Preparation and application of CAR-T targeting FOLR1

Abstract

The invention provides a preparation method and an application of a CAR-T (chimeric antigen receptor T) targeting FOLR1 (fibronectin receptor 1). Specifically, the invention provides a chimeric antigen receptor (CAR) construct of a targeted folate receptor 1 (FOLR1), a nucleic acid molecule, a carrier, a host cell, an engineered immune cell, a pharmaceutical composition, application and a preparation method thereof. The CAR and the CAR-immune cells constructed by the CAR can specifically recognize and kill tumor cells of a high-expression tumor-associated antigen FOLR1 in a targeted manner, and a new way is provided for treatment of FOLR1 positive tumors (including ovarian cancer, triple negative breast cancer, mesothelioma, non-small cell lung cancer, endometrial cancer and the like).

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the preparation and application of a FOLR1 target CAR-T. Background Art

[0002] Globally, ovarian cancer is the seventh most common cancer in women and the eighth leading cause of cancer death. Despite advances in surgical methods and chemotherapy, the 5-year survival rate is still less than 45%. More importantly, the number of cases diagnosed each year is also increasing, posing a serious threat to women's lives.

[0003] Cellular immunotherapy is a new type of precision targeted therapy and a tumor treatment model with significant efficacy, especially CAR-T. CAR-T cell therapy first collects cells from the patient's body, and then modifies them through activation and genetic engineering so that the T cells express chimeric antigen receptors (CAR) on their surface. The chimeric antigen receptor is composed of an extracellular antigen binding region, i.e., scFv that recognizes tumor-associated antigens, a transmembrane region, and an intracellular signaling domain. These CAR-T cells are then infused back into the patient's body, where they can recognize and eliminate tumor cells that express the specified antigen.

[0004] Folate receptor 1 (FOLR1), also known as folate receptor α or folate binding protein, is expressed at lower levels in normal tissues compared to tumor tissues. It is expressed on the plasma membrane and can mediate cell uptake of folic acid and affect downstream pathways to promote tumor growth. Studies have shown that FOLR1 is highly expressed in most ovarian cancers (accounting for 76% of expression in malignant ovarian cancer) and uterine cancer, endometrial cancer, pancreatic cancer, renal cancer, lung cancer and breast cancer. It can mediate cell uptake of folic acid or generate regulatory signals to promote tumor cell growth. This expression pattern of FOLR1 makes it a good target for CAR-T cell therapy of ovarian cancer.

[0005] Given that there are relatively few drugs available for the treatment of ovarian cancer, the development of a specific drug for the treatment of ovarian cancer has become an urgent matter. This field urgently needs to develop a CAR-T cell that can be efficiently modified and mobilized and reinfused to the greatest extent possible, so that it can specifically kill ovarian cancer cells. Summary of the invention

[0006] The purpose of the present invention is to provide a FOLR1-targeted CAR-T cell that can be efficiently modified and mobilized and reinfused to the greatest extent.

[0007] In a first aspect of the present invention, a chimeric antigen receptor (CAR) construct targeting folate receptor 1 (FOLR1) is provided, wherein the antigen binding domain of the CAR comprises a heavy chain variable region and a light chain variable region,

[0008] Wherein, the heavy chain variable region includes the following complementarity determining region HCDR:

[0009] HCDR1 shown in SEQ ID NO:36 or 26 or 31,

[0010] HCDR2 shown in SEQ ID NO:37 or 27 or 32, and

[0011] HCDR3 shown in SEQ ID NO:38 or 28 or 33;

[0012] Furthermore, the light chain variable region includes the following complementarity determining region LCDR:

[0013] LCDR1 shown in SEQ ID NO:39 or 29 or 34,

[0014] LCDR2 indicated by YTS or AAT, and

[0015] LCDR3 shown in SEQ ID NO:40 or 30 or 35.

[0016] In another preferred embodiment, the heavy chain variable region and the light chain variable region are selected from the following groups:

[0017] (1) The heavy chain variable region includes the following complementarity determining region HCDR:

[0018] HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, and HCDR3 shown in SEQ ID NO:38;

[0019] Furthermore, the light chain variable region includes the following complementarity determining region LCDR:

[0020] LCDR1 shown in SEQ ID NO:39, LCDR2 shown in YTS, and LCDR3 shown in SEQ ID NO:40;

[0021] (2) The heavy chain variable region includes the following complementarity determining region HCDR:

[0022] HCDR1 shown in SEQ ID NO:26, HCDR2 shown in SEQ ID NO:27, and HCDR3 shown in SEQ ID NO:28;

[0023] Furthermore, the light chain variable region includes the following complementarity determining region LCDR:

[0024] LCDR1 shown in SEQ ID NO:29, LCDR2 shown in AAT, and LCDR3 shown in SEQ ID NO:30; or

[0025] (3) The heavy chain variable region includes the following complementarity determining region HCDR:

[0026] HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, and HCDR3 shown in SEQ ID NO:33;

[0027] Furthermore, the light chain variable region includes the following complementarity determining region LCDR:

[0028] LCDR1 shown in SEQ ID NO:34, LCDR2 shown in YTS, and LCDR3 shown in SEQ ID NO:35.

