CLDN18.2 targeting chimeric antigen receptor and application thereof

Abstract

The invention relates to a CLDN18.2-targeted chimeric antigen receptor and application thereof, the chimeric antigen receptor comprises a binding domain, the binding domain comprises an antibody or an antigen binding fragment binding to CLDN18.2, and the antibody or the antigen binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL).

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a chimeric antigen receptor targeting CLDN18.2 and its application. Background Art

[0002] In adoptive cell therapy, chimeric antigen receptor T cells (CAR-T cells) are artificially modified tumor-killing cells that combine the targeted recognition function of antibodies with the tumor-killing function of T cells, which is a major breakthrough in the field of tumor immunotherapy. However, the efficacy of CAR-T on solid tumors such as gastric cancer and pancreatic cancer is still not ideal. The discovery of new CAR-T structures and new targets is the key to CAR-T's effective treatment of solid tumors.

[0003] CLDN18 belongs to the Claudins protein family. CLDN18.1 and CLDN18.2 are variable splice forms of CLDN18. In normal tissues, CLDN18.1 is mainly expressed in the lungs, and CLDN18.2 is mainly expressed in gastric mucosal epithelial cells. However, CLDN18.2 is expressed in a variety of tumor tissues, such as gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, etc., and is an ideal target for CAR-T therapy of related solid tumors. Summary of the invention

[0004] The present application provides a chimeric antigen receptor (CAR) targeting CLDN18.2 and its use, wherein the chimeric antigen receptor can obtain a higher virus titer and CAR positive rate during the preparation process, and has great advantages in the production of chimeric antigen receptors. The chimeric antigen receptor can have a significant killing effect on CLDN18.2-positive tumor cells; when treating CLDN18.2-related tumors, the chimeric antigen receptor exhibits a strong tumor suppression ability.

[0005] The antigen binding domain of the chimeric antigen receptor targeting CLDN18.2 described in the present application comprises an antigen binding protein targeting CLDN18.2, and the present application provides a murine CLDN18.2 antigen binding protein B and a humanized CLDN18.2 antigen binding protein 59B, and the proteins before and after humanization exhibit strong binding activity to human CLDN18.2. The humanized CLDN18.2 antigen binding protein exhibits more significant binding activity.

[0006] The present application provides a chimeric antigen receptor (CAR), which includes an antigen binding domain, wherein the antigen binding domain includes an antibody or antigen binding fragment that binds to CLDN18.2, wherein the antibody or antigen binding fragment includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein,

[0007] The VH comprises:

[0008] 1) a heavy chain complementarity determining region 1 (HCDR1), wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6;

[0009] 2) a heavy chain complementarity determining region 2 (HCDR2), wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0010] 3) a heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8;

[0011] The VL comprises:

[0012] 1) a light chain complementary determining region 1 (LCDR1), wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9;

[0013] 2) a light chain complementary determining region 2 (LCDR2), wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10;

[0014] 3) Light chain complementarity determining region 3 (LCDR3), wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.

[0015] In some embodiments, the heavy chain variable region (VH) further comprises heavy chain framework region 1 (H-FR1), heavy chain framework region 2 (H-FR2), heavy chain framework region 3 (H-FR3) and heavy chain framework region 4 (H-FR4).

[0016] In some embodiments, the heavy chain variable region (VH) further comprises a heavy chain framework region 1 (H-FR1), and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 20.

[0017] In some embodiments, the C-terminus of the H-FR1 is connected to the N-terminus of the HCDR1.

[0018] In some embodiments, the heavy chain variable region (VH) further comprises a heavy chain framework region 2 (H-FR2), and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 21.

[0019] In some embodiments, the H-FR2 is located between the HCDR1 and the HCDR2.

[0020] In some embodiments, the heavy chain variable region (VH) further comprises a heavy chain framework region 3 (H-FR3), and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 22.

[0021] In some embodiments, the H-FR3 is located between the HCDR2 and the HCDR3.

[0022] In some embodiments, the heavy chain variable region (VH) further comprises a heavy chain framework region 4 (H-FR4), and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 23.

[0023] In some embodiments, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3.

[0024] In some embodiments, the VH comprises heavy chain framework region 1 (H-FR1), heavy chain framework region 2 (H-FR2), heavy chain framework region 3 (H-FR3) and heavy chain framework region 4 (H-FR4), wherein:

[0025] The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:12, and the C-terminus of H-FR1 is connected to the N-terminus of the HCDR1; the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:13, and H-FR2 is located between the HCDR1 and the HCDR2; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14, and H-FR3 is located between the HCDR2 and the HCDR3; the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:15, and the N-terminus of H-FR4 is connected to the C-terminus of the HCDR3.

[0026] In some embodiments, the VH comprises heavy chain framework region 1 (H-FR1), heavy chain framework region 2 (H-FR2), heavy chain framework region 3 (H-FR3) and heavy chain framework region 4 (H-FR4), wherein:

[0027] The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:21, and the C-terminus of H-FR1 is connected to the N-terminus of the HCDR1; the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:22, and H-FR2 is located between the HCDR1 and the HCDR2; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:23, and H-FR3 is located between the HCDR2 and the HCDR3; the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:24, and the N-terminus of H-FR4 is connected to the C-terminus of the HCDR3.

[0028] In some embodiments, the light chain variable region (VL) further comprises light chain framework region 1 (L-FR1), light chain framework region 2 (L-FR2), light chain framework region 3 (L-FR3) and light chain framework region 4 (L-FR4).

[0029] In some embodiments, the light chain variable region (VL) further comprises a light chain framework region 1 (L-FR1), and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 24.

[0030] In some embodiments, the C-terminus of the L-FR1 is connected to the N-terminus of the LCDR1.

[0031] In some embodiments, the light chain variable region (VL) further comprises a light chain framework region 2 (L-FR2), and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:17.

[0032] In some embodiments, the L-FR2 is located between the LCDR1 and the LCDR2.

[0033] In some embodiments, the light chain variable region (VL) further comprises a light chain framework region 3 (L-FR3), and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 25.

[0034] In some embodiments, the L-FR3 is located between the LCDR2 and the LCDR3.

[0035] In some embodiments, the light chain variable region (VL) further comprises a light chain framework region 4 (L-FR4), and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 26.

[0036] In some embodiments, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3.

[0037] In some embodiments, the VL comprises light chain framework region 1 (L-FR1), light chain framework region 2 (L-FR2), light chain framework region 3 (L-FR3) and light chain framework region 4 (L-FR4), wherein:

[0038] The L-FR1 comprises the amino acid sequence shown in SEQ ID NO:16, and the C-terminus of L-FR1 is connected to the N-terminus of the LCDR1; the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:17, and L-FR2 is located between the LCDR1 and the LCDR2; the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:18, and L-FR3 is located between the LCDR2 and the LCDR3; the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:19, and the N-terminus of L-FR4 is connected to the C-terminus of the LCDR3.

[0039] In some embodiments, the VL comprises light chain framework region 1 (L-FR1), light chain framework region 2 (L-FR2), light chain framework region 3 (L-FR3) and light chain framework region 4 (L-FR4), wherein:

[0040] The L-FR1 comprises the amino acid sequence shown in SEQ ID NO:24, and the C-terminus of L-FR1 is connected to the N-terminus of the LCDR1; the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:17, and L-FR2 is located between the LCDR1 and the LCDR2; the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:25, and L-FR3 is located between the LCDR2 and the LCDR3; the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:26, and the N-terminus of L-FR4 is connected to the C-terminus of the LCDR3.

[0041] In some embodiments, the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 27, and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 28.

[0042] In some embodiments, the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:4.

[0043] In some embodiments, the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 28.

[0044] In some embodiments, the antigen binding fragment is selected from one or more of the following groups: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv and di-scFv.

[0045] In some embodiments, the antigen binding fragment is a scFv.

[0046] In some embodiments, the antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 29.

[0047] In some embodiments, wherein the CAR comprises a signal peptide.

[0048] In some embodiments, the signal peptide comprises a CD8α signal peptide, and the CD8α signal peptide comprises an amino acid sequence as shown in SEQ ID NO:30.

[0049] In some embodiments, wherein the CAR comprises a transmembrane domain, the transmembrane domain comprises a transmembrane domain derived from one or more of the following group proteins: CD8α, CD8β, CD28, CD3e, CD3ε, CD137, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, CD154, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64 and SLAM.

[0050] In some embodiments, the transmembrane domain includes a CD8α transmembrane domain, and the CD8α transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:32.

[0051] In some embodiments, wherein the CAR comprises a hinge region, wherein the hinge region is located between the transmembrane domain and the binding domain.

[0052] In some embodiments, the hinge region includes a hinge region derived from one or more of the following proteins: CD28, IgG1, IgG4, IgD, CD137, CD4, CD27, CD7, CD8α, CD8β, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30 and LIGHT.

[0053] In some embodiments, the N-terminus of the hinge region is connected to the C-terminus of the binding domain, and the C-terminus of the hinge region is connected to the N-terminus of the transmembrane domain.

[0054] In some embodiments, the hinge region includes a CD8α hinge region, and the CD8α hinge region comprises an amino acid sequence as shown in SEQ ID NO:31.

