Fusion Protein for Reversing Tumor Microenvironment and Use Thereof

Abstract

The present invention belongs to the field of immunotherapy, specifically relates to a fusion protein, a tumor immunosuppression-resistant CAR, an expression vector, immune cells, and uses that reverse the tumor microenvironment. The fusion protein binds to different CAR structures for various targets, such as CEA, CD19, PSCA, and BCMA, to form a tumor immunosuppression-resistant CAR, or is used in combination with these to target and kill CD47-positive tumor cells. The tumor immunosuppression-resistant CAR and immune cells can eliminate the influence of inhibitory signals in tumor tissue on CAR-T function, achieving CAR-T therapeutic efficacy while ensuring a certain level of safety.

Description

[Technical field]

[0001] The present invention belongs to the technical field of immunotherapy, specifically, to a fusion protein that reverses tumor microenvironment, a novel tumor immunosuppression-resistant CAR and expression vector, immune cells and uses thereof. [Background technology]

[0002] CAR is completely called chimeric antigen receptor, and CAR-T is chimeric antigen receptor T cell, that is, T lymphocyte expressing chimeric antigen receptor (CAR). At present, CAR-T therapy has made breakthroughs in hematological tumors, but its effect in solid tumors is not as good as that in hematological tumors. The reason is that CAR-T has difficulty entering solid tumors, and even if CAR-T cells enter solid tumors, they cannot function normally due to the tumor microenvironment, which affects the therapeutic effect of CAR-T cells on solid tumors. The tumor microenvironment is closely related to the development, growth and metastasis of tumors, and recent clinical studies have revealed that the tumor microenvironment, especially the immune microenvironment of solid tumors, has a significant impact on both the therapeutic effect and prognosis of immunotherapy (including immune cell therapy). Therefore, it is essential to solve the tumor microenvironment suppression, that is, to implement tumor microenvironment resistance, in order to treat solid tumors with CAR-T cells.

[0003] CD47, as a new immune checkpoint following PD-1 / PD-L1 and CTLA-4, suppresses the progression of innate immunity by sending a "don't eat me" signal to macrophages. Currently, tumor therapy using CAR-T targeting CD47 ScFv has been reported. CD47 can promote the formation of an immune microenvironment for tumor escape, but due to tumor heterogeneity, targeting CD47 alone has limited therapeutic efficacy, and the strong affinity of exogenous ScFv may lead to overactivation, poor persistence, and nonspecificity of CAR-T cells, which may cause potential safety risks. Therefore, it is very important to use a safer and more effective means to recognize CD47 and release the escape signal of the tumor microenvironment.

[0004] Signal regulatory protein alpha (SIRPα) is one of the ligands of CD47, can bind to CD47, and inhibit the phagocytosis of macrophages. Currently, most research on CD47 is related to SIRPα. SIRPγ can also bind to CD47, exists on the surface of T cells, the extracellular region is composed of one V domain and two C1 domains, and there is no intracellular signal, and it transmits a unidirectional signal through CD47. Therefore, there are few researchers who modify SIRPγ at present, but the inventors believe that SIRPγ is a more suitable ligand for reversing the tumor microenvironment against CD47. Therefore, the extracellular part of the SIRPγ protein was modified and designed, and a tumor immune suppression-resistant CAR was further designed.

Summary of the Invention

[0005] An object of the present invention is to provide a fusion protein capable of reversing the tumor microenvironment to target and kill tumor cells, an expression vector containing the protein, and immune cells.

[0006] To achieve the above object, the present invention adopts the following forms.

[0007] By modifying and designing the extracellular part of the SIRPγ protein, a fusion protein that reverses the tumor microenvironment and eliminates inhibitory signals in tumor tissues.

[0008] The fusion protein is a SIRPγ fusion protein including an extracellular part, a transmembrane region, and an intracellular signal region in terms of structure.

[0009] Furthermore, the transmembrane structure is derived from the transmembrane region of human CD28 or the transmembrane region derived from human CD8.

[0010] Furthermore, the amino acid sequence of the transmembrane structure is shown in SEQ ID NO:7 or SEQ ID NO:8.

[0011] Preferably, the intracellular signal region is derived from CD28 and the sequence is shown in SEQ ID NO:9 or SEQ ID NO:38.

[0012] Furthermore, the structure of the SIRPγ fusion protein is SIRPγ-CD28TM-CD28 or SIRPγ-CD8TM-4-1BB.

[0013] Furthermore, the amino acid sequence of the extracellular portion of SIRPγ is shown in SEQ ID NO:1 or a functional variant thereof.

[0014] Furthermore, the amino acid sequence of the SIRPγ fusion protein SIRPγ-CD28TM-CD28 is shown in SEQ ID NO:2 or a functional variant thereof.

[0015] Furthermore, the amino acid sequence of the SIRPγ fusion protein SIRPγ-CD8TM-4-1BB is shown in SEQ ID NO:3 or a functional variant thereof.

[0016] Furthermore, the SIRPγ fusion protein is SIRPγ-CD28TM-CD28 with a nucleotide sequence shown in SEQ ID NO:13.

[0017] Furthermore, the nucleotide sequence of the SIRPγ fusion protein SIRPγ-CD8TM-4-1BB is shown in SEQ ID NO:14.

[0018] Furthermore, immune cells containing the expression vector.

[0019] Furthermore, the immune cells are T cells, T cell precursors or NK cells.

[0020] Specifically, the T cells may be αβ T cells or γδ T cells. γδ T cells are a special subset of T cells that contain receptor γ and δ chains on the surface, accounting for 0.5-5% of all T lymphocytes. This cell population was first discovered in 1987. Although they have γδ TCR, their recognized antigen or ligand is non-MHC restricted, which is different from conventional αβ T cells. Currently, a plurality of patents such as CN107810267A and CN107771215A disclose the application of γδ T cells in cell therapy and CAR-T cell therapy.

[0021] Specifically, design the SIRPγ fusion protein and design the fusion proteins PD-1-28TM-28 and PD-1-8TM-BB of PD-1.

[0022] Specifically, when the designed SIRPγ fusion protein is used in combination with CAR1, it may be expressed alone or co-expressed with CAR1 to exert its effect. In addition, the SIRPγ fusion protein may be used alone in immunotherapy.

[0023] Specifically, in the present invention, the inventors believe that the SIRPγ protein is a more suitable ligand for relieving the tumor microenvironment against CD47. Therefore, the extracellular part of the SIRPγ protein is modified and designed to design a tumor immune suppression-resistant CAR. For this reason, the present invention applies the combination of the designed and constructed hypoxia-regulated promoter and CAR-T cell technology, and the combination of the hypoxia-regulated promoter, SIRPγ protein and CAR-T cell technology to tumor immunotherapy, that is, by further modifying the conventional CAR structure, the treatment effect of CAR-T on solid tumors and the safety of CAR-T are improved.

[0024] Furthermore, the immune cells have a chimeric antigen receptor structure that recognizes tumor antigens. The chimeric antigen receptor includes an extracellular portion that recognizes tumor antigens, a hinge region, a transmembrane region, and an intracellular signaling region. Tumor antigens include antigen molecules that can be recognized as tumor targets, such as PSCA, PSMA, CD19, BCMA, CD123, CD20, CD22, CEA, EGFR, EGFRVIII, GPC3, 5T4, CD33, Her2, GD2, CD70, CLL-1, Trop2, CD47, GPC3, CLND18.2, CD133, CS1, CD155, CD30, ROR1, MUC1, IL13RAα2, or mesothelin.

