catcagcaag 660

gacagcagca agagccaggt gtccctgaag ctgagcagcg tgaccgccgc cgacaccgcc 720

gtgtactact gcgccagagt gaacttcggc aacgccctgg actactgggg ccagggcacc 780

ctggtgaccg tgagcagcac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaagcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tttgggcccc tctggctggt 960

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 1020

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1140

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1200

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1260

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1320

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1380

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1440

cttcacatgc aggccctgcc gcctcggcga gctaaacgag gctcaggcgc gacgaacttt 1500

agtttgctga agcaagctgg ggatgtagag gaaaatccgg gtcccatgga ttggacttgg 1560

attttgttcc tcgttgccgc agcgactcgc gtccatagta actgggtgaa tgtaataagt 1620

gatttgaaaa aaattgaaga tcttattcaa tctatgcata ttgatgctac tttatatacg 1680

gaaagtgatg ttcaccccag ttgcaaagta acagcaatga agtgctttct cttggagtta 1740

caagttattt cacttgagtc cggagatgca agtattcatg atacagtaga aaatctgatc 1800

atcctagcaa acaacagttt gtcttctaat gggaatgtaa cagaatctgg atgcaaagaa 1860

tgtgaggaac tggaggaaaa aaatattaaa gaatttttgc agagttttgt acatattgtc 1920

caaatgttca tcaacacttc ttccgggggc ggcagtggag gtggcggtag cggcgggggt 1980

ggctctggtg gaggcggctc tgggggcgga agtctgcaga tcacgtgccc tccccccatg 2040

tccgtggaac acgcagacat ctgggtcaag agctacagct tgtactccag ggagcggtac 2100

atttgtaact ctggtttcaa gcgtaaagcc ggcacgtcca gcctgacgga gtgcgtgttg 2160

aacaaggcca cgaatgtcgc ccactggaca acccccagtc tcaaatgcat tagagaccct 2220

gccctggttc accaaaggcc agcgccaccc tccacagtaa cgacggcagg ggtgacccca 2280

cagccagaga gcctctcccc ttctggaaaa gagcccgcag cttcatctcc cagctcaaac 2340

aacacagcgg ccacaacagc agctattgtc ccgggctccc agctgatgcc ttcaaaatca 2400

ccttccacag gaaccacaga gataagcagt catgagtcct cccacggcac cccctctcag 2460

acaacagcca agaactggga actcacagca tccgcctccc accagccgcc aggtgtgtat 2520

ccacagggcc acagcgacac cactgtggct atctccacgt ccactgtcct gctgtgtggg 2580

ctgagcgctg tgtctctcct ggcatgctac ctcaagtcaa ggcaaactcc cccgctggcc 2640

agcgttgaaa tggaagccat ggaggctctg ccggtgactt gggggaccag cagcagagat 2700

gaagacttgg aaaactgctc tcaccaccta 2730

<210> 41

<211> 910

<212> PRT

<213> Artificial sequence

<220>

<223> 2C6.9-T-mIL-15 amino acids

<400> 41

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 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu

20 25 30

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln

35 40 45

Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln

50 55 60

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr

65 70 75 80

Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

100 105 110

Tyr Tyr Cys Gln Asn Asp Phe Ile Phe Pro Leu Thr Phe Gly Gly Gly

115 120 125

Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

145 150 155 160

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe

165 170 175

Ser Leu Thr Arg Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

180 185 190

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Glu Gly Asn Thr Asn Tyr

195 200 205

Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Ser Ser Lys

210 215 220

Ser Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

225 230 235 240

Val Tyr Tyr Cys Ala Arg Val Asn Phe Gly Asn Ala Leu Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro

260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

275 280 285

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

290 295 300

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

305 310 315 320

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

325 330 335

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

340 345 350

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

355 360 365

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala Lys Arg Gly Ser Gly

485 490 495

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

500 505 510

Pro Gly Pro Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala

515 520 525

Thr Arg Val His Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys

530 535 540

Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr

545 550 555 560

Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe

565 570 575

Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile

580 585 590

His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser

595 600 605

Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu

610 615 620

Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val

625 630 635 640

Gln Met Phe Ile Asn Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly

645 650 655

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu

660 665 670

Gln Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp

675 680 685

Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser

690 695 700

Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu

705 710 715 720

Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys

725 730 735

Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr

740 745 750

Val Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser

755 760 765

Gly Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala

770 775 780

Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser

785 790 795 800

Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly

805 810 815

Thr Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala

820 825 830

Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr

835 840 845

Val Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val

850 855 860

Ser Leu Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala

865 870 875 880

Ser Val Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr

885 890 895

Ser Ser Arg Asp Glu Asp Leu Glu Asn Cys Ser His His Leu

900 905 910

<210> 42

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> CD8 alpha Signal peptide

<400> 42

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> 43

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide-2

<400> 43

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 44

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Linker

<400> 44

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 45

<211> 45

<212> PRT

<213> Artificial sequence

<220>

<223> hinge region (CD8 alpha)

<400> 45

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> 46

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> transmembrane region (CD8TM)

<400> 46

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

20

<210> 47

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> intracellular signaling domain (4-1BB)

<400> 47

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 48

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> intracellular signaling domain (CD3 ζ)

<400> 48

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 49

<211> 154

<212> PRT

<213> Artificial sequence

<220>

<223> intracellular signaling domain (4-1BB-CD3 ζ)

<400> 49

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

35 40 45

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

50 55 60

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

65 70 75 80

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

85 90 95

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

100 105 110

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

115 120 125

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

130 135 140

Ala Leu His Met Gln Ala Leu Pro Pro Arg

145 150

<210> 50

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> P2A

<400> 50

Arg Ala Lys Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln

1 5 10 15

Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25

<210> 51

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> PD-1 scFv

<400> 51

Glu Val Gln Leu Val 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 Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Lys Tyr Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Ile Arg Leu Phe Asp Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala

130 135 140

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Pro Ser Gly Asn Val

145 150 155 160

His Asn Tyr Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys

165 170 175

Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg

180 185 190

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser

195 200 205

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His His Tyr Tyr Thr

210 215 220

Ala Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

225 230 235

<210> 52

<211> 395

<212> PRT

<213> Artificial sequence

<220>

<223> mIL-15

<400> 52

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp

20 25 30

Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp

35 40 45

Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu

50 55 60

Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr

65 70 75 80

Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly

85 90 95

Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys

100 105 110

Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe

115 120 125

Ile Asn Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln Ile Thr

145 150 155 160

Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser

165 170 175

Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys

180 185 190

Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala

195 200 205

Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp

210 215 220

Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr

225 230 235 240

Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu

245 250 255

Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala

260 265 270

Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr

275 280 285

Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser

290 295 300

Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln

305 310 315 320

Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile

325 330 335

Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu

340 345 350

Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu

355 360 365

Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg

370 375 380

Asp Glu Asp Leu Glu Asn Cys Ser His His Leu

385 390 395

<210> 53

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> IMGT 2C6.9 CDR-H2-2

<400> 53

Ile Trp Gly Glu Gly Asn Thr

1 5

<210> 54

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> primer F

<400> 54

caggtgtcgt gattcgaatt cgccaccatg gccctgccc 39

<210> 55

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> primer R

<400> 55

tcgcggcgct ggcgtcgtgg tgctgctcac ggtcaccagg 40

<210> 56

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> T2A

<400> 56

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 57

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> E2A

<400> 57

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 58

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> F2A

<400> 58

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 59

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> 1E9.2 murine heavy chain variable region

<400> 59

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Ser

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Gly Val

35 40 45

Ala Thr Ile Ser Ser Gly Asp Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Tyr Gly Asn Ala Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 60

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> 1E9.2 murine light chain variable region

<400> 60

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ser Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Tyr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 61

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> 2C6.9 murine heavy chain variable region

<400> 61

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr

20 25 30

Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Asp Gly Asn Thr Asn Phe His Ser Phe Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Val Asn Phe Gly Asn Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 62

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> 2C6.9 murine light chain variable region

<400> 62

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Glu Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Asp Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ser Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Phe Ile Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 63

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region of anti-PD-1 antibody