[0029] In another preferred embodiment, the structure of the antigen binding domain is shown in Formula A or Formula B below:

[0030] V H -V L (A)

[0031] V L -V H (B)

[0032] Where V H is the antibody heavy chain variable region;

[0033] V L is the antibody light chain variable region;

[0034] “-” represents a connecting peptide or peptide bond.

[0035] In another preferred embodiment, the V H The amino acid sequence is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or a combination thereof.

[0036] In another preferred embodiment, the V L The amino acid sequence is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or a combination thereof.

[0037] In another preferred embodiment, the V H The amino acid sequence is shown in SEQ ID NO: 7, and V L The amino acid sequence is shown in SEQ ID NO:8.

[0038] In another preferred embodiment, the V H The amino acid sequence is shown in SEQ ID NO: 9, and V LThe amino acid sequence is shown in SEQ ID NO:10.

[0039] In another preferred embodiment, the V H The amino acid sequence is shown in SEQ ID NO: 11, and V L The amino acid sequence is shown in SEQ ID NO:12.

[0040] In another preferred embodiment, the antibody heavy chain variable region and the antibody light chain variable region are connected via a connecting peptide.

[0041] In another preferred embodiment, the connecting peptide is 1-4 consecutive GGGGS sequences, preferably 2-4, and more preferably 3.

[0042] In another preferred example, the amino acid sequence of the connecting peptide is shown in SEQ ID NO:13.

[0043] In another preferred embodiment, the heavy chain variable region and light chain variable region of the antigen binding domain are derived from mouse, human, or humanized antibodies.

[0044] In another preferred embodiment, the CAR has a structure as shown in the following formula I:

[0045] L-EB-H-TM-C-CD3ζ(I)

[0046] In the formula,

[0047] Each "-" is independently a connecting peptide or a peptide bond;

[0048] L is an optional signal peptide sequence;

[0049] EB is the extracellular binding domain that targets FOLR1;

[0050] H is an optional hinge region;

[0051] TM is the transmembrane domain;

[0052] C is a co-stimulatory signal molecule;

[0053] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0054] In another preferred embodiment, the EB comprises a heavy chain variable region and a light chain variable region selected from the following groups:

[0055] (1) The heavy chain variable region includes the following complementarity determining region HCDR:

[0056] HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, and HCDR3 shown in SEQ ID NO:38;

[0057] Furthermore, the light chain variable region includes the following complementarity determining region LCDR:

[0058] LCDR1 shown in SEQ ID NO:39, LCDR2 shown in YTS, and LCDR3 shown in SEQ ID NO:40;

[0059] (2) The heavy chain variable region includes the following complementarity determining region HCDR:

[0060] HCDR1 shown in SEQ ID NO:26, HCDR2 shown in SEQ ID NO:27, and HCDR3 shown in SEQ ID NO:28;

[0061] Furthermore, the light chain variable region includes the following complementarity determining region LCDR:

[0062] LCDR1 shown in SEQ ID NO:29, LCDR2 shown in AAT, and LCDR3 shown in SEQ ID NO:30; or

[0063] (3) The heavy chain variable region includes the following complementarity determining region HCDR:

[0064] HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, and HCDR3 shown in SEQ ID NO:33;

[0065] Furthermore, the light chain variable region includes the following complementarity determining region LCDR:

[0066] LCDR1 shown in SEQ ID NO:34, LCDR2 shown in YTS, and LCDR3 shown in SEQ ID NO:35.

[0067] In another preferred example, the EB comprises the antibody heavy chain variable region shown in SEQ ID NO:7, and the antibody light chain variable region shown in SEQ ID NO:8.

[0068] In another preferred example, the EB comprises the antibody heavy chain variable region shown in SEQ ID NO:9, and the antibody light chain variable region shown in SEQ ID NO:10.

[0069] In another preferred example, the EB comprises the antibody heavy chain variable region shown in SEQ ID NO:11, and the antibody light chain variable region shown in SEQ ID NO:12.

[0070] In another preferred embodiment, L is a signal peptide of an immune cell surface molecule commonly used in the art, preferably a signal peptide of a protein selected from the following group: CD8, CD28, GM-CSF, CD4, CD137, NKG2D, or a combination thereof.

[0071] In another preferred embodiment, the L is a CD8 leader sequence (signal peptide), and its amino acid sequence is shown in SEQ ID NO:2.

[0072] In another preferred embodiment, the sequence of "H-TM-C" in Formula I can be the full-length sequence consisting of the extracellular domain (hinge region) of CD28, the transmembrane region of CD28 and the intracellular domain of CD28 (ie, Extra_CD28 TM_ICD).