[0025] Another object of the present invention is to provide a novel tumor immunosuppression-resistant CAR, an expression vector containing the CAR, and immune cells. The novel tumor immunosuppression-resistant CAR and immune cells can eliminate the influence of inhibitory signals in tumor tissues on CAR-T function, achieve the efficacy of CAR-T treatment, and moreover, ensure a predetermined safety.

[0026] To achieve the above object, the present invention adopts the following means.

[0027] The novel tumor immunosuppression-resistant CAR includes a fusion protein that reverses the tumor microenvironment described in one of the objects, and CAR1. CAR1 includes an extracellular portion that recognizes tumor antigens, a hinge region, a transmembrane region, and an intracellular signaling region.

[0028] Furthermore, the fusion protein is linked to CAR1 by a polycistronic structure. The polycistronic structure is a self-cleaving polypeptide or an internal ribosome entry site IRES. The self-cleaving polypeptide is T2A, P2A, E2A, or F2A.

[0029] Furthermore, the structure of the CAR is a ScFv-hinge-TM-CD3ζ-self-cleaving peptide-SIRPγ fusion protein or a ScFv-hinge-TM-4-1BB-CD3ζ-self-cleaving peptide-SIRPγ fusion protein.

[0030] Furthermore, the structure of the CAR is ScFv-hinge-TM-CD3ζ-self-cleaving peptide-SIRPγ-CD28TM-CD28 or ScFv -hinge-TM-4-1BB-CD3ζ-self-cleaving peptide-SIRPγ-CD28TM-CD28.

[0031] Furthermore, the CAR structure a may be a general first-generation, second-generation, or third-generation CAR structure, or a novel CAR structure such as an improved dual CAR or a regulatory CAR structure (e.g., FRB / FKBP12 regulation).

[0032] Furthermore, the sequence of the hinge region of CAR1 may be derived from IgG, CD8, CD7, or CD4, the transmembrane region of the CAR structure may be derived from CD8, CD28, CD3ε, CD4, CD16, CD137, CD80, and CD86, and the intracellular signaling region of the CAR structure may be derived from CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86, and CD127.

[0033] Furthermore, the ScFv can recognize any one of CD19, CD123, MOv-γ, PSMA, IL13Rα2, EGFRvIII, EGFR, EPCAM, GD2, MUC1, HER2, GPC3, CEA, Meso, CD133, NKG2D, CD138, LeY, k-Light, CD33, ROR1, BCMA, CD30, CD20, CD22, PSCA, CLL-1, CD70, and CD47.

[0034] Furthermore, the ScFv can recognize CD47, CEA, PSCA, CD19, or BCMA.

[0035] In some embodiments, the CAR structure has a truncated EGFRt regulatory tag; in some embodiments, the CAR structure targeting PSCA is a universal CAR structure; and in some embodiments, the CAR structure targeting PSCA has a suicide gene such as iCasp9.

[0036] In some embodiments, the CAR structure includes one or more components of natural killer cell receptors (NKRs), thereby forming an NKR-CAR. The NKR components may be transmembrane domains, hinge domains or cytoplasmic domains derived from any of the natural killer cell receptors such as killer cell immunoglobulin-like receptors (KIRs), e.g., KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1; natural cytotoxic receptors (NCRs), e.g., NKp30, NKp44, NKp46; the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME and CD2F-10; Fc receptors (FcRs), e.g., CD16, and CD64; Ly49 receptors, e.g., LY49A, LY49C. The NKR-CAR molecule can interact with an adaptor molecule or an intracellular signaling domain (e.g., DAP12).

[0037] As a preferred embodiment 1, it is a CAR structure composed of the aforementioned SIRPγ fusion protein and CAR1. In the CAR1, the amino acid sequence of the hinge is shown in SEQ ID NO: 24 or its functional variant, the amino acid sequence of the TM is shown in SEQ ID NO: 7 or SEQ ID NO: 8, the amino acid sequence of CD3ζ is shown in SEQ ID NO: 11 or its functional variant. In the fusion protein structure, the amino acid sequence of the extracellular part of SIRPγ is shown in SEQ ID NO: 1 or its functional variant, the amino acid sequence of the transmembrane region derived from human CD28 is shown in SEQ ID NO: 7, and the amino acid sequence of the intracellular signal region derived from human CD28 is shown in SEQ ID NO: 9.

[0038] Furthermore, the amino acid sequence of the ScFv is shown in SEQ ID NO: 25 or its functional variant.

[0039] Specifically, this preferred embodiment designs genetically modified T lymphocytes that express the novel immunosuppression-resistant CAR: ScFv-hinge-TM-CD3ζ-self-cleaving peptide-SIRPγ-28TM-28, ScFv-hinge-TM-CD3ζ-self-cleaving peptide-SIRPγ-8TM-BB, ScFv-hinge-TM-4-1BB-CD3ζ-self-cleaving peptide-SIRPγ-28TM-28, ScFv-hinge-TM-4-1BB-CD3ζ-self-cleaving peptide-SIRPγ-8TM-BB, and ScFv-hinge-TM-CD3ζ-self-cleaving peptide-PD-1-28TM-28 and ScFv-hinge-TM-CD3ζ-self-cleaving peptide-PD-1-8TM-BB to verify the action of the novel immunosuppression-resistant CAR.

[0040] Furthermore, the method for manufacturing the CAR structure is to co-express the CAR structure without the SIRPγ fusion protein and the SIRPγ fusion protein in the same vector-transfected immune cells, or to express the CAR structure without the SIRPγ fusion protein and the SIRPγ fusion protein in two vector-transfected immune cells respectively.

[0041] As a preferred embodiment 2, it is a CAR structure composed of the aforementioned SIRPγ fusion protein and CAR1, wherein the CAR1 includes an anti-CEA single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, CD137, and a CD3ξ dual-stimulatory signal.

[0042] Preferably, the CAR structure a is a CEA ScFv-CD8 hinge region-CD8 transmembrane region-CD137-CD3ξ structure, and its amino acid sequence includes the sequence shown in SEQ ID NO:26. The nucleic acid sequence encoding the anti-CEA single-chain antibody is shown in SEQ ID NO:36, and the nucleic acid sequence encoding the CD8 hinge region-CD8 transmembrane region-CD137-CD3ξ structure is shown in SEQ ID NO:37.

[0043] Furthermore, the nucleic acid sequence encoding the CAR structure containing a hypoxia-regulated promoter includes the sequence shown in SEQ ID NO:31.

[0044] As a preferred embodiment 3, it is a CAR structure composed of the aforementioned SIRPγ fusion protein and CAR1, wherein the CAR1 includes a CD19 single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, CD137, and a CD3ξ dual-stimulatory signal.

[0045] Preferably, the amino acid sequence of the CAR structure a is shown in SEQ ID NO:27 or a functional variant thereof.

[0046] Furthermore, the nucleic acid sequence encoding the CAR structure includes the sequence shown in SEQ ID NO:32 or SEQ ID NO:33.