<400> 63

Glu Val Gln Leu Val 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 Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Lys Tyr Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Ile Arg Leu Phe Asp Ser Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 64

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> anti-PD-1 antibody light chain variable region

<400> 64

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Pro Ser Gly Asn Val His Asn Tyr

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His His Tyr Tyr Thr Ala Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 65

<211> 256

<212> PRT

<213> Artificial sequence

<220>

<223> 1E9.2-HZ11-scfv amino acid 2

<400> 65

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 Ser 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 Phe Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Tyr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

130 135 140

Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys

145 150 155 160

Ala Ala Ser Gly Phe Ser Phe Ser Asn Ser Ala Met Ser Trp Val Arg

165 170 175

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Ser Ser Gly

180 185 190

Asp Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

195 200 205

Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu

210 215 220

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Gly Tyr Gly

225 230 235 240

Asn Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250 255

<210> 66

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<223> 2C6.9-HZ21-scfv amino acid 2

<400> 66

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 Asp 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 Phe Ile Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

130 135 140

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

145 150 155 160

Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr Gly Val Ser Trp Ile Arg

165 170 175

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Glu

180 185 190

Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser

195 200 205

Lys Asp Ser Ser Lys Ser Gln Val Ser Leu Lys Leu Ser Ser Val Thr

210 215 220

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Asn Phe Gly Asn

225 230 235 240

Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250 255

<210> 67

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region amino acid of anti-PD-L1 antibody

<400> 67

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr

20 25 30

Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Thr Gly Gly Ala Thr Asn Tyr Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Ser Asn Tyr Arg Tyr Asp Glu Pro Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 68

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> anti-PD-L1 antibody light chain variable region amino acid

<400> 68

Glu Ile Val Leu Thr Gln Ser Pro Asp Thr Leu Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Leu Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 69