[0073] In another preferred example, the amino acid sequence of the Extra_CD28 TM_ICD is shown in SEQ ID NO:1.

[0074] In another preferred embodiment, the H may be a hinge region of a protein selected from the group consisting of CD8 (including CD8α), CD28, CD137, NKG2D, or a combination thereof.

[0075] In another preferred embodiment, the H is the hinge region of CD8α, and its amino acid sequence is shown in SEQ ID NO:3.

[0076] In another preferred embodiment, the TM may be a transmembrane region of an immune cell surface molecule commonly used in the art, preferably a transmembrane region of a protein selected from the following group: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, NKG2D, DAP10, DAP12, or a combination thereof.

[0077] In another preferred embodiment, the TM includes a transmembrane region derived from CD8, and its amino acid sequence is shown in SEQ ID NO:4.

[0078] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, DAP10, DAP12, or a combination thereof.

[0079] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from 4-1BB, and its amino acid sequence is shown in SEQ ID NO:5.

[0080] In another preferred example, the amino acid sequence of CD3ζ is shown in SEQ ID NO:6.

[0081] In another preferred embodiment, the CAR has an amino acid sequence selected from the following group:

[0082] SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or a combination thereof.

[0083] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:14.

[0084] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO: 20.

[0085] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:16.

[0086] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:24.

[0087] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:18.

[0088] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:22.

[0089] In a second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the chimeric antigen receptor (CAR) construct as described in the first aspect of the present invention.

[0090] In another preferred embodiment, the nucleic acid molecule has a nucleotide sequence selected from the following group:

[0091] SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a combination thereof.

[0092] In the third aspect of the present invention, a vector is provided, wherein the vector contains the nucleic acid molecule as described in the second aspect of the present invention.

[0093] In another preferred embodiment, the vector includes DNA and RNA.

[0094] In another preferred embodiment, the vector is selected from the following group: a plasmid, a viral vector, a transposon, or a combination thereof.

[0095] In another preferred embodiment, the vector includes a DNA virus or a retroviral vector.

[0096] In another preferred embodiment, the vector is selected from the following group: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof.

[0097] In another preferred embodiment, the vector is a lentiviral vector.

[0098] In another preferred embodiment, the vector is in the form of a virus particle.

[0099] In the fourth aspect of the present invention, a host cell is provided, which contains the vector as described in the third aspect of the present invention, or an exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated into its chromosome, or it expresses the chimeric antigen receptor (CAR) construct as described in the first aspect of the present invention.

[0100] In another preferred embodiment, the host cell is an isolated cell.

[0101] In another preferred embodiment, the host cell is a genetically engineered cell.

[0102] In another preferred embodiment, the host cells include eukaryotic cells and prokaryotic cells.

[0103] In another preferred embodiment, the cell is a mammalian cell.

[0104] In another preferred embodiment, the host cell includes Escherichia coli.

[0105] In the fifth aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell expresses the chimeric antigen receptor (CAR) construct as described in the first aspect of the present invention.

[0106] In another preferred embodiment, the engineered immune cells include: T cells, NK cells, or a combination thereof.

[0107] In another preferred embodiment, the T cells include αβT cells, γδT cells, NKT cells, MAIT cells, or a combination thereof.

[0108] In another preferred embodiment, the engineered immune cells are CAR-T cells or CAR-NK cells or CAR-NKT cells, preferably CAR-T cells.

[0109] In a sixth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0110] (1) an active ingredient selected from the group consisting of a chimeric antigen receptor (CAR) construct as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, or an engineered immune cell as described in the fifth aspect of the present invention, or a combination thereof; and

[0111] (2) A pharmaceutically acceptable carrier.

[0112] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0113] In another preferred embodiment, the pharmaceutical composition is an injection or a lyophilized agent.

[0114] In another preferred embodiment, the pharmaceutical composition is formulated for intravenous administration.

[0115] In another preferred embodiment, the concentration of engineered immune cells (FOLR1 CAR-T cells) in the pharmaceutical composition is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -5×10 7 cells / ml.

[0116] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the chimeric antigen receptor (CAR) construct as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, or the engineered immune cell as described in the fifth aspect of the present invention, or a combination thereof; and 0.01 to 99.99% of a pharmaceutically acceptable carrier, and the percentages are the mass percentages of the pharmaceutical composition.

[0117] In another preferred embodiment, the pharmaceutical composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0118] In another preferred embodiment, the pharmaceutical composition further comprises a second anti-tumor active ingredient, preferably a second antibody, or a chemotherapeutic agent.

[0119] In another preferred embodiment, the chemotherapeutic agent is selected from the following group: docetaxel, carboplatin, or a combination thereof.