[0055] In some embodiments, wherein the CAR comprises one or more costimulatory domains, the costimulatory domains include costimulatory domains derived from the following group of proteins: CD28, CD137, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

[0056] In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain and / or a CD137 costimulatory domain.

[0057] In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain, and the CD28 costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:33.

[0058] In some embodiments, the costimulatory domain comprises a CD137 costimulatory domain, and the CD137 costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:35.

[0059] In some embodiments, the N-terminus of the costimulatory domain is linked to the C-terminus of the transmembrane domain.

[0060] In some embodiments, the CAR comprises one or more signaling domains, the signaling domains comprising signaling domains derived from the following group of proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12, and a domain protein comprising at least one ITAM.

[0061] In some embodiments, the signaling domain includes a CD3ζ signaling domain, and the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO:34.

[0062] In some embodiments, the N-terminus of the signaling domain is linked to the C-terminus of the costimulatory domain.

[0063] In some embodiments, it includes a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain and a signal transduction domain from N-terminus to C-terminus.

[0064] In some embodiments, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:5, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:32, the hinge region comprises the amino acid sequence shown in SEQ ID NO:31, the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:35, and the signaling domain comprises the amino acid sequence shown in SEQ ID NO:34.

[0065] In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:2.

[0066] On the other hand, the present application provides an isolated nucleic acid molecule encoding the CAR described in the present application.

[0067] In some embodiments, the nucleic acid molecule comprises the amino acid sequence shown in SEQ ID NO:1.

[0068] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0069] In some embodiments, the vectors described herein include viral vectors.

[0070] In some embodiments, the vectors described herein comprise retroviral vectors.

[0071] On the other hand, the present application provides an immune effector cell, which contains and / or expresses the CAR described in the present application, the nucleic acid molecule described in the present application, and / or the vector described in the present application.

[0072] In some embodiments, the immune effector cells described herein include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, γδT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0073] In some embodiments, the immune effector cells described herein are of human origin.

[0074] In some embodiments, the immune effector cells described herein are T cells.

[0075] On the other hand, the present application provides a method for preparing the immune effector cell described in the present application, comprising introducing the nucleic acid molecule described in the present application and / or the vector described in the present application into the immune effector cell.

[0076] On the other hand, the present application provides a method for preparing the CAR described in the present application, the method comprising culturing the immune effector cell described in the present application under the conditions of expression of the CAR described in the present application.

[0077] On the other hand, the present application provides a pharmaceutical composition comprising the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, and / or the immune effector cell described in the present application, and a pharmaceutically acceptable carrier.

[0078] On the other hand, the present application provides a use of the CAR described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, and / or the immune effector cell described in the present application in the preparation of a pharmaceutical composition.

[0079] In some embodiments, the pharmaceutical composition is used to prevent and / or treat a disease or condition associated with the expression of CLDN18.2.

[0080] On the other hand, the present application provides a CAR described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the immune effector cell described in the present application and / or the pharmaceutical composition described in the present application for use in preventing and / or treating diseases or conditions associated with the expression of CLDN18.2.

[0081] On the other hand, the present application provides a method for preventing and / or treating a disease or condition associated with the expression of CLDN18.2, comprising administering to a subject in need thereof an effective amount of the CAR described herein, the isolated nucleic acid molecule described herein, the vector described herein, the immune effector cell described herein and / or the pharmaceutical composition described herein.

[0082] In some embodiments, the disease or disorder associated with the expression of CLDN18.2 includes a disease or disorder associated with upregulated expression of CLDN18.2.

[0083] In some embodiments, the disease or disorder associated with the expression of CLDN18.2 comprises a tumor.

[0084] In some embodiments, the disease or disorder associated with the expression of CLDN18.2 comprises a CLDN18.2-positive tumor.

[0085] In some embodiments, the disease or condition associated with the expression of CLDN18.2 includes solid tumors and / or hematological tumors.

[0086] In some embodiments, the disease or condition associated with the expression of CLDN18.2 comprises a solid tumor.

[0087] In some embodiments, the disease or condition associated with the expression of CLDN18.2 includes gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, bile duct cancer, bladder cancer and / or leukemia.

[0088] On the other hand, the present application provides a method for regulating the expression of CLDN18.2 in a subject, comprising administering to the subject the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the immune effector cell described in the present application, and / or the pharmaceutical composition described in the present application.

[0089] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0091] Figure 1 Shown is a schematic diagram of the structure of 59B-CD28 CAR and 59B-CD137 CAR described in the present application: the gene encoding CAR may include a signal peptide, an antigen binding fragment of human CLDN18.2, a CD8α hinge region and a CD8α transmembrane domain, a CD137 co-stimulatory domain or a CD28 co-stimulatory domain, and a CD3ζ signaling domain.

[0092] Figure 2 Shown are the positive rates and titer detection of 59B-CD28 and 59B-CD137 after HEK293T virus infection described in the present application.

[0093] Figure 3 Shown is the positive rate detection of 59B-CD28 and 59B-CD137 after human primary T cells were infected with the virus as described in the present application, wherein UTD is untransfected T cells.

[0094] Figure 4 Shown is the positive rate detection of CAR-B infected by human primary T cells virus as described in the present application, wherein UTD is untransfected T cells.

[0095] Figure 5 Shown are the binding activity detection of the B / CLDN18.2 antibody before humanization and the binding activity detection of the 59B / CLDN18.2 antibody after humanization described in the present application.

[0096] Figure 6 Shown are the exhaustion scores and basal signal indexes of the 59B-CD28 and 59B-CD137 CAR-T cells described in this application.

[0097] Figure 7 Shown is the killing effect of CAR-T-59B-CD28 and CAR-T-59B-CD137 described in this application on tumor cells.

[0098] Figure 8 What is shown is the killing effect of CART-B described in this application on tumor cells.

[0099] Figures 9A-9C Shown is the detection of cytokine release after the CART-B cells described in this application kill tumor cells.

[0100] Fig.10 Shown are the efficacy results of CART-B described in this application on the NUGC-4-CLDN18.2 mouse model.

[0101] Fig.11 Shown are the efficacy results of CAR-T-59B-CD137 described in this application on the NUGC-4-CLDN18.2 mouse model.

[0102] Figures 12A-12B Shown are the comparative efficacy results and immunohistochemistry results of the CAR-T-59B-CD137 described in the present application and the CAR-T targeting CLDN18.2 in the prior art on the NUGC-4-CLDN18.2 mouse model. DETAILED DESCRIPTION

[0103] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0104] Definition of terms

[0105] In this application, the term "chimeric antigen receptor (Chimeric antigen receptor, CAR)" generally refers to a fusion protein recombinantly expressed in immune effector cells, comprising an extracellular binding domain capable of binding to an antigen and at least one intracellular domain that can activate immune effector cells. The chimeric antigen receptor may include a signal peptide, an antigen (eg, tumor-associated antigen) binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain. The extracellular binding domain of CAR can recognize and bind to a target antigen on the surface of a target cell (such as CLDN18.2 on the surface of a tumor cell), resulting in CAR-containing immune effector cells clustering at the target cell, while the intracellular domain can transmit signals and activate immune effector cells, triggering the proliferation of immune effector cells, the production of cytokines, and mediating cell death expressing target antigens in a manner independent of major histocompatibility (MHC). In the present application, the immune effector cells that recombinantly express CAR can be T cells, and the CAR-T cells can recognize and mediate the death of malignant cells expressing the target antigen. In some embodiments, the malignant cells can be tumor cells, such as CLDN18.2-positive tumor cells.

[0106] In this application, the term "fusion protein" generally refers to a new protein, peptide or polypeptide produced by connecting two or more genes that originally encode separate proteins, peptides or polypeptides. After expression or conjugation, the fusion protein is directly or indirectly spliced ​​together by proteins, peptides or polypeptides containing different domains to form a new protein, peptide or polypeptide containing all the functional domains of the protein, peptide or polypeptide produced by the translation of the original gene.

[0107] In the present application, the term "binding domain" generally refers to a protein, peptide or polypeptide on the extracellular domain of a chimeric antigen receptor that can bind to a target antigen on the surface of a target cell. In the present application, the binding domain may be an antigen binding fragment, for example, an antigen binding fragment of an antibody. For example, the binding of the binding domain to the target antigen is specific. In some embodiments, the binding domain may be an antigen binding fragment scFv that recognizes the target antigen, such as the scFv of the CLDN18.2 antibody. In the present application, the target antigen recognized and bound by the binding domain may be a tumor-associated antigen. The tumor-associated antigen may be a tumor-specific antigen that is expressed only in tumor cells and not present in other normal cells; it may also be expressed in tumor cells, other normal organs, tissues and / or cells (heterogeneous and allogeneic), and may also be expressed during the development and differentiation of tumor cells, other normal organs, tissues and / or cells (heterogeneous and allogeneic).

[0108] In this application, the term "signal peptide" generally refers to a peptide chain that guides protein transfer. The signal peptide can be located at the N-terminus of the CAR and can assist in the expression, secretion and membrane localization of the CAR gene. For example, the signal peptide can increase the expression efficiency of the CAR on the immune effector cell membrane. In some embodiments, the signal peptide can be a short peptide chain, which can be 5-30 amino acids in length.