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> P2A nucleotide sequence 1

<400> 69

cgggccaaga gaggatctgg agccacaaat tttagcctgc tgaaacaggc cggagatgtg 60

gaggaaaacc ctggccct 78

<210> 70

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence 2 of P2A

<400> 70

cgagctaaac gaggctcagg cgcgacgaac tttagtttgc tgaagcaagc tggggatgta 60

gaggaaaatc cgggtccc 78

<210> 71

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Signal peptide-2 nucleotide

<400> 71

atgtatagga tgcagctgct gagctgtatt gccctgagcc ttgcactggt gaccaatagc 60

<210> 72

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<223> PD-1scFv nucleotides

<400> 72

gaggtgcagc tggtgcagag cggcgccgag gtgaagaagc ctggcgccag cgtgaaggtg 60

agctgcaagg cctccggcta caccttcacc gactactaca tgaactgggt gcgagaggcc 120

cccggccagg gcctggagtg gatgggcgag atcaatccca agtacggcga taccacctac 180

aatcagaagt tcaaggaccg ggccaccctg accgtggata agtctacatc tacagcctat 240

atggagctga gcagcctgag atccgatgat accgcagtgt attattgtgc caaaggaatt 300

agactgtttg actcttgggg ccagggaacc ctagtgaccg taagcagcgg aggaggaggc 360

agcggaggag gcggatctgg cggcggcggc tccgacatcc agctgaccca gagccccagc 420

agcctgagcg ccagcgtggg cgacagggtg accatcacct gccggccctc cggcaacgtg 480

cacaattact tcgcctggta ccagcagaag cccggcaaga gccctaagct gctggtgtat 540

aatgccaaga cactggctga aggcgtgcct agtaggttta gcggcagcgg aagcggcact 600

gattatacac tgacaatttc tagcctgcag cctgaggatt ttgctacata ttattgtcat 660

cattactata cagccccttt tacatttgga ggcggaacaa aggtggagat taagtga 717

<210> 73

<211> 1188

<212> DNA

<213> Artificial sequence

<220>

<223> mIL-15 nucleotides

<400> 73

atggattgga cttggatttt gttcctcgtt gccgcagcga ctcgcgtcca tagtaactgg 60

gtgaatgtaa taagtgattt gaaaaaaatt gaagatctta ttcaatctat gcatattgat 120

gctactttat atacggaaag tgatgttcac cccagttgca aagtaacagc aatgaagtgc 180

tttctcttgg agttacaagt tatttcactt gagtccggag atgcaagtat tcatgataca 240

gtagaaaatc tgatcatcct agcaaacaac agtttgtctt ctaatgggaa tgtaacagaa 300

tctggatgca aagaatgtga ggaactggag gaaaaaaata ttaaagaatt tttgcagagt 360

tttgtacata ttgtccaaat gttcatcaac acttcttccg ggggcggcag tggaggtggc 420

ggtagcggcg ggggtggctc tggtggaggc ggctctgggg gcggaagtct gcagatcacg 480

tgccctcccc ccatgtccgt ggaacacgca gacatctggg tcaagagcta cagcttgtac 540

tccagggagc ggtacatttg taactctggt ttcaagcgta aagccggcac gtccagcctg 600

acggagtgcg tgttgaacaa ggccacgaat gtcgcccact ggacaacccc cagtctcaaa 660

tgcattagag accctgccct ggttcaccaa aggccagcgc caccctccac agtaacgacg 720

gcaggggtga ccccacagcc agagagcctc tccccttctg gaaaagagcc cgcagcttca 780

tctcccagct caaacaacac agcggccaca acagcagcta ttgtcccggg ctcccagctg 840

atgccttcaa aatcaccttc cacaggaacc acagagataa gcagtcatga gtcctcccac 900

ggcaccccct ctcagacaac agccaagaac tgggaactca cagcatccgc ctcccaccag 960

ccgccaggtg tgtatccaca gggccacagc gacaccactg tggctatctc cacgtccact 1020

gtcctgctgt gtgggctgag cgctgtgtct ctcctggcat gctacctcaa gtcaaggcaa 1080

actcccccgc tggccagcgt tgaaatggaa gccatggagg ctctgccggt gacttggggg 1140

accagcagca gagatgaaga cttggaaaac tgctctcacc acctatga 1188

Claims

1. A Chimeric Antigen Receptor (CAR) that specifically binds to CLDN18.2, comprising an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment thereof capable of specifically binding to CLDN18.2, the antibody or antigen-binding fragment thereof comprising:

(1) the following heavy chain variable region (VH) and/or light chain variable region (VL) wherein the CDRs are defined by the IMGT numbering system:

(1a) a heavy chain variable region (VH) comprising 3 CDRs as follows: the sequence is SEQ ID NO:1 or a variant thereof, having the sequence of SEQ ID NO: 2 or a variant thereof, the sequence being SEQ ID NO:3 or a variant thereof CDR-H3; and/or the presence of a gas in the gas,

a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO:4 or a variant thereof, the sequence of SEQ ID NO:5 or a variant thereof, the sequence of SEQ ID NO:6 or a variant thereof CDR-L3;

or

(1b) A heavy chain variable region (VH) comprising 3 CDRs as follows: the sequence is SEQ ID NO:7 or a variant thereof, having the sequence of SEQ ID NO: 8 or 53 or a variant thereof, having the sequence of SEQ ID NO: 9 or a variant thereof CDR-H3; and/or the presence of a gas in the gas,

a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 10 or a variant thereof, the sequence of SEQ ID NO: 11 or a variant thereof, the sequence of CDR-L2 of SEQ ID NO: 12 or a variant thereof CDR-L3;

alternatively, the first and second electrodes may be,

(2) the following heavy chain variable region (VH) and/or light chain variable region (VL) wherein the CDRs are defined by the AbM numbering system:

(2a) a heavy chain variable region (VH) comprising 3 CDRs as follows: the sequence is SEQ ID NO: 15 or a variant thereof, having the sequence of SEQ ID NO: 20 or a variant thereof, having the sequence of SEQ ID NO: 21 or a variant thereof CDR-H3; and/or the presence of a gas in the gas,

a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 26 or a variant thereof, the sequence of CDR-L1 of SEQ ID NO: 27 or a variant thereof, having the sequence of SEQ ID NO: 30 or a variant thereof CDR-L3;

or

(2b) A heavy chain variable region (VH) comprising 3 CDRs as follows: the sequence is SEQ ID NO: 31 or a variant thereof, the sequence of SEQ ID NO: 32 or 37 or a variant thereof, having the sequence of SEQ ID NO: 33 or a variant thereof CDR-H3; and/or the presence of a gas in the gas,

a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 34 or a variant thereof, the sequence of SEQ ID NO: 35 or a variant thereof, the sequence of SEQ ID NO: 36 or a variant thereof CDR-L3;

wherein the variant of any of (1a), (1b), (2a), (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

preferably, the antibody or antigen-binding fragment thereof specifically binds to human CLDN 18.2;

preferably, the antibody or antigen-binding fragment thereof further comprises Framework Regions (FRs) from human immunoglobulins.

2. The chimeric antigen receptor of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(a) SEQ ID NO:13 or a variant thereof, and/or, the VH of SEQ ID NO:14 or a variant thereof; or

(b) SEQ ID NO:16 or a variant thereof, and/or, the VH of SEQ ID NO:17 or a variant thereof;

wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or the variant has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids) to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

3. The chimeric antigen of claim 1 or 2A receptor, wherein the antibody or antigen binding fragment thereof is selected from camel Ig, IgNAR, Fab fragment, Fab 'fragment, F (ab)'₂Fragment, F (ab)'₃Fragments, Fv, single-chain antibodies (e.g. scFv, di-scFv or (scFv)₂) Minibody, diabody, triabody, tetrafunctional antibody, disulfide stabilized Fv protein (dsFv) and single domain antibody (sdAb, nanobody); preferably, the antibody or antigen-binding fragment thereof is a single chain antibody, such as an scFv, di-scFv or (scFv)₂。

4. The chimeric antigen receptor of any one of claims 1-3, wherein the extracellular antigen-binding domain is a single chain antibody comprising:

wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids) to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

preferably, the single chain antibody is scFv, di-scFv or (scFv)₂；

Preferably, the VH and VL are connected by a linker; preferably, the linker comprises a linker such as (G)_mS)_nThe sequence shown, wherein m is selected from an integer of 1 to 6; preferably, m is 3, 4, or 5; preferably, n is selected from an integer from 1 to 10; preferably, n is 2, 3, 4, 5, or 6; more preferably, the linker has the amino acid sequence of SEQ ID NO:44, or a sequence of SEQ ID NO;

preferably, the single chain antibody comprises an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in any one of SEQ ID NOs:19, 23, 65, 66; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in any one of SEQ ID NOs:19, 23, 65, 66; or (3) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions) as compared to the amino acid sequence set forth in any one of SEQ ID NOs:19, 23, 65, 66; preferably, the substitutions are conservative substitutions.

5. The chimeric antigen receptor of any one of claims 1-4, wherein the transmembrane domain is a transmembrane region of a protein selected from the group consisting of: the α, β or zeta chain of the T cell receptor, CD3 epsilon, CD3zeta, CD4, CD5, CD8 alpha, CD137, CD152, CD154 and PD-1;

preferably, the transmembrane domain is a transmembrane region of a protein selected from the group consisting of: CD8 α, CD4, PD-1, CD152 and CD 154;

preferably, the transmembrane domain comprises a sequence as set forth in SEQ ID NO:46, CD8 alpha.

6. The chimeric antigen receptor of any one of claims 1-5, wherein the spacer domain is located between the extracellular antigen-binding domain and the transmembrane domain, the spacer domain being selected from a hinge domain and/or CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG 4);

preferably, the hinge domain comprises a hinge region of CD8 a, PD-1, CD152, or CD 154; more preferably, the hinge domain comprises a sequence as set forth in SEQ ID NO:45, CD8 a, as shown.

7. The chimeric antigen receptor of any one of claims 1-6, further comprising a signal peptide at its N-terminus;

preferably, the signal peptide comprises a heavy chain signal peptide (e.g., the heavy chain signal peptide of IgG 1), a granulocyte-macrophage colony stimulating factor receptor 2(GM-CSFR2) signal peptide, or a CD8 a signal peptide; more preferably, the signal peptide is selected from the group consisting of SEQ ID NO:42, CD8 a signal peptide.