[0047] Furthermore, the method for producing the CAR structure is to co-express the CAR structure without the SIRPγ fusion protein and the SIRPγ fusion protein in the same vector-transfected immune cells, or to express the CAR structure containing the SIRPγ fusion protein and the SIRPγ fusion protein in two vector-transfected immune cells, respectively.

[0048] As a preferred embodiment 4, it is a CAR structure composed of the aforementioned SIRPγ fusion protein and CAR1, wherein the CAR structure a includes a PSCA single-chain antibody, a hinge region, a CD28 transmembrane region, CD28, CD137, and a CD3ξ triple-stimulatory signal, and the hinge region is G4H or 7H.

[0049] Preferably, the amino acid sequence of the CAR structure a is shown in SEQ ID NO:28 or its functional variant, or is shown in SEQ ID NO:29 or its functional variant. The CAR structure a in which the amino acid sequence is shown in SEQ ID NO:28 or its functional variant is a CAR structure a with a G4H hinge region, and the CAR structure a in which the amino acid sequence is shown in SEQ ID NO:29 or its functional variant is a CAR structure a with a 7H hinge region.

[0050] Furthermore, the nucleic acid sequence encoding the CAR structure includes the sequence shown in SEQ ID NO:34 or SEQ ID NO:35, and SEQ ID NO:35 includes a hypoxia-regulated promoter.

[0051] Furthermore, the method for producing the CAR structure is to co-express a CAR structure without the SIRPγ fusion protein and the SIRPγ fusion protein in the same vector-transfected immune cells, or to express a CAR structure without the SIRPγ fusion protein and the SIRPγ fusion protein in two vector-transfected immune cells respectively.

[0052] As a preferred embodiment 5, it is a CAR structure comprising the aforementioned SIRPγ fusion protein and any one of CAR1 in Preferred Embodiment 1 - Preferred Embodiment 4. The CAR structure further comprises a hypoxia promoter, and the nucleic acid sequence of the hypoxia regulatory promoter comprises the sequence shown in SEQ ID NO:30. The CAR structure targeting CD19 containing a hypoxia promoter has the role of effectively removing tumors in vivo in a hypoxic microenvironment. Moreover, the promoter induced by the hypoxic microenvironment enhances the expression of factors such as target genes and proteins in a hypoxic environment, and can improve the therapeutic effect of tumor therapeutic drugs and the effectiveness and safety of CAR-T therapy. Moreover, it can not only be effectively expressed in T lymphocytes, but also enhance the expression of CAR molecules in a hypoxic environment, endow CAR-T cells with high IFN-γ secretion ability, strengthen the killing of CAR-T cells against tumor target cells, and is useful for tumor target therapy.

[0053] Furthermore, the hypoxia regulatory promoter is formed by linking a Hifla regulatory element and a mini-promoter, and the mini-promoter is any one selected from a cytomegalovirus promoter, a promoter of HSV thymidine kinase, a promoter of simian virus 40, an adenovirus late promoter, and a synthetic promoter.

[0054] Furthermore, the method for manufacturing the CAR structure is to co-express a CAR structure not containing the SIRPγ fusion protein and the SIRPγ fusion protein in the same vector-transfected immune cells, or to express a CAR structure not containing the SIRPγ fusion protein and the SIRPγ fusion protein in two vector-transfected immune cells respectively.

[0055] The object of the present invention also provides an expression vector containing any one of the aforementioned novel tumor immunosuppression-resistant CARs (including the embodiments in Preferred Embodiment 1 - Preferred Embodiment 5 and those other than the preferred embodiments), and immune cells containing the expression vector.

[0056] Furthermore, the expression vector is any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector, and a plasmid.

[0057] Furthermore, the immune cells are T cells, T cell precursors, or NK cells.

[0058] Furthermore, the method for producing the immune cells is to co-express a CAR structure without the SIRPγ fusion protein and the SIRPγ fusion protein in the same vector-transfected immune cells, or to express the CAR structure without the SIRPγ fusion protein and the SIRPγ fusion protein in two vector-transfected immune cells, respectively.

[0059] In some embodiments, the lentiviral vector is substantially selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), maedi-visna virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0060] In some embodiments, the vector includes a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retrovirus-derived element, a promoter operably linked to a polynucleotide encoding a CAR included in the present invention, and a right (3') retroviral LTR.

[0061] In some embodiments, the CAR includes a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimal woodchuck post-transcriptional regulatory element (oPRE).

[0062] In some embodiments, the promoter of the 5'LTR is replaced with a heterologous promoter.

[0063] In some embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a Simian Virus 40 (SV40) promoter.

[0064] In some embodiments, the 5'LTR or 3'LTR is a lentiviral LTR.

[0065] In some embodiments, the 3'LTR is a self-inactivating (SIN) LTR.

[0066] In some embodiments, the nucleic acid sequence of the CAR construct includes an optimized Kozak sequence.

[0067] In some embodiments, the promoter operably linked to the polynucleotide encoding the CAR included in the present invention is selected from the group consisting of a cytomegalovirus immediate early gene promoter (CMV), an elongation factor 1α promoter (EF1-α), a phosphoglycerate kinase-1 promoter (PGK), a ubiquitin-C promoter (UBQ-C), a cytomegalovirus enhancer / chicken β-actin promoter (CAG), a polyomavirus enhancer / herpes simplex thymidine kinase promoter (MC1), a β-actin promoter (β-ACT), a simian virus 40 promoter (SV40), and a myeloproliferative sarcoma virus enhancer, with the dl587rev primer binding site replaced (MND) promoter lacking the negative control region.

[0068] In some embodiments, the vector containing the CAR may include a secreted anti-PD-1 ScFv. In some embodiments, the vector containing the CAR may include a PD-1 conjugated transduction peptide (e.g., a PD-1-CD28-CD137-CD3 signal structure). In some embodiments, the vector containing the CAR combines a plurality of CARs, for example, two CARs targeting different antigens or different recognition sites of the same antigen.

[0069] An object of the present invention is also to provide a pharmaceutical composition and its use.

[0070] To achieve the above object, the present invention adopts the following means.

[0071] The pharmaceutical composition includes the fusion protein or an expression vector containing the fusion protein, and an immune cell or an immune cell containing the novel tumor immunosuppression-resistant CAR or the novel tumor immunosuppression-resistant CAR.

[0072] In some embodiments, the fusion protein may be SIRPα or a partial domain of SIRPα, and the structure of the fusion protein may be a plurality of fusion proteins such as SIRPα-28TM-28, SIRPα-8TM-137, SIRPα-8TM-OX40, SIRPα-8TM-ICOS. In some embodiments, the immune cells expressing the above SIRPα fusion protein may be used alone, or the SIRPα fusion protein and the CAR molecule may be co-expressed in the same immune cell, or immune cells expressing the SIRPα fusion protein and the CAR molecule respectively may be mixed at a predetermined ratio.

[0073] In some specific embodiments, the activator and / or treatment may be surgery, chemotherapy, radiotherapy, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibody or other antibody therapies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines or radiation.

[0074] In some embodiments, the cells may express other activators, for example, activators that enhance CAR-expressing cell activity. The activator may be an activator that blocks inhibitory molecules. In some embodiments, inhibitory molecules such as PD1 can inhibit the ability of CAR-expressing cells to initiate an immune effector response. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFRβ) and TGFRβ. The extracellular domain of the inhibitory molecule may be fused to a transmembrane domain and an intracellular signaling domain, such as a PD1 CAR.