[0109] In the present application, the term "hinge region" generally refers to a protein, peptide or polypeptide connecting an antigen binding domain and a transmembrane domain. The hinge region may include one or more hinge regions derived from the hinge region of the following histones: CD28, IgG1, IgG4, IgD, CD137, CD4, CD27, CD7, CD8α, CD8β, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30 and LIGHT. In some embodiments, the hinge region may be a CD8α hinge region.

[0110] In this application, the term "transmembrane domain" generally refers to a domain in CAR that passes through the cell membrane, which is connected to the co-stimulatory domain in the cell and to the extracellular domain of the cell, and can play a role in transmitting signals. The transmembrane domain can anchor CAR to the cell membrane. In this application, the transmembrane domain can be derived from a natural polypeptide, or can be obtained by artificial design. For example, the transmembrane domain can include one or more transmembrane domains derived from the following histone transmembrane domains: CD8α, CD8β, CD28, CD3e, CD3ε, CD137, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, CD154, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64 and SLAM. In some embodiments, the transmembrane domain can be a CD8α transmembrane domain.

[0111] In the present application, the term "costimulatory domain" generally refers to the intracellular domain providing an immune effector cell costimulatory molecule. The costimulatory molecule is a cell surface molecule required for the effective response of lymphocytes to antigens, for example, the costimulatory domain may include one or more costimulatory domains selected from the following histone costimulatory domains: CD28, CD137, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88. In some embodiments, the costimulatory domain can be a CD137 costimulatory domain and / or a CD28 costimulatory domain.

[0112] In the present application, the terms "signaling domain" and "signaling domain" can be used interchangeably, which generally refers to a domain located inside a cell that can conduct signals, and can also be referred to as an "intracellular signaling domain". The intracellular signaling domain can target the binding domain to conduct antigen signals into the cell. For example, the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor. For example, the signaling domain may include one or more signaling domains selected from the following histone signaling domains: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV)

[0113] LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12 and a domain protein comprising at least one ITAM. In some embodiments, the signaling domain may be a CD3 zeta signaling domain.

[0114] The term "antigen" used in this application generally refers to a molecule or molecule fragment that can be specifically bound by an antibody. In some embodiments, an antigen can be used to produce an antibody that can bind to the antigen in an animal. An antigen can have one or more epitopes that can interact with different antibodies. For example, the antigen described in this application can include CLDN18.2.

[0115] In the present application, the term "CLDN18.2" generally refers to subtype 2 of the cell tight junction protein family Claudin18. The CLDN18.2 described in the present application may include a complete CLDN18.2 protein and its functional fragments, functional variants, homologs and / or proteins, peptides or polypeptides having at least one common epitope with CLDN18.2. In the present application, the "homolog" generally refers to a protein, peptide or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein, peptide or polypeptide. In the present application, the "variant" generally refers to a protein, peptide or polypeptide that has been substituted, deleted or added with one or more amino acids in the amino acid sequence of the protein, peptide or polypeptide. The functional variant can maintain the biological properties of the protein or polypeptide before the change, such as the functional variant of CLDN18.2 can be recognized and bound by the CLDN18.2 antigen binding protein.

[0116] In the present application, the term "CLDN18.2 positive tumor cell" refers to a tumor cell expressing CLDN18.2 protein on its surface. In order to determine whether a cell expresses CLDN18.2 protein on the surface, the expression of CLDN18.2 mRNA can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR), or antibodies against CLDN18.2 protein can be used to measure CLDN18.2 protein expression on the cell surface in methods such as immunohistochemistry, FACS, etc. In some embodiments, the CLDN18.2 positive tumor can be a tumor planted in a mammal, such as a tumor planted in a mouse. In the present application, the term "tumor" generally refers to or describes a physiological condition in a mammal that is generally characterized by unregulated cell growth. In the present application, a tumor may include a tumor associated with the expression of CLDN18.2. For example, a tumor may include a CLDN18.2 positive tumor.

[0117] The term "antibody" used in this application includes immunoglobulin molecules that can specifically bind to a specific antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as the immunoglobulin molecules can exhibit the desired biological activity (such as specific binding to a specific antigen) (Miller et al (2003) Jour. of Immunology 170: 4854-4861). The natural form of the antibody is usually a tetramer and is composed of two identical immunoglobulin chain pairs, each pair having a light chain and a heavy chain. In each pair, the light chain and heavy chain variable regions (VL and VH) are mainly responsible for binding to the antigen. The light chain and heavy chain variable domains respectively include three hypervariable regions (also called "complementary determining regions" or "CDRs") and a framework region interrupted by the three hypervariable regions. The constant region can be recognized by the immune system and interact with it. (See, for example, Janeway

[0118] et al., 2001, Immunol.Biology, 5th edition, Garland Publishing, New York). The antibody can be of any type (e.g., IgG, IgE, IgM, IgD or IgA), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2) or any subclass of the above antibodies. The antibody can be derived from any suitable species. In some embodiments, the antibody is human or murine, for example, the antibody can be a human, humanized or chimeric antibody. In some embodiments, the human variable region framework region of the humanized antibody can contain some non-human residues to increase the correct binding ability of the antibody. After humanization, the biological half-life of the antibody can be extended, and the ability to produce adverse immune responses after administration to the human body is reduced.

[0119] The term "antigen binding fragment" as used herein generally comprises a portion of an intact antibody, wherein the fragment retains the same specific binding ability as the intact antibody, i.e., the antigen binding fragment can bind to an antigen or antigen fragment that the intact antibody can bind to. Antigen binding fragments can be produced by recombinant DNA technology or enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen binding fragments include Fab, Fab', F(ab'), 2 、F(ab) 2 , Fv and complementarity determining region (CDR) fragments, single-chain antibody fragments (such as scFv), chimeric antibodies, diabodies and polypeptides, which contain at least a portion of an intact antibody so that the above molecules retain the same specific binding ability as the intact antibody.

[0120] In the present application, the term "nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides or ribonucleotides in an isolated form of any length. The isolated nucleic acid molecules described in the present application can be isolated from their natural environment or artificially synthesized. The nucleic acid molecules described in the present application can encode the chimeric antigen receptors described in the present application. In some embodiments, the isolated nucleic acid molecules are nucleic acid molecules prepared by DNA recombinant technology.

[0121] In the present application, the term "carrier" generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art.

[0122] The term "vector" used in the present invention generally refers to a nucleic acid delivery vehicle into which a nucleic acid molecule carrying a target gene can be inserted and the target gene can be expressed. The vector can be transformed, transduced or transfected into a host cell so that the target gene it carries can be expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage and animal viruses, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). Generally speaking, a vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, screening sequences and reporter genes. In addition, the vector may also contain a replication initiation site sequence. The vector may also include components that assist it in entering the cell, such as viral particles, liposomes or protein shells.

[0123] In this application, the term "retroviral vector" generally refers to a vector that can clone and express exogenous genes, but cannot self-package into viral particles with proliferation ability. The retroviruses mostly have reverse transcriptase, and retroviruses contain at least three genes: gag, which contains genes for proteins that make up the center and structure of the virus; pol, which contains genes for reverse transcriptase; and env, which contains genes that make up the viral shell. When a retrovirus is used as a vector, through transfection, the retroviral vector can randomly and stably integrate its own genome and the exogenous genes it carries into the host cell genome, for example, the gene encoding CAR can be integrated into the host cell.

[0124] In the present application, the term "immune effector cell" generally refers to an immune cell that participates in immune response, participates in the removal of foreign antigens and exercises effector functions. In the present application, immune effector cells can be selected from the following groups: T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, γδT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells. In some embodiments, the immune effector cell can be a human immune effector cell, such as a human T cell.

[0125] In this application, the term "T cell" refers to an immune effector cell derived from the thymus that participates in various cell-mediated immune responses. T cells may include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. For example, the T cells may include, but are not limited to, helper T cells (HTL; CD4 +T cells), cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other subset of T cells. The T cells may include, but are not limited to, T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR.

[0126] In the present application, the terms "prevention" and / or "treatment" include not only preventing and / or treating a disease, but also generally include preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the treated patient.

[0127] In the present application, the term "therapeutically effective amount" or "effective amount" generally refers to an amount of the present invention's antibody sufficient to prevent or alleviate symptoms associated with a disease or disorder (e.g., cancer). The therapeutically effective amount is related to the disease being treated, wherein those skilled in the art can easily discern the actual effective amount.

[0128] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat or monkey.

[0129] In the present application, the term "anti-tumor activity" generally refers to a decrease in the proliferation rate, viability or metastatic activity of tumor cells. For example, anti-tumor activity can be shown by a decrease in the growth rate of abnormal cells that occur during treatment, or a stable or reduced tumor size, or a longer survival period due to treatment compared to a control without treatment. This activity can be evaluated using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art for studying anti-tumor activity.

[0130] In this application, the term "comprises", "comprising" or "including" generally means including the features explicitly stated, but not excluding other elements.

