FUSION PROTEIN FOR REVERSING TUMOR MICROENVIRONMENT AND USE THEREOF

Abstract

The present application belongs to the technical field of immunotherapy, and specifically relates to a fusion protein for reversing a tumor microenvironment, a tumor immunosuppressive and resistant CAR and an expression vector, an immune cell and the use. The fusion protein is combined with different CAR structures of different targets, such as CEA, CD19, PSCA and BCMA, to form the tumor immunosuppressive and resistant CAR, or is used in combination with same to target and kill CD47 positive tumor cells; and the tumor immunosuppressive and resistant CAR and immune cells break through the influences of inhibitory signals on the function of CAR-T, which realizes the effectiveness of CAR-T treatment and can also ensure a certain degree of safety at the same time.

Claims

1-32. (canceled)

33. A fusion protein for reversing the tumor microenvironment, wherein the fusion protein is a SIRPγ fusion protein, and the structure of the SIRPγ fusion protein comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain.

34. The fusion protein according to claim 33, wherein the transmembrane domain is derived from human CD28 transmembrane domain or human CD8 transmembrane domain, preferably, the amino acid sequence of the transmembrane domain is shown in SEQ ID NO:7 or SEQ ID NO:8.

35. The fusion protein according to claim 34, wherein the intracellular signaling domain is derived from CD28 or 4-1BB; preferably, the sequence of the intracellular signaling domain is shown in SEQ ID NO:9 or SEQ ID NO:38.

36. The fusion protein according to claim 33, wherein the structure of the SIRPγ fusion protein is SIRPγ-CD28TM-CD28 or SIRPγ-CD8TM-4-1BB.

37. The fusion protein according to claim 33, wherein the amino acid sequence of the extracellular domain of SIRPγ is shown in SEQ ID NO: 1 or is a functional variant thereof.

38. The fusion protein according to claim 36, wherein the amino acid sequence of the SIRPγ fusion protein SIRPγ-CD28TM-CD28 is shown in SEQ ID NO: 2 or is a functional variant thereof; the amino acid sequence of the SIRPγ fusion protein SIRPγ-CD8TM-4-1BB is shown in SEQ ID NO: 3 or is a functional variant thereof; the nucleotide sequence of the SIRPγ fusion protein SIRPγ-CD28TM-CD28 is shown in SEQ ID NO: 13; or the nucleotide sequence of the SIRPγ fusion protein SIRPγ-CD8TM-4-1BB is shown in SEQ ID NO: 14.

39. A tumor immunosuppression-resistant CAR, wherein the CAR comprises the fusion protein according to claim 38 and CAR1, and the CARL comprises an extracellular domain recognizing a tumor antigen, a hinge region, a transmembrane domain and an intracellular signaling domain.

40. The tumor immunosuppression-resistant CAR according to claim 39, wherein the fusion protein is linked to CARL through a polycistronic structure, and the polycistronic structure is a self-cleaving polypeptide or an internal ribosome entry site IRES, and the self-cleaving polypeptide is T2A, P2A, E2A or F2A.

41. The tumor immunosuppression-resistant CAR according to claim 40, wherein the CAR structure is an ScFv-hinge-TM-CD3ζ-self-cleaving peptide-SIRPγ fusion protein or an ScFv-hinge-TM-4-1BB-CD3ζ-self-cleaving peptide-SIRPγ fusion protein; or the CAR structure is an ScFv-hinge-TM-CD3ζ-self-cleaving peptide-SIRPγ-CD28TM-CD28 or an ScFv-hinge-TM-4-1BB-CD3ζ-self-cleaving peptide-SIRPγ-CD28TM-CD28.

42. The tumor immunosuppression-resistant CAR according to claim 39, wherein in the CARL structure: the amino acid sequence of the hinge is shown in SEQ ID NO: 24 or is a functional variant thereof, and 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 is a functional variant thereof; and in the structure of the fusion protein: the amino acid sequence of the extracellular domain of SIRPγ is shown in SEQ ID NO: 1 or is a functional variant thereof; the amino acid sequence of the transmembrane domain derived from human CD28 is shown in SEQ ID NO:7; the amino acid sequence of the intracellular signaling domain derived from human CD28 is shown in SEQ ID NO:9.

43. The tumor immunosuppression-resistant CAR according to claim 39, wherein 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, and CD47.

44. The tumor immunosuppression-resistant CAR according to claim 43, wherein the amino acid sequence of the ScFv is shown in SEQ ID NO: 25 or is a functional variant thereof.

45. The tumor immunosuppression-resistant CAR according to claim 39, wherein the CAR1 comprises one of the following: a) the CAR1 comprising a CEA single chain antibody, a CD8 hinge region, a CD8 transmembrane domain, a CD137-CD3ζ dual-stimulus signal; preferably, the amino acid sequence of the CAR structure is shown in SEQ ID NO: 26 or is a functional variant thereof; orb) the CAR1 comprising a CD19 single chain antibody, a CD8 hinge region, a CD8 transmembrane domain, a CD137-CD3ζ dual-stimulus signal; preferably, the amino acid sequence of the CAR structure is shown in SEQ ID NO: 27 or is a functional variant thereof; or c) the CAR1 comprising a PSCA single-chain antibody, a hinge region, a CD28 transmembrane domain, a CD28-CD137-CD3ζ tristimulus signal; the hinge region is G4H or 7H; preferably, the amino acid sequence of the CAR structure is shown in SEQ ID NO: 28 or is a functional variant thereof; or is shown in SEQ ID NO: 29 or is a functional variant thereof.

46. The tumor immunosuppression-resistant CAR according to claim 45, wherein the CAR1 structures in a) and b) further comprise a hypoxia inducible promoter, and the nucleic acid sequence of the hypoxia inducible promoter comprises the sequence shown in SEQ ID NO:30.

47. A nucleic acid, wherein it encodes the tumor immunosuppression-resistant CAR according to claim 44, the nucleic acid sequence comprises 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.