[0075] Furthermore, use of any of the fusion proteins, any of the CAR constructs, any of the nucleic acid sequences, any of the expression vectors, or any of the immune cells in the manufacture of a tumor therapeutic agent.

[0076] Furthermore, the tumor is a malignant tumor including acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer, and skin cancer.

[0077] In the present invention, in the CAR structure, Z is the human CD3 intracellular signal CD3ζ, BB is the human 4-1BB intracellular signal, BBZ is the intracellular domain of 4-1BB ICD-CD3ζ, and 28Z is the intracellular domain of CD28ICD-CD3ζ.

[0078] In the present invention, the "functional variant" usually has substantially the same function as a certain sequence (for example, it may have the properties of the chimeric antigen receptor), and moreover, has at least 85% (for example, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity with the amino acid sequence. In some embodiments, the variant of the amino acid sequence has substantially the same function as this sequence.

[0079] In the present invention, in the CAR structure, CEAZ is a CAR structure targeting CEA that has only CD3ζ as an intracellular structure, CEA-28Z is a CAR structure targeting CEA that has a co-stimulatory signal derived from the intracellular domain of CD28 and CD3ζ in the cell, CEA-BBZ is a CAR structure targeting CEA that has a co-stimulatory signal derived from the intracellular domain of 4-1BB and CD3ζ in the cell, BCMA-BBZ is a CAR structure targeting BCMA that has a co-stimulatory signal derived from the intracellular domain of 4-1BB and CD3ζ in the cell, SIRPγ-28TM-28 is a SIRPγ fusion peptide that has only a signal derived from the intracellular domain of CD28 in the cell, and is used as an abbreviation SIRPγ-28. SIRPγ-8TM-BB is a fusion peptide that has only a signal derived from the intracellular domain of 4-1BB in the cell, and is an abbreviation SIRPγ-BB.

Advantages of the Invention

[0080] The beneficial effects of the present invention are as follows.

[0081] The fusion protein that reverses the tumor microenvironment according to the present invention can reverse the tumor microenvironment and kill CD47-positive tumor cells as targets. The novel tumor immunosuppression-resistant CAR and immune cells containing the fusion protein eliminate the influence of inhibitory signals in tumor tissues on the CAR-T function, realize the efficacy of CAR-T treatment, and moreover, can ensure a predetermined safety.

Brief Description of the Drawings

[0082]

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[0083] In FIGS. 10 to 18, 5HCEA-BBZ is a hypoxia CAR construct targeting CEA having a co-stimulatory signal derived from the intracellular domain of 4-1BB and CD3ζ intracellularly, CEA-BBZ is a CAR construct targeting CEA having a co-stimulatory signal derived from the intracellular domain of 4-1BB and CD3ζ intracellularly, CEA-28Z is a CAR construct targeting CEA having a co-stimulatory signal derived from the intracellular domain of CD28 and CD3ζ intracellularly, and SIRPγ-28TM-28 is a fusion peptide having only a signal derived from the intracellular domain of CD28 intracellularly, and hereinafter the abbreviation SIRPγ-28 is used. In FIGS. 19 to 23, 5HCD19-BBZ is a CAR construct targeting CD19 having a hypoxia promoter, a co-stimulatory signal derived from the intracellular domain of 4-1BB (abbreviation BB) and CD3ζ intracellularly, SIRPγ-28TM-28 (SIRPγ-28) is a fusion peptide having only a signal derived from the intracellular domain of CD28 intracellularly, 5HCD19-BBZ-SIRPγ-28 is a CAR of the structure 5HCD19-8H-8TM-CD137-CD3ζ, and SIRPγ-28-5HCD19-BBZ is a CAR-T obtained by expressing a CAR fusion protein containing a hypoxia promoter in two vector-transfected immune cells, respectively. In FIGS. 24 to 35, RT4-Luc-GFP are positive cells, PC-3-Luc-GFP are negative cells, SIRPγ-28TM-28 (SIRPγ-28) is a fusion peptide having only a signal derived from the CD28 intracellular domain in the cell, and PSCA-28BBZ-G4H-28TM+SIRPγ-28 or PSCA-28BBZ-7H-28TM+SIRPγ-28 are obtained by expressing PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM and the fusion protein SIRPγ-28 with SIRPγ-28 in two vector transfection immune cells, respectively. PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 or PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 are obtained by co-expressing PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM and the fusion protein SIRPγ-28 with SIRPγ-28 in the same vector transfection immune cells.

BEST MODE FOR CARRYING OUT THE INVENTION

[0084] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. Experimental methods for which specific conditions are not specified in the preferred embodiments are usually general conditions, for example, the conditions described in the Molecular Cloning Experiment Guidelines (3rd Edition, edited by J. Sambrook et al.) or the conditions recommended by the manufacturer. The examples given are only for better explaining the content of the present invention, and the content of the present invention is not limited to such examples. Substantially non-improved or adjusted embodiments made by those skilled in the art based on the above invention content also belong to the patent scope of the present invention.

[0085] In the examples of the present invention, the sequences of related vector structure elements are shown in Table 1 below.

[0086]

Table 1

[0087] In the examples of the present invention (Table 1), Z is CD3ζ, CEAZ is a CAR structure having only CD3ζ in the intracellular structure (ScFv(CEA)-hinge-TM-CD3ζ), CEA-28Z is a CAR structure targeting CEA having a co-stimulatory signal derived from the intracellular domain of CD28 and CD3ζ in the cell (ScFv(CEA)-hinge-TM-CD28-CD3ζ), CEA-BBZ is a CAR structure targeting CEA having a co-stimulatory signal derived from the intracellular domain of 4-1BB and CD3ζ in the cell (ScFv(CEA)-hinge-TM-4-1BB-CD3ζ), BCMA-BBZ is a CAR structure targeting BCMA having a co-stimulatory signal derived from the intracellular domain of 4-1BB (abbreviation BB) and CD3ζ in the cell, SIRPγ-28TM-28 is a fusion peptide having only a signal derived from the intracellular domain of CD28 in the cell, hereinafter the abbreviation SIRPγ-28 is used, SIRPγ-8TM-BB is a fusion peptide having only a signal derived from the intracellular domain of 4-1BB in the cell, hereinafter the abbreviation SIRPγ-BB is used.

[0088] In the examples of the present invention, 5HCD19-BBZ is a CAR structure targeting CD19 having a hypoxia promoter, a co-stimulatory signal derived from the intracellular domain of 4-1BB (abbreviation BB) and CD3ζ in the cell, SIRPγ-28TM-28 (abbreviation SIRPγ-28) is a fusion peptide having only a signal derived from the intracellular domain of CD28 in the cell, SIRPγ-28+5HCD19-BBZ is a CAR-T obtained by expressing a CAR fusion protein containing a hypoxia promoter in two vector-transfected immune cells respectively. 5HCD19-BBZ-P2A-SIRPγ-28 is a CAR-T obtained by co-expressing a CAR fusion protein containing a hypoxia promoter in the same vector-transfected immune cells.

[0089] In the examples of the present invention, PSCA-28BBZ-G4H-28TM+SIRPγ-28 or PSCA-28BBZ-7H-28TM+SIRPγ-28 is obtained by separately expressing PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM and the fusion protein SIRPγ-28 in two vector-transfected immune cells. PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 or PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 is obtained by co-expressing PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM and the fusion protein SIRPγ-28 in the same vector-transfected immune cells.