[0131] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. DETAILED DESCRIPTION OF THE INVENTION

[0133] B and 59B described in the present application are respectively a murine CLDN18.2 antigen binding protein and a humanized antigen binding protein. 59B-CD28, 59B-CD137 and B-CD28 are CARs with 59B and B as binding domains, respectively, and CD28 and CD137 indicate that the costimulatory domain contained in the CAR is a CD28 costimulatory domain or a CD137 costimulatory domain.

[0134] The present application provides a chimeric antigen receptor targeting CLDN18.2 and its use. The chimeric antigen receptor described in the present application exhibits one or more of the following advantages or functions: the chimeric antigen receptor has a higher viral titer and CAR positivity rate during the preparation process, which can have great advantages in the production of chimeric antigen receptors; it can have significant lethality to CLDN18.2-positive tumor cells; and it exhibits strong tumor suppression ability when treating CLDN18.2-related tumors.

[0135] The antigen binding domain of the chimeric antigen receptor targeting CLDN18.2 described in the present application comprises an antigen binding protein targeting CLDN18.2. The present application provides a humanized CLDN18.2 antigen binding protein 59B and a murine CLDN18.2 antigen binding protein B. Both the proteins before and after humanization exhibit strong binding activity to human CLDN18.2, and the humanized CLDN18.2 antigen binding protein exhibits more significant binding activity.

[0136] On the one hand, the present application provides a chimeric antigen receptor (CAR), which is based on the recognition and binding of the target antigen (CLDN18.2) and the activation of T cells, so as to achieve the immune activity fusion protein of anti-tumor cells. The main characteristic of CAR is the ability to redirect immune effector cells specifically, thereby inducing proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of cells expressing target antigens in a manner independent of major histocompatibility (MHC), and the ability of cell-specific targeting using monoclonal antibodies, soluble ligands or cell-specific co-receptors.

[0137] In the present application, the chimeric antigen receptor (CAR) may comprise a signal peptide, a binding domain of CLDN18.2, a transmembrane domain, a hinge region, a co-stimulatory domain and a signaling domain.

[0138] Binding domain

[0139] The chimeric antigen receptor (CAR) provided in the present application includes an antigen binding domain. The antigen binding domain is an extracellular domain that targets and binds to an antigen, and the extracellular binding domain includes but is not limited to an antibody or its antigen binding fragment, a bound ligand or an extracellular domain of a co-receptor, and the target antigen is a tumor-associated antigen, and the tumor-associated antigen may be a tumor-specific antigen that is expressed only in tumor cells and not present in other normal cells; it may also be an antigen that is expressed both in tumor cells and in other normal organs, tissues and / or cells (heterogeneous and allogeneic); it may also be an antigen that is expressed during the development and differentiation of tumor cells, other normal organs, tissues and / or cells (heterogeneous and allogeneic).

[0140] In the present application, the binding domain of CLDN18.2 may be greater than or equal to about 10 5 M -1 (For example, greater than or equal to about 10 5 M -1 , greater than or equal to about 10 6 M -1 , greater than or equal to about 10 7 M -1 , greater than or equal to about 10 8 M -1 , greater than or equal to about 10 9 M -1 , greater than or equal to about 10 10 M -1 , greater than or equal to about 10 11 M -1 , greater than or equal to about 10 12 M -1 , greater than or equal to about 10 13 M -1 or greater) of Ka (i.e., the equilibrium association constant for the binding interaction, which is 1 / M); or, -5 M (e.g., less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to about 10 -7 M, less than or equal to about 10 -8 M, less than or equal to about 10 -9 M, less than or equal to about 10 -10 M, less than or equal to about 10 -11M, less than or equal to about 10 -12 M, less than or equal to about 10 -13 M or less) of equilibrium dissociation constant Kd and CLDN18.2 bind or associate. In the present application, the affinity of the binding domain of CLDN18.2 and CLDN18.2 can be determined using conventional techniques in the art, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or displacement assay using a labeled ligand, or using a surface plasmon resonance device (such as Biacore T100, which can be obtained from Biacore, Inc., Piscataway, NJ) or optical biosensor technology.

[0141] In some embodiments, the CAR provided herein, the antigen binding domain may include an antibody or antigen binding fragment of CLDN18.2, wherein the antibody or antigen binding fragment may include a heavy chain variable region (VH) and a light chain variable region (VL), wherein,

[0142] VH may comprise: 1) a heavy chain complementarity determining region 1 (HCDR1), wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 6; 2) a heavy chain complementarity determining region 2 (HCDR2), wherein the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 7; 3) a heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 8;

[0143] VL may include: 1) light chain complementary determining region 1 (LCDR1), which LCDR1 may include the amino acid sequence shown in SEQ ID NO:9; 2) light chain complementary determining region 2 (LCDR2), which LCDR2 may include the amino acid sequence shown in SEQ ID NO:10; 3) light chain complementary determining region 3 (LCDR3), which LCDR3 may include the amino acid sequence shown in SEQ ID NO:11.

[0144] CDR, also known as the complementarity determining region, is a part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, comprehensive consideration of Kabat / Chothia, etc. These coding systems are known in the art. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In the present application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR after substitution, deletion and / or addition of one or more amino acids. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the CDR.

[0145] In some embodiments, the CAR provided herein, the heavy chain variable region (VH) of the antigen binding domain may further include heavy chain framework region 1 (H-FR1), heavy chain framework region 2 (H-FR2), heavy chain framework region 3 (H-FR3) and heavy chain framework region 4 (H-FR4). Wherein, the C-terminus of the H-FR1 is connected to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3.

[0146] In some embodiments, the H-FR1 may comprise an amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 20. In some embodiments, the H-FR2 may comprise an amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 21. In some embodiments, the H-FR3 may comprise an amino acid sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 22. In some embodiments, the H-FR4 may comprise an amino acid sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 23.

[0147] In some embodiments, the H-FR1 may include the amino acid sequence shown in SEQ ID NO:12, the H-FR2 may include the amino acid sequence shown in SEQ ID NO:13, the H-FR3 may include the amino acid sequence shown in SEQ ID NO:14, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO:15.

[0148] In some embodiments, the H-FR1 may include the amino acid sequence shown in SEQ ID NO:20, the H-FR2 may include the amino acid sequence shown in SEQ ID NO:21, the H-FR3 may include the amino acid sequence shown in SEQ ID NO:22, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO:23.

[0149] In some embodiments, in the CAR provided herein, the heavy chain variable region (VH) of the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 28.

[0150] In some embodiments, the light chain variable region (VL) of the antigen binding domain of the CAR provided herein may further include light chain framework region 1 (L-FR1), light chain framework region 2 (L-FR2), light chain framework region 3 (L-FR3) and light chain framework region 4 (L-FR4). Wherein, the C-terminus of the L-FR1 is connected to the N-terminus of the LCDR1, wherein the L-FR2 is located between the LCDR1 and the LCDR2, the L-FR3 is located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3.

[0151] In some embodiments, the L-FR1 may comprise an amino acid sequence as shown in SEQ ID NO: 16 or SEQ ID NO: 24. In some embodiments, the L-FR2 may comprise an amino acid sequence as shown in SEQ ID NO: 17. In some embodiments, the L-FR3 may comprise an amino acid sequence as shown in SEQ ID NO: 18 or SEQ ID NO: 25. In some embodiments, the L-FR4 may comprise an amino acid sequence as shown in SEQ ID NO: 19 or SEQ ID NO: 26.

[0152] In some embodiments, the L-FR1 may include the amino acid sequence shown in SEQ ID NO:16, the L-FR2 may include the amino acid sequence shown in SEQ ID NO:17, the L-FR3 may include the amino acid sequence shown in SEQ ID NO:18, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO:19.

[0153] In some embodiments, the L-FR1 may include the amino acid sequence shown in SEQ ID NO:24, the L-FR2 may include the amino acid sequence shown in SEQ ID NO:17, the L-FR3 may include the amino acid sequence shown in SEQ ID NO:25, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO:26.

[0154] In some embodiments, in the CAR provided herein, the light chain variable region (VL) of the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 28.

[0155] In some embodiments, the antigen binding domain of the CAR provided herein may comprise VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises the amino acid sequence shown in SEQ ID NO: 4.

[0156] In some embodiments, the antigen binding domain of the CAR provided herein may comprise VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises the amino acid sequence shown in SEQ ID NO: 28.

[0157] In some embodiments, the antigen binding domain described herein may comprise an antigen binding fragment, wherein the antigen binding fragment is selected from one or more of the following groups: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv and di-scFv.

[0158] In the present application, the CLDN18.2 binding domain may be a single-chain antibody. For example, the CD30 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises a VL domain-linker-VH domain or a VH domain-linker-VL domain from the N-terminus to the C-terminus. For example, the linker may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0159] For example, the CLDN18.2 binding domain includes but is not limited to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 29 or a functional variant thereof. The functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the CLDN18.2 binding domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the CD30 binding domain.