48. An expression vector comprising the nucleic acid according to claim 47, wherein the expression vector is any one of a lentivirus expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, DNA vector, RNA vector and plasmid.

49. An immune cell comprising the fusion protein according to claim 33.

50. The immune cell according to claim 49, wherein the immune cell is a T cell, a T cell precursor or an NK cell; preferably the immune cell is prepared by the method that the CAR structure not comprising the SIRPγ fusion protein and the SIRPγ fusion protein are co-expressed in a vector when transfecting immune cells; or the CAR structure not comprising the SIRPγ fusion protein and the SIRPγ fusion protein are expressed separately in two vectors when transfecting immune cells; and preferably the immune cell is used in the preparation of medicaments of tumors; preferably the tumors are malignant tumors, including acute lymphoid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer and skin cancer, the tumors highly express 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, and CD47.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG. 1 is a structural diagram of an immunosuppression-breaking fusion protein.

[0083] FIG. 2 is a schematic diagram of the structure of an immunosuppression-resistant CAR.

[0084] FIG. 3 is the picture of the verification of CAR construction plasmids.

[0085] FIG. 4 shows the result of target cell constructions.

[0086] FIG. 5a shows the CAR positive rate of the novel immunosuppression-resistant CAR-T.

[0087] FIG. 5b shows the expression intensity of the novel immunosuppression-resistant CAR-T.

[0088] FIG. 6a shows the in vitro function verification of the novel immunosuppression-resistant CAR-T.

[0089] FIG. 6h shows the in vitro killing rate of the novel immunosuppression-resistant CAR-T.

[0090] FIG. 7 shows the validation of the novel immunosuppression-resistant CAR-T to disrupt the tumor suppressive environment.

[0091] FIG. 8a shows the triple positive expression of CEA, PD-L1 and CD47 in NCG mice.

[0092] FIG. 8b plots the tumor volume growth curve for the tumor volume measurement data of NCG mice.

[0093] FIG. 9 shows the in vivo effectiveness of the second-generation immunosuppressive CAR.

[0094] FIG. 10 shows the average fluorescence intensity of each experimental group targeting CEA.

[0095] FIG. 11 shows the positive rate of each experimental group targeting CEA.

[0096] FIG. 12 is the in vitro expansion fold curve of CAR-T in each experimental group targeting CEA.

[0097] FIG. 13 shows the killing efficiency of CAR-T in each experimental group targeting CEA on target cells DLDL1-CEA.

[0098] FIG. 14 shows the secretion of IFN-γ in DLD1-CEA and DLD1-CEA (CD47−) cells in each experimental group targeting CEA.

[0099] FIG. 15 shows the secretion of IL-2 in DLD1-CEA and DLD1-CEA (CD47−) cells in each experimental group targeting CEA.

[0100] FIG. 16 shows the secretion of TNF-α in DLD1-CEA and DLD1-CEA (CD47−) cells in each experimental group targeting CEA.

[0101] FIG. 17 is an image of the tumor growth in in vivo hypoxia verification model mice in the control and screening groups targeting CEA.

[0102] FIG. 18 shows the bioluminescence in in vivo hypoxia verification model mice in the control and screening groups targeting CEA

[0103] FIG. 19 shows the detection of the positive rate of CAR-T in each experimental group targeting CD19 by flow cytometry.

[0104] FIG. 20 shows the cell killing of each experimental group targeting CD19.

[0105] FIG. 21 shows the secretion of IFN-γ factor in each experimental group targeting CD19.

[0106] FIG. 22 shows the in vivo bioluminescence of mice in each experimental group targeting CD19.

[0107] FIG. 23 shows tumor growth of mice in each experimental group targeting CD19.

[0108] FIG. 24 shows the secretion of IFN-γ factor by PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28.

[0109] FIG. 25 shows the secretion of IFN-γ factor by PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28.

[0110] FIG. 26 shows the secretion of IFN-γ factor by PSCA-28BBZ-G4H-28TM+SIRPγ-28.

[0111] FIG. 27 shows the secretion of IFN-γ factor by PSCA-28BBZ-7H-28TM+SIRPγ-28.

[0112] FIG. 28 shows the secretion of IFN-γ factor by PSCA-28BBZ-G4H-28TM and PSCA-28BBZ-G4H-28TM+SIRPγ-28,

[0113] FIG. 29 shows the secretion of IFN-γ factor by PSCA-28BBZ-7H-28TM and PSCA-28BBZ-7H-28TM+SIRPγ-28.

[0114] FIG. 30 shows the cell killing of PSCA-28BBZ-G4H-2-P2A-SIRPγ-28 in positive/negative cells, respectively.

[0115] FIG. 31 shows the cell killing of PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 in positive/negative cells, respectively,

[0116] FIG. 32 shows the cell killing of PSCA-28BBZ-G4H-28TM+SIRPγ-28 in positive/negative cells, respectively.

[0117] FIG. 33 shows the cell killing of PSCA-28BBZ-7H-28TM+SIRPγ-28 in positive/negative cells, respectively,

[0118] FIG. 34 shows the cell killing of PSCA-28BBZ-G4H-28TM and PSCA-28BBZ-G4H-28TM+SIRPγ-28.

[0119] FIG. 35 shows the cell killing of PSCA-28BBZ-7H-28TM and PSCA-28BBZ-7H-2-28TM+SIRPγ-28.

[0120] In FIGS. 10-18, 5HCEA-BBZ refers to a CEA-targeting CAR structure with a Hypoxia inducible promoter and a co-stimulator signal derived from the 4-1BB intracellular domain and CD3C; in its intracellular structure; CEA-BBZ refers to a CEA-targeting CAR structure with a co-stimulatory signal derived from the intracellular domain of 4-1BB and CD3ζ in its intracellular structure, CEA-28Z refers to a CEA-targeting CAR structure with a co-stimulatory signal derived from the intracellular domain of CD28 and CD3ζ in its intracellular structure, SIRPγ-28TM-28 refers to a fusion peptide with only a signal derived from the intracellular domain of CD28 in its intracellular structure, abbreviated as SIRPγ-28.