[0090] In the examples of the present invention, the lentivirus packaging method by the calcium phosphate method is used. Specifically, 293T cells are cultured to a good state using DMEM medium containing 10% FBS (w / v). The packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5 centrifuge tube at a predetermined ratio, CaCl2 and 2×HBS are added, and after uniform mixing, it is left standing at room temperature. Then, it is added to the treated 293T cell culture solution. After 3 to 5 hours, the liquid is replaced with 10 mL of DMEM medium containing 10% FBS. After 48 hours or 72 hours, the cell supernatant is collected and the virus is purified.

[0091] In the examples of the present invention, the antibody is Protein-L-PE. Protein-L can recognize the antibody light chain, and the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L. Therefore, the CAR positive rate and the CAR expression intensity can be detected using Protein-L. A GFP tag is attached to SIRPγ-28, and the expression of SIRPγ-28 is determined by the GFP positive rate.

[0092] In the embodiments of the present invention, the method for detecting the killing ability of each CAR-T against target cells is carried out using an ACEA xCELLigence RTCA MP instrument, and the experimental steps are carried out according to the instrument's operating manual. The principle of the ACEA xCELLigence RTCA MP is to record the resistance index data of tumor cells attached to the bottom of the well once every 15 minutes, and to judge the growth and death of adherent target cells based on the resistance index. The formula using the resistance index analysis result is CAR-T cell killing rate = baseline resistance index - real-time resistance index.

[0093] In the embodiments of the present invention, IFN-γ detection is carried out using a BD IFN-γ kit, and the experimental steps are carried out according to the product's operating manual. IL-2 detection is carried out using an inritrogen IL-2 kit, and the experimental steps are carried out according to the product's operating manual. TNF-α detection is carried out using a Biolegend kit, and the experimental steps are carried out according to the product's operating manual.

[0094] In the embodiments of the present invention, as a method for verifying whether the construction of the hypoxia model has been completed, PBMC is infected with a recombinant plasmid virus to construct an in vitro hypoxia cell model. After culturing for 12 - 18 h, the medium is changed, and then CoCl2 is used to induce a hypoxic environment and cultured until the Nth day. CAR expression is detected by detecting the light chain antibody of the CAR structure.

[0095] In the embodiments of the present invention, the mice used for in vivo verification are NOD.Cg-PrkdcscidII2rgtm1Sug / JicCrl, abbreviated as NOG mice, and are bred by Mamoru Ito of the Central Institute for Experimental Animals (CIEA) in Japan. Internationally, it is the most common strain in related tumor formation experiments with CAR-T in vivo.

[0096] In the embodiments of the present invention, as a method for verifying the in vivo hypoxia model, 6 - 8-week-old female NOG mice are selected. After marking the numbers on the mice's ears, target cells are subcutaneously injected into the mice's backs at a cell amount of 1×10 6 / mouse, and the tumor volume of the mice is measured on the 12th day after tumor formation.

[0097] Part 1 Example 1 Construction of plasmid Based on the CAR schematic diagram shown in Figure 1, using the SIRPγ and CD47 full-length plasmids, pL-CAG-2AGFP, pL-CAG-PD1-CD28-2ACherry, pL-CAG-PD1-BB-2Acherry as templates, the CAR structure and the corresponding single-target CAR structure shown in Figure 2 were constructed. Vectors: CEAZ-PD1-28, CEAZ-PD1-BB, CEAZ-SIRPγ-BB, CEAZ-SIRPγ-28, CEABBZ-P2A-SIRPγ-28, CEABBZ-P2A-SIRPγ-BB, BCMA-BBZ-P2A-SIRPγ-28 and single-target CAR: CEAZ, PD1-28, PD1-BB, SIRPγ-28TM-28, SIRPγ-8TM-BB were constructed. The results of enzyme cleavage, sequencing, and alignment verification showed that, as shown in Figure 3, the results of enzyme cleavage identification of the recombinant plasmids were as follows: 1-3: The pL-CAG-CEAZ-PD1-28 plasmid was successively the original plasmid, CEAZ (1371bp), PD1-28 (783bp); 4-6: The pL-CAG-CEAZ-PD1-BB plasmid was successively the original plasmid, CEAZ (1371bp), PD1-BB (798bp); 7-9: The pL-CAG-CEAZ-SIRPγ-28 plasmid was successively the original plasmid, CEAZ (1371bp), SIRPγ-28 (1374bp); 10-12: The pL-CAG-CEAZ-SIRPγ-BB plasmid was successively the original plasmid, CEAZ (1371bp), SIRPγ-BB (1368bp); M1 was the DL5000 DNA molecular weight marker, M2 was the DL15000 DNA molecular weight marker; 13-15: The pL-CAG-SIRPγ-28-2AGFP plasmid was successively the original plasmid, SIRPγ-28 (1292bp), 2AGFP (785bp); 16-18: The pL-CAG-SIRPγ-BB-2AGFP plasmid was successively the original plasmid, SIRPγ-BB (1286bp), 2AGFP (785bp); 19-20: The pL-CAG-CD47 plasmid was successively CD47 (974bp), the original plasmid; M3 was the DL5000 DNA molecular weight marker. The construction was successful.

[0098] Example 2 Construction of Target Cells CEA, PD-L1, and CD47 antigen viruses were produced by the calcium phosphate method and used to infect CHO cells respectively to construct CHO-CEA cells, CHO-CEA-PD-L1 cell line, and CHO-CEA-CD47 cell line. Signal regulatory protein α (SIRPγ) is one of the ligands of CD47 and can bind to CD47, so CD47-positive target cells are used to evaluate CEAZ-SIRPγ-BB, CEAZ-SIRPγ-28, SIRPγ, SIRPγ-28, and SIRPγ-BB. After subculturing the three cell lines for 10 generations and detecting their positive rates, as shown in Figure 4, the positive rate of CHO-CEA was 97.1%, the double positive rate of CHO-CEA-CD47 was 97.6%, and the double positive rate of CHO-CEA-PD-L1 was 87%, meeting the experimental requirements, indicating that the cell lines were successfully constructed and could be used for subsequent CAR-T drug efficacy evaluation as target cells.

[0099] Example 3 Production of Lentivirus and Infection of T Lymphocytes Lentivirus was packaged by the calcium phosphate method to obtain five single-expression CARs (CEAZ, PD1-28, PD1-BB, SIRPγ-28, SIRPγ-BB) and six novel immunosuppression-resistant CARs (CEAZ-PD1-28, CEAZ-PD1-BB, CEAZ-SIRPγ-28, CEAZ-SIRPγ-BB, CEABBZ-SIRPγ-28, CEABBZ-SIRPγ-BB) virus particles in Example 1. Lymphocytes were separated by gradient centrifugation. After centrifugation, the second-layer white lymphocyte layer was washed with physiological saline, and RPMI 1640 complete medium containing 10% FBS was added for culture to obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 h, and then the activated PBMCs were infected at a predetermined multiplicity of infection (MOI). On the 12th day after virus infection, the positive rate of CAR-T was detected. The detection method was flow cytometry detection, and the antibody was Protein-L-PE. Protein-L could recognize the antibody light chain, and the light chain of the ScFv sequence in the CAR antigen recognition region could be recognized by Protein-L. Therefore, the CAR positive rate and CAR expression intensity could be detected by Protein-L. As a result, as shown in Fig. 5a, the immunosuppression-release fusion protein could be expressed on the T cell surface. As shown in Fig. 5b, the novel immunosuppression-resistant CAR was successfully expressed.