[0160] Signal peptide

[0161] The present application provides that CAR may include a signal peptide. For example, the signal peptide may include a signal peptide derived from CD8α. In some embodiments, the CAR provided herein includes a CD8α signal peptide, wherein the CD8α signal peptide includes an amino acid sequence shown in SEQ ID NO: 30 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide that has been substituted, deleted or added with one or more amino acids in the CD8α signal peptide; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the CD8α signal peptide.

[0162] Hinge area

[0163] The CAR provided herein may include a "hinge region" or a "hinge domain". The hinge region may connect two adjacent domains of the CAR, and may be located at any one or more positions between the extracellular binding domain, the transmembrane domain, the co-stimulatory domain, and / or the signaling domain of the CAR. For example, the hinge region may connect the extracellular antigen binding domain and the transmembrane domain. The N-terminus of the hinge region may be connected to the C-terminus of the binding domain, and the C-terminus of the hinge region may be connected to the N-terminus of the transmembrane domain. The connection may be directly connected by a peptide bond, or the connection may be connected by a linker. For example, the C-terminus of the extracellular binding domain is connected to the N-terminus of the hinge region by a peptide bond.

[0164] The CAR described herein may comprise 1, 2, 3, 4 or 5 or more hinge regions. The length of the hinge region may be about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any amino acids in between. The hinge region may be natural or synthetic. The hinge region may comprise a naturally occurring immunoglobulin hinge region or a variant of an immunoglobulin hinge region.

[0165] The CAR provided herein may comprise a hinge region derived from one or more of the following histones: CD28, IgG1, IgG4, IgD, CD137, CD4, CD27, CD7, CD8α, CD8β, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30 and LIGHT.

[0166] The CAR provided in the present application may include a hinge region, wherein the hinge region may include a CD8α hinge region. In the present application, the CD8α hinge region may include an amino acid sequence as shown in SEQ ID NO: 31 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide that has been substituted, deleted or added with one or more amino acids in the CD8α hinge region; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the transmembrane domain.

[0167] Transmembrane domain

[0168] The CAR provided herein may include a transmembrane domain. The transmembrane domain enables the fusion of the extracellular binding domain portion and the intracellular co-stimulatory domain, and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain described herein may be natural or synthetic.

[0169] The CAR provided herein may include a transmembrane domain derived from one or more of the following histones: CD8α, CD8β, CD28, CD3e, CD3ε, CD137, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, CD154, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64 and SLAM. The CAR provided herein may include a transmembrane domain, and the transmembrane domain may include a transmembrane domain derived from CD8α. In the present application, the CD8α transmembrane domain may comprise an amino acid sequence as shown in SEQ ID NO: 32 or a functional variant thereof. The functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the CD8α transmembrane domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the CD8α transmembrane domain.

[0170] Costimulatory domain

[0171] The CAR provided herein may include a co-stimulatory domain. The co-stimulatory domain is an intracellular domain that provides a co-stimulatory molecule for immune effector cells. The co-stimulatory molecule is a cell surface molecule required for the effective response of lymphocytes to antigens.

[0172] The CAR provided herein may include one or more costimulatory domains. In some embodiments, the CAR provided herein may include a costimulatory domain.

[0173] The CAR provided herein may comprise a co-stimulatory domain derived from the following histones: CD28, CD137, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

[0174] The CAR provided in the present application may include a costimulatory domain, wherein the costimulatory domain may include a CD28 costimulatory domain and / or a CD137 costimulatory domain. In the present application, the CD28 costimulatory domain may include an amino acid sequence as shown in SEQ ID NO:33 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide that is substituted, deleted or added with one or more amino acids in the CD28 costimulatory domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with a CD28 costimulatory domain. In the present application, the CD137 costimulatory domain may include an amino acid sequence as shown in SEQ ID NO:35 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the CD137 costimulatory domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the CD137 costimulatory domain.

[0175] In the CAR provided by the present application, the N-terminus of the co-stimulatory domain can be connected to the C-terminus of the transmembrane domain.

[0176] Signaling domain

[0177] The CAR provided herein may include a signaling domain. The signaling domain is an intracellular signaling domain that participates in conducting information of effective CAR binding target antigens into the interior of immune effector cells to induce the function of immune effector cells, and the function may include cell activation, cytokine production, cell proliferation and / or cytotoxic activity, including releasing cytotoxic factors to target cells bound by CAR, or other cellular responses triggered by antigens bound to extracellular CAR domains.

[0178] The CAR provided herein may comprise one or more signaling domains. In some embodiments, the CAR provided herein may comprise a signaling domain.

[0179] The CAR provided in the present application may comprise a signaling domain derived from the following group of proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12 and a domain protein comprising at least one ITAM.

[0180] The CAR provided in the present application may include a signaling domain, and the signaling domain includes a CD3ζ signaling domain. In the present application, the CD3ζ signaling domain may include an amino acid sequence as shown in SEQ ID NO: 34 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide that has been substituted, deleted or added with one or more amino acids in the CD3ζ signaling domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the CD3ζ signaling domain.

[0181] In the CAR provided by the present application, the N-terminus of the signaling domain can be connected to the C-terminus of the co-stimulatory domain.

[0182] Chimeric Antigen Receptor

[0183] The chimeric antigen receptor (CAR) provided by the present application may include a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain from the N-terminus to the C-terminus of the CAR.

[0184] The CAR provided in the present application may comprise a signal peptide sequence of the amino acid sequence shown in SEQ ID NO: 30, a CLDN18.2 binding domain of the amino acid sequence shown in SEQ ID NO: 5, a hinge region of the amino acid sequence shown in SEQ ID NO: 31, a transmembrane domain of the amino acid sequence shown in SEQ ID NO: 32, a costimulatory domain of the amino acid sequence shown in SEQ ID NO: 35, and an intracellular signal transduction domain of the amino acid sequence shown in SEQ ID NO: 34.

[0185] The CAR provided in the present application may comprise a signal peptide sequence of the amino acid sequence shown in SEQ ID NO: 30, a CLDN18.2 binding domain of the amino acid sequence shown in SEQ ID NO: 5, a hinge region of the amino acid sequence shown in SEQ ID NO: 31, a transmembrane domain of the amino acid sequence shown in SEQ ID NO: 32, a costimulatory domain of the amino acid sequence shown in SEQ ID NO: 33, and an intracellular signal transduction domain of the amino acid sequence shown in SEQ ID NO: 34.

[0186] The CAR provided in the present application may comprise a signal peptide sequence of an amino acid sequence as shown in SEQ ID NO: 29, a CLDN18.2 binding domain of an amino acid sequence as shown in SEQ ID NO: 5, a hinge region of an amino acid sequence as shown in SEQ ID NO: 31, a transmembrane domain of an amino acid sequence as shown in SEQ ID NO: 32, a co-stimulatory domain of an amino acid sequence as shown in SEQ ID NO: 35, and an intracellular signal transduction domain of an amino acid sequence as shown in SEQ ID NO: 34.

[0187] The CAR provided in the present application may comprise a signal peptide sequence of the amino acid sequence shown in SEQ ID NO: 29, a CLDN18.2 binding domain of the amino acid sequence shown in SEQ ID NO: 5, a hinge region of the amino acid sequence shown in SEQ ID NO: 31, a transmembrane domain of the amino acid sequence shown in SEQ ID NO: 32, a costimulatory domain of the amino acid sequence shown in SEQ ID NO: 33, and an intracellular signal transduction domain of the amino acid sequence shown in SEQ ID NO: 34.

[0188] In the present application, the CAR may comprise an amino acid sequence as shown in SEQ ID NO: 2, 39 or 41 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the CAR; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with CAR.

[0189] In the present application, the CAR may have one or more effects selected from the following: (1) excellent expression efficiency; (2) cells expressing the chimeric antigen receptor have significant anti-tumor activity; (3) cells expressing the chimeric antigen receptor have sustained anti-tumor activity; (4) cells expressing the chimeric antigen receptor have significant cell activation ability; and (5) cells expressing the chimeric antigen receptor have significant cytokine secretion ability.

[0190] Nucleic acids, vectors and cells

[0191] In one aspect, the present application provides an isolated nucleic acid molecule encoding the CAR described in the present application.

[0192] The present application provides one or more nucleic acid molecules, which may encode the CAR or its functional fragment described in the present application. For example, each of the one or more nucleic acid molecules may encode a complete CAR or a portion of a functional fragment thereof. In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence shown in SEQ ID NO: 1, 38 or 40 or a functional variant thereof.

[0193] The nucleic acid molecules described in the present application can be isolated. For example, they can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning recombination, (iii) purified, such as by enzyme cutting and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. The isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA technology. In the present application, nucleic acids encoding the antibodies and their antigen-binding fragments can be prepared by a variety of methods known in the art, including but not limited to, using restriction fragment manipulation or overlapping extension PCR using synthetic oligonucleotides, and specific operations can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

[0194] On the other hand, the present application provides a vector comprising a nucleic acid molecule encoding the CAR described in the present application.

[0195] The present application provides one or more vectors, which contain one or more nucleic acid molecules described in the present application. Each vector may contain one or more of the nucleic acid molecules. In addition, the vector may also contain other genes, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The expression control sequence may be an adjustable element. The specific structure of the expression control sequence may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that participate in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcription control functionally connected to a nucleic acid. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoter, CMV promoter and EF1a promoter. One or more nucleic acid molecules described in the present application may be operably linked to the expression control element. The vector may include a viral vector, such as a retroviral vector.