[0121] In FIGS. 19-23, 5HCD19-BBZ refers to a CD19-targeting CAR structure with a Hypoxia inducible promoter, a co-stimulatory signal derived from the intracellular domain of 4-1BB (abbreviated as BB) and CD3ζ in its intracellular structure; SIRPγ-28TM-28 (SIRPγ-28) refers to a fusion peptide with an only intracellular signal from CD28; 5HCD19-BBZ-SIRPγ-28 refers to a CAR with the following structure: 5HCD19-8H-8TM-CD137-CD3ζ; SIRPγ-28-5HCD19-BBZ refers to a CAR-T obtained by transfecting immune cells with the CAR comprising a hypoxia inducible promoter and the fusion protein expressed by two vectors respectively.

[0122] In FIGS. 24-35, RT4-Luc-GFP is a positive cell, and PC-3-Luc-GFP is a negative cell; SIRPγ-28TM-28 (SIRPγ-28) refers to a fusion peptide with only the signal derived from the intracellular domain of CD28 in its intracellular structure; PSCA-28BBZ-G4H-28TM+SIRPγ-28 or PSCA-28BBZ-7H-28TM+SIRPγ-28 refers to the products obtained by transfecting immune cells with two vectors expressing PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM with the fusion protein SIRPγ-28 separately. PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 or PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 refers to the products obtained by transfecting immune cells with a vector co-expressing PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-28TM with the fusion protein SIRPγ-28.

DETAIL DESCRIPTION OF THE INVENTION

[0123] Hereinafter, preferred examples of the present application will be described in detail with reference to the drawings. In the preferred examples, the experimental methods that do not specify specific conditions are usually in accordance with conventional conditions, such as the conditions described in the Molecular Cloning: A Laboratory Manual (third edition, J. Sambrook et al.), or as recommended by the manufacturer. The listed examples are used to better illustrate the content of the present application, but the content of the present application is not limited to the listed examples. Therefore, those skilled in the art make non-essential improvements and adjustments to the embodiments according to the above-mentioned contents of the application, resulting in which still belong to the protection scope of the present application.

[0124] In the examples of the present application, the sequences involving vector structure elements are shown in Table 1 below

TABLE-US-00001 TABLE 1 Sequences of the vector structure elements. CAR structure element Nucleotide sequence Amino acid sequence SIRPγ SEQ ID NO: 12 SEQ ID NO: 1 hinge SEQ ID NO: 24 CD28TM SEQ ID NO: 18 SEQ ID NO: 7 CD8TM SEQ ID NO: 19 SEQ ID NO: 8 CD28 ICD SEQ ID NO: 20 SEQ ID NO: 9 4-1BB ICD SEQ ID NO: 21 SEQ ID NO: 10 CD3ζ SEQ ID NO: 22 SEQ ID NO: 11 CEA ScFv SEQ ID NO: 25 CEAZ SEQ ID NO: 15 SEQ ID NO: 4 CEA-BB-CD3ζ SEQ ID NO: 23 SIRPγ-28TM-28 SEQ ID NO: 13 SEQ ID NO: 2 SIRPγ-8TM-BB SEQ ID NO: 14 SEQ ID NO: 3 CEAZ-P2A-SIRPγ-28 SEQ ID NO: 16 SEQ ID NO: 5 CEAZ-P2A-SIRPγ-BB SEQ ID NO: 17 SEQ ID NO: 6

[0125] In the examples of the present application (in Table 1): Z refers to CD3ζ, CEAZ refers to the CAR structure (ScFv(CEA)-hinge-TM-CD3ζ) with only the CD3ζ in the intracellular structure, and CEA-28Z refers to a CEA-targeting CAR structure (ScFv(CEA)-hinge-TM-CD28-CD3ζ) with a co-stimulatory signal derived from the intracellular domain of CD28 and CD3; in its intracellular structure, CEA-BBZ refers to a CEA-targeting CAR structure (ScFv(CEA)-hinge-TM-4-1BB-CD3ζ) with a co-stimulatory signal derived from the intracellular domain of 4-1BB and CD3ζ in its intracellular structure, BCMA-BBZ refers to a BCMA-targeting CAR structure with a co-stimulatory signal derived from the intracellular domain of 4-1BB and CD3ζ in its intracellular structure; SIRPγ-28TM-28 refers to a fusion peptide with only a signal derived from the intracellular domain of the CD28 in its intracellular structure, abbreviated as SIRPγ-28; SIRPγ-8TM-BB refers to a fusion peptide with only a signal derived from the intracellular domain of 4-1BB in its intracellular structure, abbreviated as SIRPγ-BB.

[0126] In the examples of the present application, 5HCD19-BBZ refers to a CD19-targeting CAR structure with a Hypoxia inducible promoter, a co-stimulatory signal derived from the intracellular domain of the 4-1BB (abbreviated as BB) and the CD3ζ in its intracellular structure; SIRPγ-28TM-28 (abbreviated as SIRPγ-28) refers to a fusion peptide with only a signal derived from the intracellular domain of the CD28 in its intracellular structure; SIRPγ-28+5HCD19-BBZ refers to a CAR-T obtained by transfecting immune cells with two vectors expressing the CAR comprising a Hypoxia inducible promoter and the fusion proteins separately. 5HCD19-BBZ-P2A-SIRPγ-28 refers to a CAR-T obtained by transfecting immune cells with a vector co-expressing the CAR comprising a hypoxia inducible promoter and the fusion protein.

[0127] In the examples of the present application, PSCA-28BBZ-G4H-28TM+SIRPγ-28 or PSCA-28BBZ-7H-28TM+SIRPγ-28 refers to the products obtained by transfecting immune cells with two vectors expressing PSCA-28BBZ-G414-28TM or PSCA-28BBZ-7H-28TM with the fusion protein SIRPγ-28 separately. PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 or PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 refers to the products obtained by transfecting immune cells with a vector co-expressing PSCA-28BBZ-G4H-28TM or PSCA-28BBZ-7H-281M with the fusion protein SIRPγ-28.