[0120] In another preferred embodiment, when the pharmaceutical composition is administered, the effector-target ratio of FOLR1 CAR-T cells to tumor cells is (0.1-20):1, such as (10-20):1, (1-20):1, (1-10):1, (1-5):1, 3:1, (0.1-1):1.

[0121] In the seventh aspect of the present invention, there is provided a chimeric antigen receptor (CAR) construct as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, or a use of an engineered immune cell as described in the fifth aspect of the present invention for preparing a drug or preparation for treating a tumor.

[0122] In another preferred example, the tumor comprises a FOLR1-positive tumor.

[0123] In another preferred embodiment, the tumor is a solid tumor.

[0124] In another preferred embodiment, the tumor includes: ovarian cancer, triple-negative breast cancer, mesothelioma, non-small cell lung cancer, endometrial cancer, or a combination thereof.

[0125] In another preferred embodiment, the tumor is ovarian cancer.

[0126] In an eighth aspect of the present invention, a method for preparing the engineered immune cell according to the fifth aspect of the present invention is provided, the method comprising the steps of:

[0127] (a) providing immune cells to be modified; and

[0128] (b) transducing the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention into the immune cell, thereby obtaining the engineered immune cell.

[0129] In another preferred embodiment, the engineered immune cells are CAR-T cells or CAR-NK cells.

[0130] In another preferred embodiment, the method further comprises the step of testing the function and effectiveness of the obtained engineered immune cells.

[0131] In the ninth aspect of the present invention, a method for treating tumors or cancer is provided, comprising administering to a subject in need of treatment an effective amount of a chimeric antigen receptor (CAR) construct as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, a cell as described in the fourth aspect of the present invention, an immune cell as described in the fifth aspect of the present invention, or a pharmaceutical composition as described in the sixth aspect of the present invention.

[0132] In another preferred example, the tumor or cancer comprises a FOLR1-positive tumor.

[0133] In another preferred embodiment, the tumor is a solid tumor.

[0134] In another preferred embodiment, the tumor or cancer includes: ovarian cancer, triple-negative breast cancer, mesothelioma, non-small cell lung cancer, endometrial cancer, or a combination thereof.

[0135] In another preferred embodiment, the tumor or cancer is ovarian cancer.

[0136] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 Schematic diagrams of the CAR structures of F5-28, F5-BB, F10-28, F10-BB, F19-28, and F19-BB are shown.

[0138] Figure 2 Anti-FOLR1 scFv F5, ​​F10, F9 and FLOR1 protein (left, Figure 2 A) and ovarian cancer cell line SKOV3 (right, Figure 2 B) Binding detection

[0139] Figure 3 The killing test of F5-28, F5-BB, F10-28, F10-BB, F19-28 and F19-BB on tumor cell lines SKOV3 (FOLR1+) and ES-2 (FOLR1-) cells is shown.

[0140] Figure 4 The detection of IFN-γ in the supernatant of tumor cell lines SKOV3 (FOLR1+) and ES-2 (FOLR1-) cells by F5-28, F5-BB, F10-28, F10-BB, F19-28 and F19-BB is shown.

[0141] Figure 5 The tumor inhibition test of F5-28, F5-BB, and F10-28 in the mouse CDX model is shown. A, schematic diagram of the tumor inhibition test; B, statistical graph of tumor volume measurement after reinfusion of CAR-T; C, statistical graph of tumor volume measurement on the left and right sides of the mice after rechallenge at D31 and subcutaneous inoculation of SKOV3 on the left side of the mice.

[0142] Figure 6The tumor suppressive effect of F5-BB on ovarian cancer PDX model is shown.

[0143] Figure 7 The cell killing effects of CAR Mov19, F5-28, F10-28, and F19-28 are compared ( Figure 7 A) and comparison of IFN-γ in cell killing supernatant ( Figure 7 B). DETAILED DESCRIPTION

[0144] After extensive and in-depth research and a large amount of screening, the inventors have developed a CAR-immune cell (such as a CAR-T cell) targeting the FOLR1 target. Through the screening and careful design of the CAR element structure (including but not limited to the screening of scFv suitable for CAR expression and tumor killing), the CAR-immune cells of the present invention can kill tumor cells expressing FOLR1 with high specificity without killing normal cells, thereby improving safety. At the same time, the CAR-immune cells of the present invention have a high killing rate for tumor cells and can induce the release of high levels of IFN-γ, thereby improving the effectiveness of treatment. The present invention provides a new approach for the treatment of FOLR1-positive tumors (including ovarian cancer, triple-negative breast cancer, mesothelioma, non-small cell lung cancer, endometrial cancer, etc.). On this basis, the present invention is completed.

[0145] the term

[0146] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0147] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0148] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0149] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0150] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0151] Chimeric Antigen Receptor (CAR)-Immune Cells

[0152] As used herein, the terms "chimeric antigen receptor (CAR)-immune cell", "CAR-immune cell", and "immune cell of the present invention" are used interchangeably and all refer to the specific CAR-immune cell targeting FOLR1 described in the first aspect of the present invention.