8. The chimeric antigen receptor of any one of claims 1-7, wherein the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain;

preferably, the intracellular signaling domain comprises, in order from N-terminus to C-terminus, a costimulatory signaling domain and a primary signaling domain;

preferably, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain;

preferably, the primary signaling domain comprises an Immunoreceptor Tyrosine Activation Motif (ITAM);

preferably, the primary signalling domain comprises an intracellular signalling domain of a protein selected from: CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CDs, CD22, CD79a, CD79b, or CD66 d; more preferably, the primary signaling domain comprises a sequence as set forth in SEQ ID NO:48, the intracellular signaling domain of CD3 ζ;

preferably, the co-stimulatory signaling domain comprises an intracellular signaling domain selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD270(HVEM), CD278(ICOS), or DAP 10;

preferably, the co-stimulatory signaling domain is selected from the intracellular signaling domain of CD28 or the intracellular signaling domain of CD137(4-1BB) or a combination of fragments of both, wherein the intracellular signaling domain sequence of CD137(4-1BB) is as set forth in SEQ ID NO:47 is shown;

more preferably, the intracellular signaling domain sequence is as set forth in SEQ ID NO: shown at 49.

9. The chimeric antigen receptor of any one of claims 1-8, wherein the chimeric antigen receptor comprises, in order from its N-terminus to its C-terminus, the signal peptide, extracellular antigen-binding domain, spacer domain, transmembrane domain, intracellular signaling domain;

preferably, the signal peptide comprises the heavy chain signal peptide of IgG1 or the CD8 a signal peptide (e.g., the signal peptide of the sequence shown in SEQ ID NO: 42);

preferably, the spacer domain comprises a hinge region (e.g., of the sequence shown as SEQ ID NO: 45) of CD8 (e.g., CD8 a);

preferably, the transmembrane domain comprises the transmembrane region of CD8 (e.g., CD8 α) (e.g., the transmembrane region of the sequence shown in SEQ ID NO: 46);

preferably, the intracellular signaling domain comprises a primary signaling domain comprising an intracellular signaling domain of CD3 ζ (e.g., as set forth in SEQ ID NO: 48) and a costimulatory signaling domain comprising an intracellular signaling domain of CD137 (e.g., as set forth in SEQ ID NO: 47); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the amino acid sequence of SEQ ID NO: 49;

preferably, the chimeric antigen receptor has an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in any one of SEQ ID NOs:25, 29; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:25, 29; or (3) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions) as compared to the amino acid sequence set forth in any of SEQ ID NOs:25, 29; preferably, the substitutions are conservative substitutions.

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-9;

preferably, the sequence of the isolated nucleic acid molecule is selected from the group consisting of: (1) 24, 28 or a degenerate variant thereof; (2) a sequence that is substantially identical to a sequence set forth in any of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in any of (1), or a sequence having one or more nucleotide substitutions to a sequence set forth in any of (1)).

11. A nucleic acid construct comprising the first nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-9, and further comprising a second nucleotide sequence encoding an additional biologically active molecule;

preferably, the first and second nucleotide sequences are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A); preferably, the self-cleaving peptide is P2A (e.g., P2A of the sequence shown as SEQ ID NO: 50);

preferably, the first nucleotide sequence is selected from the group consisting of: (1) 24, 28 or a degenerate variant thereof; (2) a sequence that is substantially identical to a sequence set forth in any of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in any of (1), or a sequence having one or more nucleotide substitutions to a sequence set forth in any of (1));

preferably, the additional biologically active molecule is selected from one or more of the following components: an immune checkpoint inhibitor (e.g., an anti-PD-1, PD-L1, CTLA-4, or LAG-3 antibody or antigen-binding fragment thereof), a cytokine (e.g., IL-15, IL-7, IL-12, IL-18, or IL-21), or a membrane-chimeric polypeptide (e.g., mIL-15, mIL-7, mIL-12, mIL-18, or mIL-21);

preferably, the further biologically active molecule further comprises at its N-terminus a signal peptide-2; preferably, the signal peptide-2 is different from the signal peptide comprised in the chimeric antigen receptor; preferably, the signal peptide-2 at the N-terminus of the further biologically active molecule is the IL2 signal peptide (e.g., the IL2 signal peptide of the sequence shown as SEQ ID NO: 43).