[0128] In the examples of the present application, the method for packaging lentivirus by calcium phosphate method is specifically: 293T cells are cultured to a better state with DMEM medium comprising 10% FBS (w/v), the packaging plasmids (RRE: REV: 2G) and the expression plasmid are added to a 1.5 centrifuge tube according to a certain ratio, and CaCl.sub.2) and 2×HBS are added. After being well mixed, cells are added to the treated 293T cell culture medium after being left to stand at room temperature. After 3-5 hours, the medium is changed again to 10 mL DMEM medium with 10% FBS, and the cell supernatant is collected after 48 h or 72 h, and the virus is purified.

[0129] In the examples of the present application, the antibody is: Protein-L-PE. Protein-L can recognize the light chain of the antibody, and the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L. So the Protein-L can be used to detect CAR positive rate and CAR expression intensity. SIRPγ-28 is tagged with GFP, and its expression is determined by the GFP positive rate.

[0130] In the examples of the present application, the method for detecting the killing ability of different CAR-Ts on target cells is as follows: using the ACEA xCELLigence RICA MP instrument, and the experimental steps are performed according to the instructions of the instrument. The principle of ACEA xCELLigence RICA MP is using the resistance index data to record the tumor cells attached to the bottom of the well every 15 minutes, and the proliferation or death of the adherent target cells is determined according to the resistance index. The results are analyzed by using the resistance index, the formula is: CAR-T cell killing rate=baseline resistance index−real-tune resistance index.

[0131] In the examples of the present application, IFN-γ is detected with BD IFN-γ kit, and the experimental steps are carried out according to the product instructions; IL-2 is detected with Invitrogen IL-2 kit, and the experimental steps are carried out according to the product instructions; TNF-α, is detected with the Biolegend kit, and the experimental steps are carried out according to the product instructions.

[0132] In the examples of the present application, the method for verifying whether the construction of the hypoxia model has been achieved is as follows: an in vitro hypoxic cell model is constructed by infection of activated PBMC with recombinant plasmid virus, and after culturing for 12-18 hours, the medium is changed; then a hypoxic environment is induced by CoCl2, and, the expression of CAR is detected by detecting the light chain antibody on the CAR structure on day N of the culture.

[0133] In the examples of the present application, the mice used for in vivo verification are NOD. Cg-PrkdescidII2rgtm1Sug/JicCrl is abbreviated as NOG mice, which are bred by Mamoru Ito of the Central institute of Experimental Animals (CIEA), Japan, and are the most common strain of CAR-T related tumorigenesis in vivo experiment in the world.

[0134] In the examples of the present application, the method for verifying with the hypoxia model in vivo is as follows: 6-8-week old female NOG mice are selected, after marking the ear numbers, on the back of the mouse, target cells are injected subcutaneously at an amount of 1×10{circumflex over ( )}6 cells/mouse, the tumor volume of mice is measured on the 12th day after tumor formation.

[0135] Part I

Example 1 Plasmid Construction

[0136] Based on the CAR pattern shown in FIG. 1, using SIRPγ and CD47 full-length plasmids, pL-CAG-2AGFP, pL-CAG-PD1-CD28-2ACherry, and pL-CAG-PD1-BB-2ACherry as templates, the CAR structure shown in FIG. 2 and the corresponding single-target CAR structure were constructed. The 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 CARS: CEAZ, PD1-28, PD1-BB, SIRPγ-28TM-28, SIRPγ-28TM-1BB were obtained by construction, After verification with enzyme digestion and sequencing and alignment, the results were shown in FIG. 3. The enzyme digestion identification of the recombinant plasmid: 1-3: pL-CAG-CEAZ-PD1-28 plasmid, the order is: original plasmid, CEAZ (1371 bp), PD1-28 (783 bp); 4-6: pL-CAG-CEAZ-PD1-BB plasmid, the order is: original plasmid, CEAZ (1371 bp), PDT-BB (798 bp); 7-9: pL-CAG-CEAZ-SIRPγ-28 plasmid, the order is: original plasmid, CEAZ (1371 bp), SIRPγ-28 (1374 bp); 10-12: pL-CAG-CEAZ-SIRPγ-BB plasmid, the order is: original plasmid, CEAZ (1371 bp), SIRPγ-BB (1368 bp); M1: DL5000 DNA molecular weight standard; M2: DL15000 DNA molecular weight standard; 13-15: pL-CAG-SIRPγ-28-2AGFP plasmid, the order is: original plasmid, SIRPγ-28 (1292 bp), 2AGFP (785 bp) 16-18: pL-CAG-SIRPγ-1BB-2AGFP plasmid, the order is: original plasmid, SIRPγ-BB (1286 bp), 2AGFP (785 bp) 19-20: pL-CAG-CD47 plasmid, the order is: CD47 (974 bp), the original plasmid; M3: DL5000 DNA molecular weight standard; the construction was successful.

Example 2 Target Cell Construction

[0137] Viruses with CEA, PD-L1 and CD47 antigens were prepared by calcium phosphate method, and CHO cells were infected to construct CHO-CEA cells, CHO-CEA-PD-L1 cell lines and CHO-CEA-CD47 cell lines, respectively. Signal regulatory protein gamma (SIRPγ) was one of the ligands of CD47 and can bind to CD47, so CD47-positive target cells could be used to evaluate CEAZ-SIRPγ-BB, CEAZ-SIRPγ-28, SIRPγ, SIRPγ-28, and SIRPγ-BB.

[0138] The positive rate of the three cell lines was detected after ten subcultures. The results were shown in FIG. 4. The positive rate of CHO-CEA was 97.1%, the double-positive rate of CHO-CEA-CD47 was 97.6%, and the positive rate of CHO-CEA-PD-L1 was 87%, which meets the experimental requirements, indicating that the cell lines had been successfully constructed and could be used as target cells for subsequent CAR-T efficacy evaluation.