[0153] The CAR-immune cells of the present invention, except for the specific extracellular binding domain, have the structure of conventional chimeric antigen receptors in the art.

[0154] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0155] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0156] As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is usually a scFv (single-chain variable fragment). The size of a scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the scFv comprises an antibody that specifically recognizes FOLR1, preferably a single-chain antibody.

[0157] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0158] Carrier

[0159] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0160] The present invention also provides vectors into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0161] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0162] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0163] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0164] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0165] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0166] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0167] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0168] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0169] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0170] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0171] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0172] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0173] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0174] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0175] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0176] preparation

[0177] The present invention provides a preparation containing the CAR-T cells described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0178] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0179] Therapeutic applications

[0180] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding an expression cassette of the present invention. The transduced T cells can target the tumor cell marker FOLR1, can be used for autologous and allogeneic tumor treatment, can be prepared on a large scale, have uniform and stable quality, and can be used for any patient at any time.

[0181] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cell of the present invention to the mammal.

[0182] In one embodiment, the present invention includes a type of cell therapy in which T cells are genetically modified to express the CAR of the present invention, and the CAR-T cells are infused into a recipient in need thereof. The infused cells are capable of killing the recipient's tumor cells. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0183] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-B7-H3 CAR-T cells cause a specific immune response to cells expressing B7-H3.

[0184] Although the data disclosed herein specifically discloses a lentiviral vector comprising an anti-FOLR1 scFv, a human Fc hinge region, ICOS transmembrane and intracellular regions, and 4-1BB and CD3ζ signaling domains, the present invention should be construed to include any number of variations to each of the construct components.

[0185] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors.Cancers may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors.The types of cancers treated with the CAR of the present invention include, but are not limited to, cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas.Also include adult tumors / cancers and childhood tumors / cancers.

[0186] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

[0187] The CAR-immune cells of the present invention can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0188] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0189] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0190] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0191] The present invention provides a method for treating tumors, which comprises administering a therapeutically effective amount of the CAR-immune cells of the present invention to a subject in need thereof.

[0192] The CAR-immune cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0193] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0194] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6 The T cell composition can also be administered at these doses multiple times. The cells can be administered by using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0195] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0196] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, at the same time, or after), including but not limited to treatment with the following agents: the agents such as bevacizumab, megestrol acetate dispersible tablets, paclitaxel injection, ifosfamide, ifosfamide for injection for the treatment of ovarian cancer patients. In a further embodiment, the CAR-immune cells of the present invention may be used in combination with: chemotherapy, radiation, immunosuppressants, such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In a further embodiment, the cell composition of the present invention is administered to patients in combination with bone marrow transplantation, chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), and cyclophosphamide (e.g., before, at the same time, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0197] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The CAR-immune cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0198] The main advantages of the present invention include:

[0199] 1) The present invention provides the structure and construction method of a CAR-T cell vector targeting FOLR1, wherein the structure comprises a unique anti-FOLR1 single-chain antibody; the antibody can confer new antigen specificity to T cells, specifically killing ovarian cancer cells with high expression of FOLR1 without killing normal cells.

[0200] 2) The modified T cells are independent of MHC I and can effectively avoid the immune escape mechanism of downregulation of MHC expression in tumor cells, thereby effectively killing tumor cells.

[0201] 3) Through the screening and careful design of the CAR element structure (including but not limited to the screening of scFv suitable for CAR expression and tumor killing), the CAR-immune cells of the present invention can kill FOLR1-expressing tumor cells with high specificity without killing normal cells, thereby improving safety. At the same time, the CAR-immune cells of the present invention have a high killing rate for tumor cells and can induce the release of high levels of IFN-γ, thereby improving the effectiveness of treatment.

[0202] The following embodiments are used to further illustrate the invention: The following embodiments are illustrative and are only used for the present invention and are not used to limit the scope of protection of the present invention. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods used in the present invention, unless otherwise specified, are conventional methods in the art; the reagents in the present invention, unless otherwise specified, are conventional reagents in the art.

[0203] Table 1 Sequences of the present invention

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] Example 1: Detection of the killing effect of CAR-T prepared with different scFv on tumor cells

[0213] 1.1 Detection of binding of different scFvs to FOLR1 by ELISA

[0214] FOLR1 was diluted to 0.1 μg / mL with coating solution, added to 96-well plate, 100 μl per well, coated at room temperature for 1 hour, the supernatant was washed off, 100 μl 5% BSA in TBST was added for blocking, and after standing at room temperature for 1 hour, different scFvs were added in gradient dilutions, namely F5 scFv, F10 scFv and F19 scFv (in 1% BSA in TBST). After incubation at room temperature for 1 hour, the plate was washed 3 times, the secondary antibody rabbit anti-human IgG Fc was diluted to 0.2 μg / mL, added to 96-well plate, 100 μL per well, incubated at room temperature for 1 hour, the plate was washed 3 times, and color reading was performed.