[0196] On the other hand, the application provides an immune effector cell cell, which may include and / or express the CAR described in the application, the nucleic acid molecules described in the application, and / or the vector described in the application. The nucleic acid molecules described in the application may be introduced into the cell by methods known in the art, such as electroporation, liposome transfection, viral infection, etc.

[0197] The immune effector cells provided by the present application contain and / or express the CAR described in the present application, the nucleic acid molecule described in the present application, and / or the vector described in the present application.

[0198] The immune effector cells provided by the application include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, γδT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells. In some embodiments, the immune effector cells provided by the application can be T cells. In some embodiments, the immune effector cells provided by the application can be human-derived.

[0199] Pharmaceutical composition

[0200] On the one hand, the present application provides a pharmaceutical composition comprising the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, and / or the immune effector cell described in the present application.

[0201] In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delay agents compatible with drug administration, usually safe and nontoxic. The acceptable components of the composition are preferably nontoxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions. In certain embodiments, the pharmaceutical composition may also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable preparations of preservatives.

[0202] In some embodiments, the pharmaceutical composition may further comprise instructions for use.

[0203] In the present application, the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration and / or administration via a subcutaneous depot.

[0204] use

[0205] On the other hand, the present application provides the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, and / or the use of the immune effector cell described in the present application in the preparation of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition is used to prevent and / or treat a disease or condition related to the expression of CLDN18.2.

[0206] On the other hand, the present application provides the use of the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the immune effector cell described in the present application and / or the pharmaceutical composition described in the present application in preventing and / or treating diseases or conditions related to the expression of CLDN18.2.

[0207] On the other hand, the present application provides a method for preventing and / or treating a disease or condition associated with the expression of CLDN18.2, comprising administering to a subject in need thereof an effective amount of the CAR described herein, the nucleic acid molecule described herein, the vector described herein, the immune effector cell described herein and / or the pharmaceutical composition described herein.

[0208] The diseases or conditions associated with the expression of CLDN18.2 described herein may include diseases or conditions associated with upregulated expression of CLDN18.2. The diseases or conditions associated with the expression of CLDN18.2 described herein may include tumors associated with the expression of CLDN18.2. For example, the tumors associated with the expression of CLDN18.2 described herein may include CLDN18.2-positive tumors. For example, the CLDN18.2-positive tumors contain tumor cells expressing CLDN18.2. The diseases or conditions associated with the expression of CLDN18.2 described herein may include solid tumors and / or hematological tumors associated with the expression of CLDN18.2. The diseases or conditions associated with the expression of CLDN18.2 described herein may include gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, bile duct cancer, bladder cancer and / or leukemia.

[0209] Preparation method

[0210] On the one hand, the present application provides a method for preparing the immune effector cells described in the present application, comprising introducing the nucleic acid molecules described in the present application and / or the vectors described in the present application into the immune effector cells.

[0211] In the present application, the method for preparing the immune effector cells described in the present application may include the step of obtaining the immune effector cells from a subject. For example, the T lymphocytes can be obtained from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumor. In addition, the method may further include the step of selecting a specific cell subset from the immune effector cells. For example, a specific T cell subset can be selected by the specific expression of CD3, CD28, CD4, CD8, CD45RA and CD45RO.

[0212] On the other hand, the present application provides a method for preparing the CAR described in the present application, the method comprising culturing the immune effector cells described in the present application under the conditions of CAR expression described in the present application.

[0213] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0214] Example

[0215] Example 1 Preparation of CAR cells targeting CLDN18.2

[0216] 1.1 Construction of CLDN18.2 CAR gene vector

[0217] In this example, a retroviral vector capable of expressing the CLDN18.2 CAR gene was constructed.

[0218] First, the pSF Lenti expression plasmid pSF-Genome-SFFV-EGFP-KanR (WuXi Biologics Cat#Q6974) was modified by replacing the SFFV promoter and EGFP with the EF1a promoter and the multiple restriction site MCS to obtain the main plasmid of the retroviral vector pSF-EF1a-MCS-KanR.

[0219] Secondly, the CLDN18.2 CAR gene was constructed on the modified retroviral vector pSF-EF1a-MCS-KanR. The gene structures of each CLDN18.2 CAR are as follows: CAR-B (ZG59) ScFv-CD8-CD28-CD3ζ (hereinafter referred to as 59B-CD28) contains CD8α signal peptide, CLDN18.2 (ZG59B) targeting binding domain, CD8α hinge, CD8α transmembrane domain, CD28 costimulatory domain and CD3ζ signaling domain ( Figure 1 ); CAR-B (ZG59) ScFv-CD8-CD137-CD3ζ (hereinafter referred to as 59B-CD137) contains CD8α signal peptide, CLDN18.2 (ZG59B) targeting binding domain, CD8α hinge, CD8α transmembrane region, CD137 costimulatory domain and CD3ζ signaling domain ( Figure 1 ); CAR-BScFv-CD8-CD28-CD3ζ (hereinafter referred to as B-CD28 or CAR-B) contains CD8α signal peptide, CLDN18.2 (B) targeting binding domain, CD8α hinge, CD8α transmembrane region, CD137 co-stimulatory domain and CD3ζ signaling domain.

[0220] 1.2 CLDN18.2 CAR lentivirus preparation

[0221] Four plasmid systems were used to package the vectors. HEK293T cells in the logarithmic growth phase were inoculated in a 15 cm cell culture dish at a density of 1.3 x 10 7cells / 30 ml culture medium / dish, the culture medium is DMEM (VivaCell, Cat#C3113-0500) containing 10% FBS. After culturing for 24 hours, when the cell density reaches 80%-90%, it can be used for transfection and replaced with serum-free culture medium 2 hours before transfection. In a 1.5 ml low-adsorption EP tube, 18.75 μg of pSF-EF1a-MCS-KanR vector plasmid carrying the CLDN18.2CAR gene, 7.5 μg of pSF-Rev-KanR (WuXi Bioscience Cat#Q6972) vector plasmid, 15 μg of pSF-VSV-G-KanR (WuXi Bioscience Cat#Q6973) vector plasmid, 18.75 μg of pSF-GagPol-KanR (WuXi Bioscience Cat#Q6975) vector plasmid and 180 uL of PEI were added and mixed evenly, and the total volume was adjusted to 1 ml. After incubation at room temperature for 15 minutes, it was added dropwise to the culture medium inoculated with HEK 293T cells, mixed well and placed at 37 ° C, 5% CO 2 Culture in a cell culture incubator. 6 hours after transfection, discard the supernatant, wash with PBS and replace with 30 ml of fresh DMEM medium containing 10% FBS, and continue to culture in a 37°C, 5% CO2 incubator. 48 hours after transfection, collect the supernatant in a 50 ml centrifuge tube, centrifuge at 4°C, 4000g for 10 minutes to remove cell debris, filter the supernatant with a 0.45μm filter and collect it in a 40 ml ultracentrifuge tube, centrifuge at 4°C, 20000rpm for 3 hours, discard the supernatant, add virus preservation solution, gently blow and repeatedly resuspend, and after sufficient dissolution, centrifuge at 10000rpm for 5 minutes, aliquot the supernatant and store at -80°C. 10μl of virus dilution solution infects 2x10 4 HEK293T cells (100 μl) were added, and the cells were collected after 48 hours, washed twice with FACS buffer (PBS containing 2% FBS), and then stained with Anti-G4S n(B02H1) mAb (PE) (Heyousheng, Cat# GS-ARPE100) at 4°C for 30 minutes. After the staining, the cells were washed twice with FACS buffer (PBS solution containing 2% FBS), and the cells were resuspended in 200ul FACS buffer and the expression of CAR was detected by flow cytometry.

[0222] The virus sample titer was calculated according to the formula IU / ml=(F×Ci / V)×D, where F is the cell positivity rate, Ci is the total number of cells during conduction, V is the volume of the inoculated virus, and D is the viral vector dilution multiple.

[0223] The experimental results show that ( Figure 2), under the same experimental conditions, the virus titers of the 59B-CD137 and 59B-CD28 experimental groups were 1.96E8 TU / ml and 7.66E7 TU / ml, respectively.

[0224] Example 2 Preparation of CLDN18.2 CAR-T cells and detection of expression levels

[0225] 2.1 Preparation of CLDN18.2 CAR-T cells

[0226] By EasySep TM Human T Cell Isolation Kit (Stem Cell, Cat#17951) operation process: Human primary T cells were isolated from human peripheral blood mononuclear cells, added to CD3 / CD28 Dynabeads (Gibco, Cat#11131D) and activated for 48 hours before CAR virus infection (MOI of 15). After 48 hours of infection, CLDN18.2 CAR-T cells were collected.