[0100] Example 4 Verification of the in vitro function of the novel immunosuppression-resistant CAR-T Using Control T cells that did not express the immunosuppression-release fusion protein as a control, the functions of the immunosuppression-release fusion proteins SIRPγ-28TM-28 and SIRPγ-8TM-BB were verified. The target cells were a CHO cell line that expressed CD47. As a result, as shown in Fig. 6a, both the SIRPγ-28TM-28 and SIRPγ-8TM-BB fusion proteins had a killing effect on CD47-positive target cells. The CEAZ group was used as a positive control, the Control-T group as a negative control, the CEAZ-PD1-28, CEAZ-PD1-BB, CEAZ-SIRPγ-28, and CEAZ-SIRPγ-BB groups as experimental groups, and CHO-CEA-CD47 and CHO-CEA-PD-L1 as target cells to verify the in vitro efficacy of the novel immunosuppression-resistant CAR-T. As a result, as shown in Figure 6b and Table 2 below, the cell killing rate in the CEAZ-PD1-28 and CEAZ-SIRPγ-28 groups was significantly higher than that in the CEAZ group, while the target cell killing rate in the CEAZ-PD1-BB and CEAZ-SIRPγ-BB groups was not significantly improved compared to the CEAZ group. Furthermore, when the CEAZ-PD1-28 and CEAZ-PD1-BB groups were co-cultured with CHO-CEA-CD47, or when the CEAZ-SIRPγ-28 and CEAZ-SIRPγ-BB groups were co-cultured with CHO-CEA-PD-L1, no significant difference in killing efficiency was observed compared to the CEAZ group, indicating that the novel immunosuppression-resistant CAR-T group has killing specificity, and that CEAZ-PD1-28 and CEAZ-SIRPγ-28 have significant killing effects on target cells.

[0101] [Table 2]

[0102] The function of BCMA-BBZ-P2A-SIRPγ-28 was examined using K562-BCMA as the target cells and the Control-T group as a negative control, and the results showed that BCMA-BBZ-P2A-SIRPγ-28 showed excellent killing function.

[0103] Example 5: Verification of release of tumor suppressive environment by novel immunosuppression-resistant CAR-T After CAR-T cells infiltrate into tumor tissues, they are easily affected by the tumor immunosuppressive environment, highly express exhaustion molecules such as PD1, LAG-3, and Tim-3, have a reduced function of effectively killing tumor cells, and the apoptosis of CART cells themselves increases. To verify whether the novel immunosuppression-resistant CAR-T can relieve the tumor-suppressive microenvironment signals, when culturing CAR-T cells of the CEAZ, CEA-28Z, CEA-BBZ, CEAZ-PD1-28, CEAZ-PD1-BB, CEAZ-SIRPγ-28, CEAZ-SIRPγ-BB, and Control-T groups until Day7, co-culture the CAR-T cells and DLD-1-CEA-Luc-GFP cells in a 12-well cell culture plate. After 48h, collect the CAR-T cells, label them with anti-human CD3, PL, PD1, LAG-3, and Tim-3 flow cytometry antibodies, then perform flow cytometry detection, analyze the detection results, and evaluate their effects and capabilities. For the CEAZ-PD1-28 and CEAZ-PD1-BB groups, due to the exogenous expression of PD1, the PD1 positive rates of these groups cannot be used to evaluate the degree of exhaustion of CAR-T cells. As shown in the detection results of exhaustion molecules, Figure 7 and Table 3 below, for CD3+PL+, the expression levels of PD1, LAG-3, and Tim-3 in CEAZ-SIRPγ-28 are lower than those in the CEAZ group, indicating that the degree of exhaustion of CEAZ-SIRPγ-28 is lower after antigen stimulation.

[0104]

Table 3

[0105] Similarly, the expression of exhaustion molecules in BCMA-BBZ-P2A-SIRPγ-28 was also low after BCMA antigen stimulation.

[0106] Example 6 Verification of the in vivo function of the novel immunosuppression-resistant CAR-T The mice used for in vivo verification were NCG mice. Thirty NCG mice were injected with DLD-1-CEA-Luc-GFP (triple positive expression of CEA, PD-L1, and CD47, see Figure 8a) to induce cancer. When the tumors grew to the size of mung beans and became measurable, the tumor size was measured. During the experiment, in the mice of the experimental group, listlessness approaching death, hemiplegia or general paralysis were observed, the body weight decreased by 20% (compared with that before the experiment), and the tumor volume reached 1500 mm 3 or more, and the experiment was terminated. Five days after cancer induction, the fluorescence values of the mouse tumors were measured, and the mice were randomly grouped according to the imaging fluorescence values of the living body so that there was no significant difference in the body weight and fluorescence intensity values of the mice in each group, and the average body weight value was calculated. On the 6th day, 100 μL of CAR-T cells (containing 3×10 6 ) were reinjected, and untransfected T cells administered with the same total cell number were used as the control group. As a result of creating a tumor volume increase curve from the measured data of the tumor volume of NCG mice, from 27 to 30 days, CEAZ-SIRPγ-28 showed a significant inhibitory effect on the tumors, and the results are shown in Figure 8b. Furthermore, the in vivo efficacy of the immunosuppressive CAR designed by combining the second-generation CAR and the fusion protein, namely the CEABBZ-P2A-SIRPγ-28 structure, was verified. Similarly, NCG mice were injected with DLD-1-CEA-Luc-GFP cells to induce cancer. Thirteen days after cancer induction, CAR-T cells were reinjected at a total cell number of 8×10 6 . As a result, as shown in Figure 9, the immunosuppressive CAR-T (CEABBZ-P2A-SIRPγ-28) of the present invention can effectively exert its function in vivo, and the effect is superior to that of CEABBZ with the second-generation CAR structure as the control group.

[0107] Second part Experimental part of the CAR structure (the amino acid sequence of CAR1 is 27) targeting CEA

[0108] Example 7 Construction of a plasmid targeting CEA (1) Construction of the experimental group plasmid Using the mini promoter miniCMV, a hypoxia-inducible sequence 5HRE-CMVmini promoter with a nucleotide sequence shown in SEQ ID NO:1 was synthesized. Subsequently, the 5HRE-CMVmini promoter, lentiviral expression vector, and the CAR structure of CEAScFv-CD8 hinge region-CD8 transmembrane region-CD137-CD3ξ-P2A-SIRPγ-CD28 (5HCEA-BBZ-P2A-SIRPγ-28) were each cleaved by double enzyme digestion, the fragments were recovered, the gene fragments were ligated, transformed, monoclonal was collected, and a recombinant plasmid PBKL1-5H1P-CEA-OPRE (SIRPγ fusion protein) containing the SIRPγ fusion protein of the CEA-targeted CAR-T cell preparation was constructed. In this step, the CAR and the fusion protein were co-expressed in the same vector-transfected immune cells, or the CAR and the fusion protein were each expressed in individual vector-transfected immune cells. The CAR structure containing the fusion protein was constructed by two methods. Here, the one obtained by expressing the CAR and the fusion protein in individual vector-transfected immune cells is represented by the abbreviation "5HCEA-BBZ+SIRPγ-28". (2) Construction of control group plasmid According to the method of (1) in Example 1, 5HCEA-BBZ-8H-8 was constructed.