[0215] The results are as follows Figure 2 As shown in (A): F5 scFv has the strongest binding ability to FOLR1 protein, F10 scFv has the second strongest binding ability to FOLR1 protein, and F19 scFv has the weakest binding ability to FOLR1 protein.

[0216] 1.2 Detection of binding of different scFvs to SKOV3 using FACS

[0217] SKOV3 cells (human ovarian cancer cells) were washed and dispensed into 96-well plates, with 1×10 6 , add different scFvs with gradient dilution, namely F5 scFv, F10 scFv and F19 scFv, resuspend the cells, incubate at 4℃ for 40min, wash the cells 3 times, add secondary antibody and incubate at 4℃ for 20min, wash 3 times, and detect on the machine.

[0218] The results are as follows Figure 2 (B) shows that F5 scFv has the strongest binding ability to SKOV3 cells, while F10 scFv and F19scFv have relatively weak binding ability to SKOV3 cells.

[0219] 1.3 Virus packaging

[0220] According to Figure 1The structures shown in the figure were synthesized and cloned into the virus to transfer to the lentiviral vector pCDH. The vector can be used after sequencing. The density of 293T cells (DMEM containing 10% FBS) cultured in a 10cm culture dish was controlled at 70%-80%. Plasmids pCDH, pLP1, pLP2, and pLP-VSVG were added to 2mL of Opti-Mem at a ratio of 6.4μg:1.3μg:4μg:1.6μg and mixed to prepare a mixture of 3 transfection plasmids. Then mix it with X-tremeGENEHP DNA Transfection Reagent. Generally speaking, the ratio of DNA to Transfection Reagent is 1:3 (1000μl per 10cm culture dish). Incubate at room temperature for 15min.

[0221] The mixture was added dropwise to the prepared 293 culture dish. After culturing in a cell culture incubator for 6 hours, the culture medium was cleaned and replaced with 15 mL of DMEM complete medium. After culturing for 72 hours, the virus was collected.

[0222] Centrifuge the virus supernatant at about 500g for 5 minutes to remove cell debris and collect the supernatant. Then filter the recovered supernatant with a 0.45μm filter membrane. Then concentrate it 15 times for later use. If it is not used temporarily, it should be promptly packaged and frozen in liquid nitrogen, and transferred to a -80℃ refrigerator as soon as possible.

[0223] 1.4 CAR-T cell preparation

[0224] PBMC cells were cultured with X-VIVO 15 (LONZA, 04-418Q) at an initial cell density of 1×10 6 cells / mL.

[0225] CD3 and CD28 antibodies were added to a final concentration of 50 ng / mL and 100 ng / mL, respectively, and 300 IU / mL of IL-2 was added to activate T cell proliferation. After 48 hours of cell activation, an appropriate amount of the virus prepared in Example 1.3 was added to infect T cells. The cells were centrifuged at 800 g for 90 min and placed in a cell culture incubator for culture.

[0226] After 24 h of lentiviral infection, the cell suspension was aspirated and plated at 1×10 6 Fresh X-VIVO 15 medium (containing 300 IU / mL of IL-2) was added to the concentration of 1×10 cells / mL. The cell density was observed every day and T cell culture medium was added in time to maintain the T cell density at 1×10 6 The cells / mL were about 100. The cells were expanded for 5-10 days to complete the preparation of CAR-T cells.

[0227] 1.5 Verification of cell killing effect

[0228] Construct Hibit+ expressing target cells ES-2-Hibit and SKOV3-Hibit. When plating in 96-well plates, add 4E3 target cells / well, add CAR-T cells according to different E:T ratios, and set up the maximum release group (only target cells, but lysed) and spontaneous group (only target cells). Cultivate in the culture phase at 37℃5% CO2. When the preset incubation time of 24h or 48h is reached, then follow The killing activity of CAR-T cells was detected according to the instructions of the HiBiT Extracellular Detection System kit. The method refers to the instructions of the kit. The cytotoxic effect is calculated as follows:

[0229] Cytotoxicity % = (experimental group - spontaneous) / (maximum release group - spontaneous) × 100%

[0230] The results of cell killing assays were as follows Figure 3 As shown: the three prepared CAR-Ts, F5, F10, and F19 scFv, can kill the target cell SKOV3 to varying degrees, but not ES-2, indicating that these CAR-Ts have good specificity.

[0231] 1.6 Detection of IFN-γ in cell killing supernatant

[0232] The supernatant after killing detection was taken for IFN-γ detection according to the method of Human IFN-gamma Quantikine ELISA Kit (R&D system).