Example 3 Preparation of Lentivirus and Infection of T Lymphocytes

[0139] The lentivirus was packaged by the calcium phosphate method to obtain 5 virus particles with single-expressed CARs (CEAZ, PD1-28, PD1-BB, SIRPγ-28, SIRPγ-BB) and 6 virus particles with novel immunosuppression-resistant CARs (CEAZ-PD1-28, CEAZ-PD1-BB, CEAZ-SIRPγ-28, CEAZ-SIRPγ-BB, CEABBZ-SIRPγ-28, CEABBZ-SIRPγ-BB).

[0140] Gradient centrifugation was used to separate lymphocytes; after centrifugation, the second layer with white lymphocytes was taken, washed with physiological saline, and cultured by adding RPMI 1640 complete medium comprising 10% FBS to obtain human PBMC cells. The obtained PBMC cells were activated by anti-CD3 and CD28 monoclonal antibodies for 24 hours, the activated PBMCs were infected with a certain multiplicity of infection (MOI), and the positive rate of CAR-T was detected on the 12th day of virus infection. The detection method was flow cytometry, the antibody was: Protein-L-PE. Protein-L could recognize the light chain of the antibody, and the light chain of the ScFv sequence of the CAR antigen recognition region could be recognized by Protein-L, so Protein-L could be used to detect CAR positive rate and the density of CAR expression.

[0141] The results were shown in FIG. 5a: the immunosuppression-disrupting fusion protein could be successfully expressed on the surface of T cells, as shown in FIG. 5b, the novel immunosuppression-resistant CARs were successfully expressed.

Example 4 In Vitro Function Verification of Novel Immunosuppression-Resistant CAR-T

[0142] Control T cells that did not express the immunosuppression-disrupting fusion protein were used as a control to verify the functions of the immunosuppressive-disrupting fusion proteins SIRPγ-28TM-28 and SIRPγ-8TM-BB. The target cells were the CHO cell line expressing CD47. The results were shown in FIG. 6a, SIRPγ-28TM-28 and SIRPγ-8TM-BB fusion proteins had killing effects on CD47-positive target cells.

[0143] Taking CEAZ group as the positive control and Control-T group as the negative control, CEAZ-PD1-28, CEAZ-PD1-BB, CEAZ-SIRPγ-28, CEAZ-SIRPγ-BB groups were set as experimental groups, and CHO-CEA-CD47 and CHO-CEA-PD-L1 were set as target cells to verify the in vitro effectiveness of the novel immunosuppression-resistant CAR-Ts. The results were shown in FIG. 6h and Table 2 below, the cell killing efficiency of the CEAZ-PD1-28 and CEAZ-SIRPγ-28 groups were significantly higher than that of the CEAZ group, while the killing efficiency of the CEAZ-PD1-BB and CEAZ-SIRPγ-BB groups was not significantly enhanced compared with that of CEAZ group. In addition, in CEAZ-PD1-28 and CEAZ-PD1-BB groups, in the case of co-culturing with CIO-CEA-CD47, and in CEAZ-SIRPγ-28 and CEAZ-SIRPγ-BB groups, in the case of co-culturing with CHO-CEA-PD-L1, there was no significant difference in the killing efficiency compared with that of the CEAZ group, indicating that the novel immunosuppression-resistant CAR-Ts had specific killing, and CEAZ-PD1-28 and CEAZ-SIRPγ-28 had significant killing effects on target cells.

TABLE-US-00002 TABLE 2 In vitro killing efficiency of immunosuppression-resistant CAR-T Target cell CHO-CEA-CD47 CHO-CEA-PDL1 Structure (specific lysis %) (specific lysis %) CEA-Z 52.95458 ± 16.246  42.37838 ± 17.43639 CEA-Z-PD1-28  26.8274 ± 13.62647 66.58775 ± 16.8083  CEA-Z-PD1-BB 25.88708 ± 19.55145 21.18065 ± 18.72001 CEA-Z-SIRPγ-28 62.89653 ± 15.15207 19.26225 ± 24.32014 CEA-Z-SIRPγ-BB 19.08425 ± 19.57656 6.92085 ± 4.83563

[0144] Using K562-BCMA as the target cell and Control-T group as the negative control, the function of BCMA-BBZ-P2A-SIRPγ-28 was verified, and the result showed BCMA-BBZ-P2A-SIRPγ-28 could show good killing function.

Example 5 Validation of a New Type of Immunosuppression-Resistant CAR-T to Disrupt the Tumor Suppressive Environment

[0145] After CAR-T′ cells infiltrated tumor tissues, they were often affected by tumor immunosuppressive microenvironment, and highly expressed attenuating molecules PD1, LAG-3, and Tim-3 a, thereby the effective function of killing tumor cells was weakened, and the apoptosis of CAR-T cells themselves was increased. In order to verify whether the novel immunosuppression-resistant CAR-T could break the tumor suppressive microenvironment signal, in CEAZ, CEA-28Z, CEA-BBZ, CEAZ-PD1-28, CEAZ-PD1-BB, CEA-SIRPγ-28, CEAZ-SIRPγ-BB and Control-T groups, when CAR-T cells were cultured to Day 7, CAR-T cells and DLD-1-CEA-Luc-GFP cells were co-cultured in a 12-well cell culture plate, and CAR-T cells were collected after 48 hours, labeled with anti-human CD3, PL, PD1, LAG-3, Tim-3 flow cytometry antibody, and then detected by flow cytometry, and the detection results were analyzed to evaluate their effect ability. Since Pal was exogenously expressed in the CEAZ-PD1-28 and CEAZ-PD1-BB groups, the PD1 positivity rates of these two groups were not used to evaluate the degree of CAR-T cell exhaustion. The results of exhausted molecular detection were shown in FIG. 7 and Table 3 below: Under the condition of CD3+, the expression levels of PD1, LAG-3 and Tim-3 in CEAZ-SIRPγ-28 group were lower than those in CEAZ group, indicating that the degree of exhaustion in CEAZ-SIRPγ-28 after antigen stimulation was lower.