[0109] Example 8 Model verification of in vitro function of CEA-targeted plasmid 5HCEA-BBZ-8H-8, 5HCEA-BBZ-P2A-SIRPγ-28, and SIRPγ-28 were each set as a hypoxia model for verification in the experimental group by adding CoCl2. As a result, as shown in Figures 10 and 11, for 5HCEA-BBZ-8H-8, 5HCEA-BBZ-P2A-SIRPγ-28, and SIRPγ-28, the mean fluorescence intensity and positive rate were similar. As shown in Figure 12, 5HCEA-BBZ-P2A-SIRPγ-28 was more excellent in terms of the amplification multiple.

[0110] Example 9 Verification of the effectiveness of CEA-targeted CAR-T (1) Killing efficiency of target cells against DLDL1-CEA CEA-positive DLD1-CEA and DLD1-CEA(CD47-) cells were used as target cells respectively. After hypoxic treatment of effector cells (ordinary CAR-T cells and CAR-T cells expressing a hypoxic promoter-containing CAR), they were plated on target cells at an effector-target ratio of 1:1, and the target cell killing ability of each CAR-T was detected. After 24 h of CAR-T addition, as shown in the results of the killing efficiency of each group of CAR-T against target cells DLDL1-CEA in Figure 13 and Table 4 below, 5HCEA-BBZ-8H-8, 5HCEA-BBZ+SIRPγ-28, and 5HCEA-BBZ-P2A-SIRPγ-28 have high killing functions, and the SIRPγ-28 group also has a killing function.

[0111]

Table 4

[0112] (2) Detection of IFN-γ, IL-2, and TNF-α secretion Following (1), after 24-hour killing, cell supernatants were collected, and the secretion abilities of IFN-γ, IL-2, and TNF-α after target cell stimulation by CAR-T cells were detected. For the collected supernatants, the secretion of IFN-γ and IL-2 was detected by ELISA method using kits. As shown in the detection results in Figures 14, 15, and 16, when the target cells are DLD1-CEA, 5HCEA-BBZ-8H-8 has low secretion of IFN-γ, IL-2, and TNF-α, and 5HCEA-BBZ-P2A-SIRPγ-28 has higher secretion of IFN-γ, IL-2, and TNF-α than the 5HCEA-BBZ-8H-8 group. When the target cells are DLD1-CEA(CD47-) cells, IFN-γ, IL-2, and TNF-α are either low or undetectable. 5HCEA-BBZ-P2A-SIRPγ-28 of the present invention is more advantageous for the proliferation of CAR-T and the secretion of tumor killing-related factors, indicating that the effectiveness of CAR-T can be reliably improved.

[0113] Verification of the in vivo function of a plasmid containing the SIRPγ fusion protein DLD1-CEA-Luc-GFP cells were used as the tumor-forming target cells for in vivo verification, and a human CEA+ solid tumor-bearing cancer model was constructed. According to tumor volume, the mice were randomly divided into a Control T (CT) group, a 5HCEA-BBZ-8H-8 group, and a 5HCEA-BBZ-P2A-SIRPγ-28 group, with the control group being the Control T group. On the 12th day after tumor formation, the corresponding CAR-T cells of each group of mice were injected via the tail vein at 1*10 7 Copies / mouse. In the Control T group, the same number of T lymphocytes were reinjected on the 8th to 12th days. The tumor volume of the mice in each group was measured once every 3 days. As a result of the experiment, as shown in Figures 17 and 18, 5HCEA-BBZ-8H-8 had a significantly improved in vivo efficacy compared to the mice in the 5HCEA-BBZ-P2A-SIRPγ-28 group, and the 5HCEA-BBZ-8H-8 group also had a significant elimination effect on tumors.

[0114] The experimental part of the CD19-targeted CAR structure Example 9 Construction of a plasmid targeting CD19 Using the SIRPγ and CD47 full-length plasmids, pL-CAG-2AGFP, pL-CAG-PD1-CD28-2ACherry, and pL-CAG-PD1-BB-2Acherry as templates, a CD19-targeted CAR structure was constructed. Vectors: SIRPγ-28, 5HCD19-BBZ, and 5HCD19-BBZ-SIRPγ-28 were constructed. The results of verification by comparison through sequencing were successful.

[0115] Example 10 Production of lentivirus and infection of T lymphocytes Lentivirus was packaged by the calcium phosphate method, and three virus particles (SIRPγ-28, 5HCD19-BBZ, 5HCD19-BBZ-P2A-SIRPγ-28) of Example 1 were obtained. Lymphocytes were separated by gradient centrifugation. After centrifugation, the second-layer white lymphocyte layer was washed with physiological saline, and RPMI 1640 complete medium containing 10% FBS was added for culture to obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 h, then the activated PBMC were infected at a predetermined multiplicity of infection (MOI), and the positive rate of CAR-T was detected by flow cytometry on the 8th day of virus infection. The results are shown in Figure 19 and Table 5 below.

[0116]

Table 5

[0117] Example 11 In vitro Pharmacodynamic Evaluation Targeting CD19 Using Control T as the control group and SIRPγ-28 group, 5HCD19-BBZ group, 5HCD19-BBZ-P2A-SIRPγ-28 group as the experimental groups, Nam6-Luc-GFP (CD19 positive) and K562-Luc-GFP (CD19 negative) as target cells, the in vitro efficacy was verified by in vitro killing and in vitro factor secretion. The results, as shown in Figure 20 and Table 6 below, are that those obtained using the same co-expression vector transfection immune cells (5HCD19-BBZ-P2A-SIRPγ-28) and those obtained using two vector co-transfection immune cells expressed respectively (SIRPγ-28 + 5HCD19-BBZ) had significantly higher in vitro killing than the SIRPγ-28 group and the 5HCD19-BBZ group, and there was no killing of negative cells. As shown in Figure 21 and Table 7, those obtained using the same co-expression vector transfection immune cells (5HCD19-BBZ-P2A-SIRPγ-28) and those obtained using two vector co-transfection immune cells expressed respectively (SIRPγ-28 + 5HCD19-BBZ) had significantly higher factor secretion than the SIRPγ-28 group and the 5HCD19-BBZ group.

[0118]

Table 6

[0119]

Table 7

[0120] Example 12 In Vivo Pharmacodynamic Evaluation Targeting CD19 Nalm6-Luc-GFP cells were subcutaneously injected (s.c.) into NCG mice (female, 6 weeks old) at a dose of 1×10 6 Cells / mouse to establish an in vivo tumor-bearing model. Eight days after tumor-bearing, CAR-T of each group (Control T, 5HCD19-BBZ, 5HCD19-BBZ-P2A-SIRPγ-28) was injected into the tail vein (i.v.) at a dose of 1×10 7 CAR-T Cells / mouse. In vivo tumor growth was observed by bioluminescence imaging, and the therapeutic effect of each CAR-T on lymphoma was evaluated in vivo. As a result, as shown in FIGS. 22 and 23, compared with the Control T group and the 5HCD19-BBZ group, 5HCD19-BBZ-P2A-SIRPγ-28 had an obvious in vivo antitumor effect and could clearly remove tumors.