[0227] 2.2 Detection of CLDN18.2 CAR-T cell positivity and expression level

[0228] The collected CLDN18.2 CAR-T cells were washed twice with FACS buffer, and then Anti-G4Sn (B02H1) antibody (PE) (Heyousheng, Cat# GS-ARPE100) was added and stained at 4°C for 30 minutes. After staining, the cells were washed twice with FACS buffer, and the cells were resuspended in 200ul FACS buffer for flow cytometry detection of CAR expression. The experimental results are shown in Figure 3 and Figure 4 The results showed that compared with the negative control group, the CAR positivity rates of CART-CD137 and CART-59B-CD28 were 76.6% and 47.1%, respectively, and the CAR positivity rate of CART-B was 42.1%.

[0229] Example 3 Antigen Binding Activity of Different Anti-CLDN18.2 Sequences

[0230] This example compares the antigen binding activity of different CLDN18.2 CAR-T cells when the ZG59B sequence is used as the antigen binding domain of CAR and the B sequence is used as the antigen binding domain of CAR. The CDR sequence of the ZG59B sequence and the B sequence are the same but the FR sequence is different, wherein the ZG59B sequence is the sequence after humanization of the B sequence.

[0231] Coat the plate one day in advance, dilute CLDN18.2-His to 1ug / ml with 1xELISA coating solution, take the ELISA enzyme-labeled plate, add 100ul / well, and coat overnight at 4℃; take out the plate, equilibrate to room temperature, and prepare PBST (0.05% Tween-20in 1x PBS) and blocking solution (2% BSAin PBST); discard the supernatant, add 300ul / well PBST to wash the plate, and repeat 6 times; add 300ul / well blocking solution, block at 37℃ for 2 hours; prepare diluent (0.5% BSAin PBST), and use the diluent to dilute the humanized antibody purified protein to 1ug / ml, and perform 5-fold gradient dilution, with a total of 8 concentration points; after blocking, wash the plate 6 times with PBST, add 100ul / well of diluted antibody or humanized antibody supernatant, and incubate at 37℃ for 2 hours; dilute the detection antibody Goat anti human IgG with diluent 1:10000 Fc (HRP); after incubation, wash the plate 6 times with PBST, add 100ul / well of detection antibody, and incubate at 37°C for 1 hour; after incubation with detection antibody, wash the plate 6 times with PBST, add 100ul / well of 1x TMB, and develop color at room temperature in the dark for about 3 minutes; add 50ul / well ELISA stop solution to stop color development, and read the OD450 value with a multifunctional microplate reader.

[0232] The experimental results show that ( Figure 5 ), both the sequences before and after humanization showed strong binding activity to human CLDN18.2-His, and were positively correlated with the concentration, with no significant difference before and after humanization (EC50 before humanization was 1.624 ng / ml; EC50 after humanization was 1.734 ng / ml).

[0233] Example 4 In vitro functional evaluation of CLDN18.2 CAR-T

[0234] This example studies the basal signal index and exhaustion score of CLDN18.2 CAR-T with different CAR structures, and evaluates and compares the killing effect of CAR-T cells on CLDN18.2-positive tumor cells.

[0235] 4.1. Calculation of Basal Signaling Index and Depletion Score

[0236] Jurkat cells were infected with viruses expressing different CAR structures, and the basal signal index of the cells was detected on the 3rd day after infection. After gating PE-positive cells, the mean fluorescence intensity (MFI) of Anti-G4Sn(B02H1) mAb (PE) (Heyousheng, Cat#GS-ARPE100) and CD69 (BD, Cat#740460) in the cells were measured.

[0237] The CAR basal signal index calculation formula is as follows:

[0238] CAR Basal Signal Index = MFICD69:MFIGFP

[0239] To calculate the exhaustion score, primary T cells activated 48 hours after infection with viruses expressing different CAR structures were gated for PE-positive cells on day 10 after infection. Flow cytometry was used to detect the expression of PD-1 (BD, Cat#563245), Tim-3 (BD, Cat#742857) and Lag-3 (BD, Cat#565716) on the cell surface in Anti-G4S n(B02H1) mAb (PE) (Heyousheng, Cat#GS-ARPE100) positive cells.

[0240] The CAR exhaustion score was assessed using the following formula:

[0241] CAR exhaustion score = (MFI PD-1 / MFI GFP +MFI Lag-3 / MFI GFP +MFI Tim-3 / MFI GFP ) / 3

[0242] The exhaustion score of each CAR-T cell was calculated by taking the average expression of three exhaustion markers, PD-1 (BD, Cat#563245), Tim-3 (BD, Cat#742857), and Lag-3 (BD, Cat#565716), and directly compared.

[0243] The experimental results are as follows Figure 6 The results showed that there was a significant positive correlation between the exhaustion score and the basal signal index, and both CART-59B-CD28 and CART-59B-CD137 had lower exhaustion scores and basal signal indexes.

[0244] 4.2 Evaluation of the killing effect on tumor cells

[0245] In this example, the effect of different effector-target ratios on the killing rate of tumor cells was studied. The effector-target ratio (CAR-T:Tumor cell) was 5:1, 3:1, 1:1, 1:3 and 1:5. NUGC-4, CLDN18.2 CAR-T and UTD cells were collected and adjusted to an appropriate cell density with Assay buffer (RPMI1640 containing 10% FBS). 100ul / well (cell number was 5e3 / well) of tumor cells and 100ul / well of CAR-T or UTD cells were added to a 96-well cell culture plate. After the cells were co-incubated overnight, an equal volume of Luciferase detection reagent (Vkeybio, A2000102N) was added. After incubation at room temperature for 5 minutes, the luminescent signal value was read and calculated according to the formula: Killing rate (%) = (1-Lum treated / Lum control )*100, the killing rate of tumor cells in each treatment group was calculated, and the line graph of effect-target ratio (X axis) and cell killing rate (Y axis) was drawn using GraphPad Prism software, n≥3. The results are shown in Figure 7 and Figure 8 shown.

[0246] The experimental results showed that CART-59B-CD28, CART-59B-CD137 and CART-B all showed strong killing effects on tumor cells. The killing effect was more obvious when the effector-target ratio of CART-59B-CD28 and CART-59B-CD137 was 5:1 and 3:1. At the same effector-target ratio, there was no significant difference in the tumor cell killing effect of CART-59B-CD28 and CART-59B-CD137.

[0247] 4.3 Evaluation of CLDN18.2 CAR-T cell factor release

[0248] This experiment uses the ELISA method to detect the levels of key cytokines IL-2 and IFN-γ released into the cell supernatant during the killing of tumor cells by CART-B cells. The experiment was carried out according to the ELISA protocol (Biolegend, Cat#431801, #430101), and the cell supernatant was diluted at different times to be within the detection range. GraphPad Prism software was used for data processing to draw a standard curve of cytokine concentration (X axis) and OD_blanked (Y axis), where OD_blanked = OD 样品孔 –OD 空白孔 , and the release level of cytokines in the supernatant of each cell was calculated, n≥3.

[0249] The experimental results showed that CART-B showed a strong ability to secrete cytokines IFN-γ and IL-2 when killing tumor cells, and CART-B also showed a strong cytokine release ability for different tumor cells ( Figures 9A-9B ). After humanization, CART-59B-CD28 and CART-59B-CD137 showed a strong ability to secrete cytokines IFN-γ and IL-2 when killing tumor cells ( Fig. 9C ).

[0250] Example 5 In vivo efficacy experiment of CLDN18.2 CART cells

[0251] 5.1 Efficacy results of NUGC-4-CLDN18.2 mouse model

[0252] 32 M-NSG mice (6-8 weeks, female) were subcutaneously inoculated with NUGC-4-CLDN18.2 cells (cell number: 5E6 / mouse + 50% Matrigel), and the survival status and subcutaneous tumor formation of the mice were observed every other day after inoculation. When the average tumor volume reached 100mm 3 At the time of the experiment, 16 mice were enrolled and divided into 2 experimental groups, with 8 mice in each group. Including Group 1 UTD: 9E5 cells / 200ul / mouse, iv (hereinafter referred to as Group 1), Group 2 CAR-T: 5E5 CAR + cells / 200ul / cell, iv (hereinafter referred to as Group 2). This experiment used CAR-T cells + is 55%.

[0253] Experimental animal grouping

[0254] Table 1 Animal groups of NUGC-4-CLDN18.2 efficacy experiment

[0255]

[0256] On the 36th day after the mice were given the drug, the average tumor volume of the control group Group 1 was 1924.68±165.14mm 3 Group 2 (5e5 CAR + ) The average tumor volume was 615.26±199.99mm 3 The tumor volume inhibition rate (TGI) was 71.76%. Compared with the tumor volume of the control group, there was a significant difference (P<0.0001).

[0257] On the 36th day after the start of administration, the average weight changes of mice in Group 1 and Group 2 were 1.8% and 8.3%, respectively. On the 36th day after administration, one mouse in the control group Group 1 died abnormally, and two mice were killed due to excessive tumor volume.

[0258] The experimental results show that ( Fig.10 ), CLDN18.2 CAR-T was able to better control the tumor size compared to the control group (T cells without CAR), showing a stronger anti-tumor effect in mice.