TABLE-US-00003 TABLE 3 Validation of immunosuppression-resistant CAR-T against tumor suppressive environment Expression value of exhausted molecules Structure PD1 LAG-3 TIM-3 CEAZ 27.93333 ± 11.55783 37.36667 ± 12.72019 41.26667 ± 19.85254 CEAZ-PD1-28 50.56667 ± 28.00042 26.02667 ± 29.26886 34.11333 ± 40.76119 CEAZ-PD1-BB 40.93333 ± 36.89205   31.36 ± 37.05815 39.86667 ± 41.51293 CEAZ-SIRPγ-28 11.27667 ± 7.901559 12.28667 ± 7.190447 27.06667 ± 16.64041 CEAZ-SIRPγ-BB 29.49667 ± 29.36888   30.2 ± 25.03937   36.44 ± 38.31466 Control-T 11.65667 ± 13.05796   19.05 ± 14.40113 17.20667 ± 8.675606

[0146] Likewise, BCMA-BBZ-P2A-SIRPγ-28 also showed lower expression of exhausted molecules after stimulation with BCMA antigen.

Example 6 In Vivo Functional Verification of Novel Immunosuppression-Resistant CAR-T

[0147] The mice used for in vivo validation were NCG mice. Thirty NCG mice were selected and injected with DLD-1-CEA-Luc-GFP (CEA, PD-1.1, CD47 triple positive expression, as shown in FIG. 8a) for tumor bearing. When the tumor grew to a measurable size as luting bean, the tumor size is measured. During the experiment, when the mice in the experimental group appeared any situation of: sluggish and dying, half- or whole-body paralysis, lost 20% of the body weight (compared with that before the start of the experiment), and the tumor volume ≥1500 mm.sup.3, the experiment was terminated.

[0148] The tumor fluorescence value of mice was measured on Day 5 after tumor bearing, and the fluorescence value of in vivo imaging was used to randomly group the mice to ensure that there was no significant difference in the body weight and fluorescence value of the mice in each group, and the average body weight was calculated. On Day 6, CAR-T cells were re-infused in a volume of 100 μL (comprising 3×10.sup.6 effective CAR-T cells), and untransfected T cells with the same total cell number were given as a control group. The tumor volume growth curve was drawn based on tumor volume measurement data of NCG mice, and it was found that on Day 27-30, CEAZ-SIRPγ-28 had a relatively obvious inhibitory effect on the tumor, and The results were shown in FIG. 8b.

[0149] Further, the in vivo effectiveness of an immunosuppressive CAR (i.e., the CEABBZ-P2A-SIRPγ-28 structure) designed by combining the second-generation CAR with the fusion protein was verified. NCG mice were also injected with DLD-1-CEA-Luc-GFP cells to bear tumors. After 13 days of tumor bearing, CAR-T cells were reinfused, and the total number of cells was 8×10.sup.6. The results were shown in FIG. 9 that the immunosuppressive CAR-T of the present application (CEABBZ-P2A-SIRPγ-28) could function well in vivo, and the effect was obviously better than that of CEABBZ with the second-generation CAR structure of the control group.

[0150] Part 11 the experimental part of the CEA-targeting CAR structure (the amino acid sequence of CARL is SEQ ID NO: 27)

Example 7 Construction of Plasmid Targeting CEA

[0151] (1) Plasmid Construction of Experimental Group

[0152] The hypoxia inducible promoter sequence 5HRE-CMVmini promoter was synthesized from the mini promoter miniCMV, and its nucleotide sequence was shown in SEQ ID NO: 1. Then the 5HRE-CMVmini promoter, lentiviral expression vector, CEAScFv-CD8 hinge region-CD8 transmembrane region-CD137-CD3ζ-P2A-SIRPγ-CD28 (5HCEA-BBZ-P2A-SIRPγ-28) CAR structure were cleaved by double enzyme digestion and recovered, the gene fragments were connected and transformed, and single clones were picked, and the recombinant plasmid PBKL1-5H1P-CEA-CPRE (SIRPγ fusion protein) comprising SIRPγ fusion protein of the CEA-targeting CAR-T cell preparation was constructed. In this step, the structure of our CAR comprising fusion protein can be obtained by two methods: the CAR and the fusion protein were co-expressed in one vector when transfecting immune cells or the CAR and the fusion protein were expressed separately in two vectors when transfecting immune cells. Wherein the CAR and the fusion protein were expressed separately in two vectors when transfecting immune cells to obtain the product abbreviated as “5HCEA-BBZ+SIRPγ-28”.

[0153] (2) Construction of Plasmid in Control Group

[0154] 5HCEA-BBZ-8H-8 was constructed according to the method of (1) in Example 1.

Example 8 In Vitro Function Validation of Plasmid Targeting CEA in Models

[0155] 5HCEA-BBZ-8H-8, 5HCEA-BBZ-P2A-SIRPγ-28, SIRPγ-28 plus CoCl2 were experimental verification hypoxia models. The results were shown in FIG. 10 and FIG. 11. The average fluorescence intensity and positive rate of 5HCEA-BBZ-8H-8, 5HCEA-BBZ-P2A-SIRPγ-28, and SIRPγ-28 are similar, as shown in FIG. 12, 5HCEA-BBZ-P2A-SIRPγ-28 had more advantages in amplification.

Example 9 Validation of the Effectiveness of CAR-T Targeting CEA

[0156] (1) Killing Efficiency of Target Cell DLDL1-CEA

[0157] CEA-positive DLD1-CEA and DLD1-CEA(CD47−) cells were used as target cells, respectively. After hypoxia treatment of effector cells (conventional CAR-T cells and CAR-T cells with Hypoxia inducible promoter CAR expression), they were plated on target cells according to the effector-target ratio of 1:1 and the killing ability of different CAR-Ts on target cells were detected.