12. The nucleic acid construct of claim 11, wherein the additional biologically active molecule is selected from the group consisting of an immune checkpoint inhibitor that is an anti-PD-1 or PD-L1 antibody or antigen binding fragment thereof (e.g., scFv);

preferably, the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region of any one of the following groups: (1) a heavy chain variable region and/or a light chain variable region of Nivolumab or a variant thereof, (2) a heavy chain variable region and/or a light chain variable region of Pembrolizumab or a variant thereof, (3) a heavy chain variable region and/or a light chain variable region of Atezolizumab or a variant thereof, (4) a heavy chain variable region and/or a light chain variable region of Durvalumab or a variant thereof, (5) a heavy chain variable region and/or a light chain variable region of Avelumab or a variant thereof, (6) a VH having a sequence shown in SEQ ID NO:67 or a variant thereof and/or a VL having a sequence shown in SEQ ID NO:68 or a variant thereof, (7) a VH having a sequence shown in SEQ ID NO:63 or a variant thereof and/or a VL having a sequence shown in SEQ ID NO:64 or a variant thereof;

preferably, the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof is a single chain antibody;

preferably, the single chain antibody comprises an amino acid sequence selected from the group consisting of: (1) 51, SEQ ID NO; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO 51; (3) has one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:51 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions;

preferably, the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) 38 or a degenerate variant thereof; (2) a sequence that is substantially identical compared to the sequence set forth in (1), e.g., a sequence that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in (1).

13. The nucleic acid construct of claim 11, wherein the additional biologically active molecule is selected from the group consisting of a membrane-chimeric polypeptide of mIL-15;

preferably, the membrane-chimeric polypeptide mIL-15 sequence comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 52; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO 52; (3) has one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:52 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions;

preferably, the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) 40 or a degenerate variant thereof; (2) a sequence that is substantially identical compared to the sequence of (1), e.g., a sequence that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of (1).

14. A vector comprising the isolated nucleic acid molecule of claim 10, or the nucleic acid construct of any one of claims 11-13;

optionally, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, or a viral vector;

preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.

15. An engineered immune cell expressing the Chimeric Antigen Receptor (CAR) of any one of claims 1-9;

optionally, the immune cell further expresses an additional biologically active molecule selected from one or more of the following components: an antibody or antigen-binding fragment thereof that specifically binds an immune checkpoint (e.g., an anti-PD-1, PD-L1, CTLA-4, or LAG-3 antibody or antigen-binding fragment thereof), a cytokine (e.g., IL-15, IL-7, IL-12, IL-18, IL-21), or a membrane-chimeric polypeptide (e.g., mIL-15, mIL-7, mIL-12, mIL-18, mIL-21);

preferably, the membrane-chimeric polypeptide is mIL-15; preferably, the membrane-chimeric polypeptide mIL-15 has a sequence as shown in SEQ ID NO: shown at 52.

16. The engineered immune cell of claim 15, which expresses the chimeric antigen receptor of any one of claims 1-9 and an anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof;

preferably, the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region selected from the group consisting of:

17. The engineered immune cell of claim 15 or 16, the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof being a single chain antibody;

preferably, the single chain antibody is scFv, di-scFv or (scFv)₂；

Preferably, the VH and VL are connected by a linker; preferably, the linker comprises a linker such as (G)_mS)_nThe sequence shown, wherein m is selected from an integer of 1 to 6; preferably, m is 3, 4, or 5; preferably, n is selected fromAn integer of 1 to 10; preferably, n is 2, 3, 4, 5, or 6; preferably, the linker has the amino acid sequence of SEQ ID NO:44, or a sequence of SEQ ID NO;

preferably, the single chain antibody comprises an amino acid sequence selected from the group consisting of: (1) 51, SEQ ID NO; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO 51; (3) has one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:51 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

18. The immune cell of any one of claims 15-17, wherein the immune cell comprises the isolated nucleic acid molecule of claim 10, or the nucleic acid construct of any one of claims 11-13 or the vector of claim 14.