[0121] Part 4 Experimental Part of PSCA-Targeted CAR Structure Example 13 Construction of PSCA-Targeted Plasmid and Infection of T Cells (1) Construction of Plasmid The CAR structure of the lentiviral expression vector, PSCA ScFv-G4H hinge region-CD28 transmembrane region-CD28-CD137-CD3ξ-P2A-SIRPγ-28 (PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28), was cut by double enzyme digestion respectively, the fragments were recovered, the gene fragments were ligated, transformed, and monoclonal was collected. The lentiviral expression vector, the CAR structure of PSCA ScFv-7H hinge region-CD28 transmembrane region-CD28-CD137-CD3ξ-P2A-SIRPγ-28 (PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28) was cut by double enzyme cleavage respectively, the fragments were recovered, the gene fragments were ligated, transformed, and monoclonal antibodies were collected. (2) Infection of T cells T cells were infected with the obtained plasmid to obtain CAR-T cells.

[0122] Example 14 IFN-γ factor secretion targeting PSCA Using Control T as the control group, and PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28, PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28, PSCA-28BBZ-G4H-28TM+SIRPγ-28, PSCA-28BBZ-7H-28TM+SIRPγ-28, PSCA-28BBZ-G4H-28TM, PSCA-28BBZ-7H-28TM as the experimental groups respectively, RT4-Luc-GFP (PSCA positive) was used as the target cell, and the in vitro efficacy was verified by in vitro factor secretion. The results are shown in Tables 8 - 13 and Figures 24 - 29. PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 or PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 obtained using the same co-expression vector transfected immune cells, and PSCA-28BBZ-G4H-28TM+SIRPγ-28 or PSCA-28BBZ-7H-28TM+SIRPγ-28 obtained using the two vector co-transfected immune cells expressing respectively had much higher IFN-γ factor secretion than the control group and the PSCA-28BBZ-G4H-28TM group or PSCA-28BBZ-7H-28TM group, and there was no killing effect on negative cells.

[0123]

Table 8

[0124]

Table 9

[0125]

Table 10

[0126]

Table 11

[0127]

Table 12

[0128]

Table 13

[0129] Example 14 Cell killing targeting PSCA PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28, PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28, PSCA-28BBZ-G4H-28TM+SIRPγ-28, PSCA-28BBZ-7H-28TM+SIRPγ-28, PSCA-28BBZ-G4H-28TM, and PSCA-28BBZ-7H-28TM were respectively set up as experimental groups, RT4-Luc-GFP (PSCA positive) and PC-3-Luc-GFP (PSCA negative) were respectively used as target cells, and the results are shown in Tables 14 to 19 and Figures 30 to 35. PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 or PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 obtained using the same co-expression vector transfection immune cells, and PSCA-28BBZ-G4H-28TM+SIRPγ-28 or PSCA-28BBZ-7H-28TM+SIRPγ-28 obtained using the two vector co-transfection immune cells expressing respectively were far superior to the control group and the PSCA-28BBZ-G4H-28TM group or the PSCA-28BBZ-7H-28TM group in in vitro killing, and there was no killing of negative cells.

[0130]

Table 14

[0131]

Table 15

[0132]

Table 16

[0133]

Table 17

[0134]

Table 18

[0135]

Table 19

[0136] Note that the above embodiments are merely for explaining the technical means of the present invention and are not intended to be limiting. Although the present invention has been described in detail with reference to preferred embodiments, as will be apparent to those skilled in the art, without departing from the gist and scope of the technical means of the present invention, modifications and equivalent substitutions can be made to the technical means of the present invention, and all of these shall be included in the scope of the claims of the present invention.

Claims

Claim 1: A SIRPγ fusion protein and CAR1, comprising: The CAR1 comprises a ScFv that recognizes a tumor antigen, a hinge region, a transmembrane region, and an intracellular signaling region; The structure of the SIRPγ fusion protein is SIRPγ-CD28TM-CD28; The SIRPγ fusion protein is linked to CAR1 by a self-cleaving polypeptide; The self-cleaving polypeptide is T2A, P2A, E2A or F2A, and is a tumor immunosuppression-resistant CAR. Claim 2: The tumor immunosuppression-resistant CAR according to claim 1, wherein the amino acid sequence of the extracellular portion of the SIRPγ is shown in SEQ ID NO:

1. Claim 3: The amino acid sequence of the SIRPγ fusion protein SIRPγ-CD28TM-CD28 is shown in SEQ ID NO: 2; or The nucleotide sequence of the SIRPγ fusion protein SIRPγ-CD28TM-CD28 is shown in SEQ ID NO: 13, and is the fusion protein according to claim 1. Claim 4: The structure of the CAR is: ScFv-hinge-TM-CD3ζ-self-cleaving peptide-SIRPγ-CD28TM-CD28, or ScFv-hinge-TM-4-1BB-CD3ζ-self-cleaving peptide-SIRPγ-CD28TM-CD28, and is the tumor immunosuppression-resistant CAR according to claim 1. Claim 5: The ScFv recognizes any one or more of CD19, CD123, Mov-γ, PSMA, IL13Rα2, EGFRvIII, EGFR, EpCAM, GD2, MUC1, HER2, GPC3, CEA, Meso, CD133, NKG2D, CD138, LeY, k-Light, CD33, ROR1, BCMA, CD30, CD20, CD22, PSCA, CLL-1, CD70, CD47, and is the tumor immunosuppression-resistant CAR according to claim 4. Claim 6: The amino acid sequence of the ScFv is shown in SEQ ID NO: 25, or The CAR1 is as follows: a) CAR1 comprises a CEA single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, CD137, and a CD3ξ dual-stimulatory signal; b) The CAR1 comprises a CD19 single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, CD137, and a CD3ξ dual-stimulatory signal, and c) The CAR1 includes a single-chain antibody of PSCA, a hinge region, a transmembrane region of CD28, CD28, CD137 and a triple-stimulatory signal of CD3ξ, and the hinge region is G4H or 7H. The tumor immunosuppression-resistant CAR according to claim 5, comprising one selected from the group consisting of.

7. A nucleic acid molecule encoding the tumor immunosuppression-resistant CAR according to claim 6, wherein the nucleotide sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO:

35. Nucleic acid molecule.

8. An expression vector comprising the nucleic acid molecule according to claim 7 or its complementary sequence.

9. An immune cell comprising the tumor immunosuppression-resistant CAR according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the expression vector according to claim 8.

10. When transfecting immune cells, co-express the CAR structure without the SIRPγ fusion protein and the SIRPγ fusion protein in the same vector. The method for producing the immune cell according to claim 9.

11. A pharmaceutical composition comprising the tumor immunosuppression-resistant CAR according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, or the immune cell according to claim 9.

12. Use of the pharmaceutical composition according to claim 11 for the manufacture of a tumor therapeutic agent, wherein the tumor is acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer and skin cancer, and the tumor is CD19, CD123, Mov-γ, PSMA, IL13Rα2, EGFRvIII, EGFR, EpCAM, GD2, MUC1, HER2, GPC3, CEA, Meso, CD133, NKG2D, CD138, LeY, k-Light, CD33, ROR1, BCMA, CD30, CD20, CD22, PSCA, CLL-1, CD70, CD47. Use that highly expresses one or more of them.

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