[0158] 24 h after adding CAR-T, the killing efficiency results of CAR-T on target cells DLDL1-CEA in each group are shown in FIG. 13 and Table 4 below. 5HCEA-BBZ-8H-8, 5HCEA-BBZ+SIRPγ-28 and 5HCEA-BBZ-P2A-SIRPγ-28 had strong killing functions, and in the SIRPγ-28 group, it also had a killing function.

TABLE-US-00004 TABLE 4 The killing efficiency of each group of CAR-T targeting CEA on target cells DLDL1-CEA Structure Specific Lysis (%) 5HCEA-BBZ+SIRPγ-28 84.4752 5HCEA-BBZ-P2A-SIRPγ-28 99.4934 5HCEA-BBZ-8H-8 99.7738 SIRPγ 45.2293 Control T 29.721 Medium 0

[0159] (2) Detection of IFN-γ, IL-2, and TNF-α Secretion

[0160] Followed by (1), the cell supernatant was collected 24 hours after killing, and the secretion capacity of IFN-γ, IL-2, and TNF-α was detected after CAR-T cells were stimulated by target cells. The collected supernatant was used to detect the secretion of IFN-γ and IL-2 by ELISA method using a kit.

[0161] The results were shown in FIG. 14, FIG. 15, and FIG. 16. When the target cell was DLD1-CEA, in 5HCEA-BBZ-8H-8 group, IFN-γ, IL-2, and TNF-α were secreted less, and in 5HCEA-BBZ-P2A-SIRPγ-28 group, IFN-γ, IL-2, and TNF-α were secreted much higher than that of 5HCEA-BBZ-8H-8 group, and when the target cells were DLD1-CEA (CD47−) cells, the secretion levels of IFN-γ. IL-2, and TNF-α were low or unable to reach the detection line. The 5HCEA-BBZ-P2A-SIRPγ-28 of the present application is more conducive to the proliferation of CAR-T and the secretion of tumor-killing-related factors, indicating that it can indeed improve the effectiveness of CAR-T.

Example 8 In Vivo Function Verification of Plasmids Comprising SIRPγ Fusion Protein

[0162] DLD1-CEA-Luc-GFP cells were selected as the tumorigenic target cells used for in vivo validation to construct a human CEA+ solid tumor-bearing model.

[0163] According to tumor volume, patients were randomly divided into Control 1 (CT) group, 5HCEA-BBZ-8H-8 group, 5HCEA-BBZ-P2A-SIRPγ-28 group, and the control group was Control T group. On the 12th day of tumor formation, the corresponding CAR-T cells 1*10{circumflex over ( )}7 Copies/mice were injected into the tail vein of mice in different groups; the Control T group was infused with the same total number of 1 lymphocytes on the 12th day. The tumor volume of each group of mice was measured every three days. The experimental results were shown in FIG. 17 and FIG. 18. It could be seen that the in vivo effectiveness on mice of group 5HCEA-BBZ-8H-8 was significantly improved compared with that of group 5HCEA-BBZ-P2A-SIRPγ-28, and in the 5HCEA-BBZ-8H-8 group, it also had a significant elimination effect on tumors.

[0164] Part III Experimental Part of the CAR Structure Targeting CD19

Example 9 Construction of Plasmid Targeting CD19

[0165] Using SIRPγ and CD47 full-length plasmids, pL-CAG-2AGFP, pL-CAG-PD1-CD28-2ACherry, pL-CAG-PD1-BB-2ACherry as templates and a CD19-targeting CAR structure to construct vectors: SIRPγ-28, 5HCD19-BBZ, and 5HCD19-BBZ-SIRPγ-28. After verification by sequencing and alignment, the structures were successfully constructed.

Example 10 Preparation of Lentivirus and Infection of T Lymphocytes

[0166] The lentivirus was packaged in the calcium phosphate method to obtain three viral particles (SIRPγ-28, 5HCD19-BBZ, 5HCD19-BBZ-P2A-SIRPγ-28) in Example 1.

[0167] The lymphocytes were separated by gradient centrifugation. After centrifugation, the second layer (the white lymphocyte layer) was taken, washed with physiological saline, and cultured by adding RPMI 1640 complete medium comprising 10% FBS to obtain human PBMC cells. After the obtained PBMC cells were activated by anti-CD3 and CD28 monoclonal antibodies for 24 hours, the activated PBMCs were infected by a certain 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 were as shown in FIG. 19 and Table 5 below.

TABLE-US-00005 TABLE 5 The positive rate of CAR-T targeting CD19 in each experimental group detected by flow cytometry Structure % of CD3+ T Cells Control T 0.26 SIRPγ-28 51.25 5HCD19-BBZ 53.15 5HCD19-BBZ-P2A-SIRPγ-28 54.89 SIRPγ-28+5HCD19-BBZ 18.04

Example 11 In Vitro Pharmacodynamic Evaluation of CAR-T Targeting CD19

[0168] Control T was used as the control group, and the experimental groups were set as SIRPγ-28 group, 5HCD19-BBZ group, and 5HCD19-BBZ-P2A-SIRPγ-28 group. Nam6-Luc-GFP (CD19 positive) and K562-Luc-GFP (CD19 negative) were used as target cells, and the effectiveness was verified with in vitro killing and in vitro factor secretion. The results were shown in FIG. 20 and Table 6 below, the in vitro killing effect of the product (5HCD19-BBZ-P2A-SIRPγ-28) obtained by transfecting immune cells with one co-expression vector and the product (SIRPγ-28+5HCD19-BBZ) obtained by co-transfecting immune cells with two separate expression vectors were significantly higher than that of the SIRPγ-28 group and the 5HCD19-BBZ group, and they did not kill the negative cells.