[0233] The results are as follows Figure 4 As shown: In the supernatant of the three prepared CAR-T F5, F10, and F19 scFv co-incubated with SKOV3, the release of IFN-γ can be detected, and the trend is consistent. Among them, in the experimental group with an effector-target ratio of 1:1, in the CAR structure using the 4-1BB intracellular stimulation domain, the IFN-γ release of F5-BB is higher than that of other groups; in the CAR structure using the CD28 intracellular stimulation domain, the IFN-γ release of F5-28 is also higher than that of other groups.

[0234] Example 2: Functional verification of CAR-T cells prepared with different scFvs in vivo

[0235] 2.1 Construction of SKOV3 mouse subcutaneous model and evaluation of the killing effects of F5-BB, F5-28 and F10-28 on it

[0236] SKOV-3 cells were cultured in McCoy's 5a medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a 37°C, 5% CO2 incubator. Cells in the logarithmic growth phase will be used to establish an in vivo transplant tumor model.

[0237] Take cells in the logarithmic growth phase, add appropriate amount of HBSS to resuspend, count, and adjust the cell density to 8×10 7 cells / mL and placed on ice for later use. 0.2 mL of tumor cell suspension (containing 50% Matrigel) was subcutaneously inoculated on the right side of each NCG mouse, i.e., 8×10 6 cells / mouse. The tumor volume was 91.23-185.66mm 3 A total of 96 mice were randomly divided into 12 groups for pharmacodynamics, with 8 mice in each group. The day of grouping and medication was day 0 (D0). 4E6 CAR-T was injected through the tail vein on D0, and the changes in mouse body weight and tumor volume were recorded twice a week.

[0238] The mice were subcutaneously inoculated on the left side on D31 for a Rechallenge test, and the changes in mouse body weight and tumor volume were recorded twice a week after injection.

[0239] The results are as follows Figure 5 As shown: Compared with the control group UTD-T cells (untransduced T cells), F5-BB, F10-28 and F5-28 all showed good anti-tumor activity, among which F5-BB had the best anti-tumor activity, followed by F10-28. After rechallenge, both still showed good anti-tumor activity.

[0240] 2.2 Detection of the tumor inhibition effect of F5-BB CAR-T on mouse ovarian cancer PDX model

[0241] Two cases of ovarian cancer, LD1-0032-200756 and LD1-0032-200675, were revived in PDX and NCG mice, with each model revived in two mice. When the tumor grew to 500-800mm 3 The tumor tissue was surgically removed aseptically, and non-tumor tissue and necrotic tissue were removed in a biosafety cabinet. The exfoliated tumor tissue was evenly cut into tumor pieces of approximately 3 mm × 3 mm × 3 mm (approximately 50-90 mg) and inoculated subcutaneously on the right side of the NCG mice. The mice were then observed after inoculation and their weight changes and tumor growth were monitored. When the tumor grew to 100-200 mm 3 When the tumor volume and body weight were measured, 12 tumor-bearing mice were selected and randomly divided into 2 groups according to the tumor volume, with 6 mice in each group. The density of CAR-T (Td 50%) cell suspension was adjusted to 7.5×10 7cells / mL of cell suspension, 0.2 mL was injected into the tail vein of each animal, that is, 1.5×10 7 The changes in mouse body weight and tumor volume were recorded twice a week.

[0242] The results are as follows Figure 6 As shown: F5-BB showed good tumor inhibitory effect on both PDX models.

[0243] Based on the above results, the in vitro test results show that the CAR-T constructed by the three scFvs F5, F10, and F19 can specifically kill tumor cells SKOV3 (FOLR1+), but not ES-2 (FOLR1-). The in vivo test results show that the CAR-T constructed using F5 and F10 scFvs have good tumor inhibition activity.

[0244] Comparative Example 1: Comparison of the technical effects of CAR and CAR of this application

[0245] In this comparative example, a scFv different from the CAR of the present application and the same hinge region, transmembrane region and / or intracellular stimulation domain as the CAR of the present application were used to construct a comparative CAR. The technical effects of the comparative CAR and the CAR of the present application were compared, including but not limited to comparison of cell killing effects, comparison of IFN-γ in cell killing supernatants, in vivo functional verification, etc.

[0246] For example, the experimental method for comparing the cell killing effect is: based on the comparison of the CAR structure and the CAR structure of the present application, CAR-T cells are prepared respectively. Based on the method in Example 1, the cell killing effect is detected and compared.

[0247] The experimental method for comparing IFN-γ in the cell killing supernatant is as follows: based on the method in Example 1, the supernatants in which the comparison CAR-T and the CAR-T of the present application were co-incubated with target cells were tested and compared for IFN-γ.

[0248] The experimental method for in vivo functional verification is: based on the method in Example 2, the comparison CAR-T and the CAR-T of the present application are administered to model mice respectively, and the changes in mouse body weight and tumor volume are recorded.

[0249] Taking CAR Mov19 as an example for illustration, the only difference between CAR Mov19 and F5-28, F10-28, and F19-28 is that CAR Mov19 uses Mov19 scFv.