[0259] 5.2 Efficacy results of humanized NUGC-4-CLDN18.2 mouse model

[0260] NOG mice (6-8 weeks, female) were subcutaneously inoculated with NUGC4-CLDN18.2 cells (cell number: 5E6 / mouse + 30% Matrigel), and the survival status and subcutaneous tumor formation of the mice were observed every other day after inoculation. When the average tumor volume reached 100mm 3 At the time of the experiment, 24 mice were enrolled and divided into 3 experimental groups, with 8 mice in each group. Including Group 1 UTD: 1.3E7 cells / 200ul / mouse, iv (hereinafter referred to as Group 1); Group 2 59B-CD137: 5E6 CAR + cells / 200ul / cell, iv (hereinafter referred to as Group 2); Group 3 59B-CD137:1E6 CAR + cells / 200ul / cell, iv (hereinafter referred to as Group 3). This experiment used 59B-CD137 cell CAR + It is 55.1%.

[0261] Experimental animal grouping

[0262] Table 2 Animal groups of NUGC-4-CLDN18.2 efficacy experiment

[0263]

[0264] The experimental results show that ( Fig.11 ), 59B-CD137 had significant tumor inhibition effects at both dosages, and was dose-dependent.

[0265] Example 6 Comparison of in vivo efficacy of CLDN18.2 CART cells

[0266] Comparison of drug efficacy in NUGC-4-CLDN18.2 mouse model

[0267] NOG mice (6-8 weeks, female) were subcutaneously inoculated with NUGC4-CLDN18.2 cells (cell number: 5E6 / mouse + 30% Matrigel), and the survival status and subcutaneous tumor formation of the mice were observed every other day after inoculation. When the average tumor volume reached 100mm 3 At the time of the experiment, 24 mice were enrolled and divided into 3 experimental groups, with 8 mice in each group. Including Group 1 UTD: 1.3E7 cells / 200ul / mouse, iv (hereinafter referred to as Group 1); Group 2 59B-CD137: 5E6 CAR + cells / 200ul / cell, iv (hereinafter referred to as Group 2); Group 3 Prior art CAR-T cells targeting CLDN18.2 (the amino acid sequence of the prior art CAR is shown in SEQ ID NO: 42): 5E6 CAR + cells / 200ul / cell, iv (hereinafter referred to as Group 3). This experiment used 59B-CD137 cell CAR + 55.1%; Existing technology CAR-T cell CAR targeting CLDN18.2 + It is 37.6%.

[0268] On the 11th day after administration, the mice were dissected when their condition was relatively poor, and the organs were taken for histochemistry (IHC). Experimental conditions: hCD3 was used as the primary antibody, and Wonfo secondary antibody was used as the secondary antibody. Experimental samples: mouse spleen, liver, kidney, and tumor.

[0269] Experimental animal grouping

[0270] Table 3 Animal groups of NUGC-4-CLDN18.2 efficacy experiment

[0271]

[0272] The experimental results show that ( Figures 12A-12B ), CLDN18.2 CAR-T cells were able to better control the size of tumors compared to the control group, showing a stronger anti-tumor effect in mice. At the same time, at the same dose, 59B-CD137 cells showed a stronger tumor infiltration effect than the existing CAR-T cells targeting CLDN18.2.

Claims

1. A chimeric antigen receptor (CAR), comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody or antigen binding fragment that binds to CLDN18.2, wherein the antibody or antigen binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein, The VH comprises: 1) a heavy chain complementarity determining region 1 (HCDR1), wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6; 2) a heavy chain complementarity determining region 2 (HCDR2), wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7; 3) a heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8; The VL comprises: 1) a light chain complementary determining region 1 (LCDR1), wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9; 2) a light chain complementary determining region 2 (LCDR2), wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10; 3) Light chain complementarity determining region 3 (LCDR3), wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

11.

2. The CAR of claim 1, wherein the VH comprises a heavy chain framework region 1 (H-FR1), a heavy chain framework region 2 (H-FR2), a heavy chain framework region 3 (H-FR3), and a heavy chain framework region 4 (H-FR4), wherein: The H-FR1 comprises an amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 20, and the C-terminus of H-FR1 is connected to the N-terminus of the HCDR1; the H-FR2 comprises an amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 21, and H-FR2 is located between the HCDR1 and the HCDR2; the H-FR3 comprises an amino acid sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 22, and H-FR3 is located between the HCDR2 and the HCDR3; the H-FR4 comprises an amino acid sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 23, and H- The N-terminus of FR4 is connected to the C-terminus of HCDR3.

3. The CAR of claim 1, wherein the VL comprises a light chain framework region 1 (L-FR1), a light chain framework region 2 (L-FR2), a light chain framework region 3 (L-FR3), and a light chain framework region 4 (L-FR4), wherein: The L-FR1 comprises an amino acid sequence as shown in SEQ ID NO: 16 or SEQ ID NO: 24, and the C-terminus of L-FR1 is connected to the N-terminus of LCDR1; the L-FR2 comprises an amino acid sequence as shown in SEQ ID NO: 17, and L-FR2 is located between LCDR1 and LCDR2; the L-FR3 comprises an amino acid sequence as shown in SEQ ID NO: 18 or SEQ ID The amino acid sequence shown in SEQ ID NO: 25, L-FR3 is located between the LCDR2 and the LCDR3; the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 26, and the N-terminus of L-FR4 is connected to the C-terminus of the LCDR3.

4. The CAR according to claim 1, wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 27, and / or; the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO:

28.

5. The CAR according to claim 1, wherein the antigen binding fragment is selected from one or more of the following groups: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv and di-scFv.

6. The CAR according to claim 1, wherein the antigen binding fragment comprises SEQ ID NO: 5 or SEQ ID The amino acid sequence shown in NO:

29.

7. The CAR according to any one of claims 1-6, comprising a signal peptide, wherein the signal peptide includes a CD8α signal peptide, and the CD8α signal peptide comprises an amino acid sequence as shown in SEQ ID NO:

30.

8. The CAR according to any one of claims 1-6, comprising a hinge region, wherein the N-terminus of the hinge region is connected to the C-terminus of the binding domain, the C-terminus of the hinge region is connected to the N-terminus of the transmembrane domain, the hinge region includes a CD8α hinge region, and the CD8α hinge region comprises the amino acid sequence shown in SEQ ID NO:

31.

9. The CAR according to any one of claims 1-6, comprising a transmembrane domain, wherein the transmembrane domain includes a CD8α transmembrane domain, and the CD8α transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

32.

10. The CAR according to any one of claims 1 to 6, comprising one or more co-stimulatory domains, the co-stimulatory domains comprising a CD28 co-stimulatory domain and / or a CD137 co-stimulatory domain, the CD28 co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 33, the CD137 co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 35, and the N-terminus of the co-stimulatory domain is connected to the C-terminus of the transmembrane domain.

11. The CAR according to any one of claims 1-6, comprising one or more signaling domains, the signaling domains comprising a CD3ζ signaling domain, the CD3ζ signaling domain comprising an amino acid sequence as shown in SEQ ID NO: 34, and the N-terminus of the signaling domain is connected to the C-terminus of the co-stimulatory domain.

12. The CAR according to any one of claims 1-6, comprising a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain and a signaling domain.

13. The CAR according to any one of claims 1-6, comprising the amino acid sequence shown in SEQ ID NO: 2, 39 or 41.

14. An isolated nucleic acid molecule encoding the CAR of any one of claims 1-13. The nucleic acid molecule according to claim 14 , comprising the nucleotide sequence shown in SEQ ID NO: 1, 38 or 40.

16. A vector comprising the nucleic acid molecule according to any one of claims 14-15.

17. The vector of claim 16, which comprises a viral vector.

18. The vector according to any one of claims 16-17, which comprises a retroviral vector.

19. An immune effector cell comprising and / or expressing the CAR of any one of claims 1 to 13, claims 14 to 15 The isolated nucleic acid molecule of any one of claims 1 to 18, and / or the vector of any one of claims 16 to 18.

20. The immune effector cell of claim 19, comprising T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, γδT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

21. The immune effector cell of claim 19, wherein the immune effector cell is a T cell.

22. The immune effector cell according to any one of claims 19-21, wherein the immune effector cell is of human origin.

23. A method for preparing the immune effector cell of any one of claims 19-22, comprising introducing into the immune effector cell the isolated nucleic acid molecule of any one of claims 14-15, and / or the vector of any one of claims 16-18.

24. A pharmaceutical composition comprising the CAR of any one of claims 1-13, the isolated nucleic acid molecule of any one of claims 14-15, the vector of any one of claims 16-18, and / or the immune effector cell of any one of claims 19-22, and a pharmaceutically acceptable carrier.

25. Use of the CAR of any one of claims 1-13, the isolated nucleic acid molecule of any one of claims 14-15, the vector of any one of claims 16-18, and / or the immune effector cell of any one of claims 19-22 in the preparation of a pharmaceutical composition.

26. The use according to claim 25, wherein the pharmaceutical composition is used to treat tumors.

27. The use according to claim 25, wherein the pharmaceutical composition is used to treat CLDN18.2-positive tumors.

28. The use according to claim 27, wherein the CLDN18.2-positive tumors include gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, bile duct cancer, bladder cancer and / or leukemia.

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