19. The immune cell of any one of claims 15-18, further comprising one or more of the following characteristics:

(i) the immune cell further has a binding specificity other than a CLDN18.2 target; preferably, the immune cell also has binding specificity for a target selected from the group consisting of: CD19, CD20, CD22, CD33, CD123, or CD 138;

(ii) the immune cell further comprises a knockout of one or more endogenous genes, wherein the endogenous genes are selected from the group consisting of: genes encoding TCR α, TCR β, CD52, Glucocorticoid Receptor (GR), deoxycytidine kinase (dCK), or an immune checkpoint protein (e.g., PD-1).

20. The immune cell of any one of claims 15-19, wherein the immune cell is derived from a T lymphocyte, an NK cell, a monocyte, a macrophage, or a dendritic cell, and any combination thereof; preferably, the immune cells are obtained from a patient; optionally, the immune cell is obtained from a healthy donor.

21. A method of making an immune cell of any one of claims 15-20, comprising: (1) providing immune cells from a patient or a healthy donor; (2) obtaining an immune cell capable of expressing a CLDN 18.2-specific binding chimeric antigen receptor or an immune cell co-expressing a CLDN 18.2-specific binding chimeric antigen receptor and a biologically active molecule; wherein step (2) comprises introducing the isolated nucleic acid molecule of claim 10 or the nucleic acid construct of any one of claims 11-13 or the vector of claim 14 into the immune cell of step (1).

22. An immune cell composition comprising the engineered immune cell of any one of claims 15-20; optionally, the composition further comprises an unmodified and/or unsuccessfully modified immune cell; preferably, the number of engineered immune cells is 10% to 100%, more preferably 40% to 80% of the total number of cells of the immune cell composition.

23. A kit comprising the isolated nucleic acid molecule of claim 10, the nucleic acid construct of any one of claims 11-13, or the vector of claim 14;

optionally, the kit is for preparing a Chimeric Antigen Receptor (CAR) according to any one of claims 1 to 9 or an immune cell according to any one of claims 15 to 20.

24. A pharmaceutical composition comprising an isolated nucleic acid molecule according to claim 10, a nucleic acid construct according to any one of claims 11 to 13, or a vector according to claim 14, or an immune cell according to any one of claims 15 to 20, or an immune cell composition according to claim 22, and a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug with anti-tumor activity;

preferably, the additional pharmaceutically active agent is an interferon, interleukin-2, or a chemotherapeutic drug;

preferably, the additional pharmaceutically active agent is selected from: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, leucovorin, paclitaxel and albumin-bound paclitaxel.

25. Use of the isolated nucleic acid molecule of claim 10, or the nucleic acid construct of any one of claims 11-13, or the vector of claim 14, or the immune cell of any one of claims 15-20, or the immune cell composition of claim 22, or the pharmaceutical composition of claim 24, in the manufacture of a medicament for preventing and/or treating and/or adjunctively treating a tumor, and/or delaying tumor progression, and/or reducing or inhibiting tumor recurrence in a subject;

preferably, the tumor is selected from the group consisting of a solid tumor, a hematologic tumor, and a metastatic, refractory or recurrent lesion of the cancer;

preferably, the tumor or cancer is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, 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, bile duct cancer, gall bladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia;

preferably, the tumor is selected from the group consisting of gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer);

preferably, the tumour is selected from the group consisting of gastric cancer, gastric adenocarcinoma or gastroesophageal junction (GEJ) adenocarcinoma, for example locally advanced unresectable or metastatic gastric cancer, gastric adenocarcinoma or gastroesophageal junction (GEJ) adenocarcinoma;

preferably, the tumor is CLDN18.2 positive, more preferably, the tumor is HER2 negative;

preferably, the subject is a human.