[0250] Mov19 scFv has HCDR1 as shown in SEQ ID NO: 43, HCDR2 as shown in SEQ ID NO: 44, HCDR3 as shown in SEQ ID NO: 45, LCDR1 as shown in SEQ ID NO: 46, LCDR2 as shown in RAS, and LCDR3 as shown in SEQ ID NO: 47. The VH sequence of Mov19 scFv is shown in SEQ ID NO: 41, and the VL sequence is shown in SEQ ID NO: 42.

[0251] (1) Comparison of cell killing effects

[0252] like Figure 7 As shown in A, although the comparative CAR-T (i.e., Mov19) can also kill target cells, the killing rate is not as good as the CAR-T of the present application (i.e., F5-28, F10-28, and F19-28).

[0253] (2) Comparison of IFN-γ in cell killing supernatants

[0254] like Figure 7 As shown in B, although the comparative CAR-T (i.e., Mov19) can also detect the release of IFN-γ, the level of IFN-γ is not as good as that of the CAR-T of the present application (i.e., F5-28, F10-28, and F19-28).

[0255] In addition to the comparative CAR Mov19, the inventors also constructed comparative CARs using other scFvs, and their technical effects were not as good as the CAR-T of the present application.

[0256] Comparative Example 2: Comparison of the technical effects of CAR and CAR of this application

[0257] In this comparative example, the same scFv as the CAR of the present application (i.e., F5 scFv, F10 scFv, or F19scFv) and hinge regions, transmembrane regions, and / or intracellular stimulation domains commonly used in the art and different from the CAR of the present application were used to construct a comparative CAR. The technical effects of the comparative CAR and the CAR of the present application were compared, including but not limited to comparison of cell killing effects, comparison of IFN-γ in cell killing supernatants, in vivo functional verification, etc. Each experimental method refers to Comparative Example 1.

[0258] Similarly, the results show that: although the comparative CAR-T can also kill target cells, the killing rate is not as good as the CAR-T of the present application; or, although the comparative CAR-T can also detect the release of IFN-γ, the IFN-γ level is not as good as the CAR-T of the present application; or, although the comparative CAR-T can also kill tumor cells, its tumor inhibition activity is not as good as the CAR-T of the present application, and the change in tumor volume is not as significant as the CAR-T of the present application.

[0259] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A chimeric antigen receptor (CAR) construct targeting folate receptor 1 (FOLR1), characterized in that: The antigen binding domain of the CAR comprises a heavy chain variable region and a light chain variable region, Wherein, the heavy chain variable region includes the following complementarity determining region HCDR: HCDR1 shown in SEQ ID NO:36 or 26 or 31, HCDR2 shown in SEQ ID NO:37 or 27 or 32, and HCDR3 shown in SEQ ID NO:38 or 28 or 33; Furthermore, the light chain variable region includes the following complementarity determining region LCDR: LCDR1 shown in SEQ ID NO:39 or 29 or 34, LCDR2 indicated by YTS or AAT, and LCDR3 shown in SEQ ID NO:40 or 30 or 35.

2. The CAR construct according to claim 1, wherein The CAR has a structure as shown in the following formula I: L-EB-H-TM-C-CD3ζ(I) In the formula, Each "-" is independently a connecting peptide or a peptide bond; L is an optional signal peptide sequence; EB is the extracellular binding domain that targets FOLR1; H is an optional hinge region; TM is the transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

3. The CAR construct according to claim 1, wherein The CAR has an amino acid sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or a combination thereof.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the chimeric antigen receptor (CAR) construct of any one of claims 1-3.

5. A carrier, characterized in that The vector contains the nucleic acid molecule as claimed in claim 4.

6. A host cell, characterized in that The host cell contains the vector as claimed in claim 5, or an exogenous nucleic acid molecule as claimed in claim 4 is integrated into its chromosome, or it expresses the chimeric antigen receptor (CAR) construct as claimed in any one of claims 1-3.

7. An engineered immune cell, characterized in that: The engineered immune cells express the chimeric antigen receptor (CAR) construct described in any one of claims 1-3.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (1) an active ingredient selected from the group consisting of a chimeric antigen receptor (CAR) construct as described in any one of claims 1 to 3, a nucleic acid molecule as described in claim 4, a vector as described in claim 5, a host cell as described in claim 6, or an engineered immune cell as described in claim 7, or a combination thereof; and (2) A pharmaceutically acceptable carrier.

9. Use of the chimeric antigen receptor (CAR) construct according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the host cell according to claim 6, or the engineered immune cell according to claim 7, characterized in that: Used for preparing drugs or preparations for treating tumors.

10. A method for preparing the engineered immune cell according to claim 7, characterized in that: The method comprises the steps of: (a) providing immune cells to be modified; and (b) transducing the nucleic acid molecule according to claim 4 or the vector according to claim 5 into the immune cell, thereby obtaining the engineered immune cell.

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