[0169] As shown in FIG. 21 and Table 7, the factor secretion of the product (5HCD19-BBZ-P2A-SIRPγ-28) obtained by transfecting immune cells with one co-expression vector and the product (SIRPγ-28+5HCD19-BBZ) obtained by co-transfecting immune cells with two separate expression vectors were significantly higher than that of SIRPγ-28 group and 5HCD19-BBZ group,

TABLE-US-00006 TABLE 6 Cell killing of each experimental group targeting CEA Structure Specific Lysis (%) Control T 37.2867 SIRPγ-28 28.6995 5HCD19-BBZ 84.3876 5HCD19-BBZ-P2A-SIRPγ-28 97.5187 SIRPγ-28+5HCD19-BBZ 97.4608

TABLE-US-00007 TABLE 7 The secretion of IFN-γ factor in each experimental group targeting CEA Structure IFN-γ (pg/ml) Control T 135 SIRPγ-28 453.97 5HCD19-BBZ 17296.67 5HCD19-BBZ-P2A-SIRPγ-28 21820 SIRPγ-28+5HCD19-BBZ 14363.33

[0170] Example 12 In vim pharmacodynamic evaluation when targeting CD19 NCG mice (female, 6 weeks old) were selected and injected subcutaneously (s.c.) Nalm6-Luc-GFP cells to establish an in vivo tumor-bearing model, and 8 d after tumor-bearing, the dose of 1×10.sup.7 CAR-T Cells/mouse was injected into the tail vein (i.v.) to administer CAR-T to different groups (Control 5HCD19-BBZ, 5HCD19-BBZ-P2A-SIRPγ-28). The tumor growth in vivo was observed by in vivo imaging, and the therapeutic effects of different CAR-Ts on lymphoma were evaluated in vivo. The results were shown in FIG. 22 and FIG. 23, and compared with the Control T group and the 5HCD19-BBZ group, 5HCD19-BBZ-P2A-SIRPγ-28 had significant anti-tumor effect in vivo, and could significantly eliminate the tumor.

[0171] Part IV Experimental part of the CAR structure targeting PSCA

Example 13 Construction of Plasmid Targeting PSCA and Infection of T Cells

[0172] (I) Plasmid Construction

[0173] The lentiviral expression vector and PSCA ScFv-G4H hinge region-CD28 transmembrane region-CD28-CD137-CD3P2A-SIRPγ-28 (PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28) CAR structure were cleaved by double enzyme digestion and recovered, the gene fragments were connected and transformed, and single clones were picked.

[0174] The lentivirus expression vector and PSCA ScFv-7H hinge region-CD28 transmembrane region-CD28-CD137-CD3ζ (PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28) CAR structure were cleaved by double enzyme digestion and recovered, and the gene fragments were ligated and transformed and single clones were picked.

[0175] (2) Infection of Cells

[0176] T cells were infected with the obtained plasmid to obtain CAR-T cells.

Example 14 IFN-γ Factor Secretion of that Targeting PSCA

[0177] Control T was used as the control group, and in the experimental groups, PSCA-28BBZ-G-4HH-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 set as target cells, and RT4-Luc-GFP (PSCA positive) was the target cell, the in vitro effect was verified by in vitro factor secretion. The results were shown in Tables 8 to 13 and FIGS. 24 to 29. The IFN-γ factor secretions of the products PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 or PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 obtained by transfecting immune cells with one co-expression vector and the products PSCA-28BBZ-G4H-28TM+SIRPγ-28 or PSCA-28BBZ-7H-28TM+SIRPγ-28 obtained by co-transfecting immune cells with two separate expression vectors were significantly higher than that of the control group and PSCA-28BBZ-G4H-28TM group or PSCA-28BBZ-7H-28TM group and, and they did not kill the negative cells.

TABLE-US-00008 TABLE 8 PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 secreted IFN-γ factor Structure IFN-γ (pg/ml) PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 4060.33 Control T 234.23

TABLE-US-00009 TABLE 9 PSCA-28BBZ-7H-28TM-P2A-SIRP-28 secreted IFN-γ factor Structure IFN-γ (pg/ml) PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 4024.67 Control T 234.23

TABLE-US-00010 TABLE 10 PSCA-28BBZ-G4H-28TM+SIRPγ-28 secreted IFN-γ factor Structure IFN-γ (pg/ml) PSCA-28BBZ-G4H-28TM+SIRPγ-28 6862.33 Control T 234.23

TABLE-US-00011 TABLE 11 PSCA-28BBZ-7H-28TM+SIRPγ-28 secreted IFN-γ factor Structure IFN-γ (pg/ml) PSCA-28BBZ-7H-28TM+SIRPγ-28 8550.67 Control T 234.23

TABLE-US-00012 TABLE 12 PSCA-28BBZ-G4H-28TM, PSCA-28BBZ-G4H-28TM+SIRPγ-28 secreted IFN-γ factor Structure IFN-γ (pg/ml) PSCA-28BBZ-G4H-28TM 6998.67 PSCA-28BBZ-G4H-28TM+SIRPγ-28 6862.33 Control T 234.23

TABLE-US-00013 TABLE 13 PSCA-28BBZ-7H-28TM, PSCA-28BBZ-7H-28TM+SIRPγ-28 secreted IFN-γ factor Structure IFN-γ (pg/ml) PSCA-28BBZ-7H-28TM 6660.33 PSCA-28BBZ-7H-28TM+SIRPγ-28 8550.67 Control T 234.23

Example 14 Cell Killing of that Targeting PSCA

[0178] The experimental groups were set up with PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28, PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28, PSCA-28BBZ-G-4H-28TM+SIRPγ-28, PSCA-28BBZ-7H-28TM+SIRPγ-28, PSCA-28BBZ-G4H-28TM, PSCA-28BBZ-7H-28TM, and using RT4-Luc-GFP (PSCA positive) and PC-3-Luc-GFP (PSCA negative) as target cells respectively. The results were shown in Table 14-Table 19 and FIG. 30-FIG. 35. The in vitro killing of the product PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 or PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 obtained by transfecting immune cells with one co-expression vector and the product PSCA-28BBZ-G4H-28TM+SIRPγ-28 or PSCA-28BBZ-7H-28TM-1-SIRPγ-28 obtained by co-transfecting immune cells with two separate expression vectors were significantly higher than that of the control group and PSCA-28BBZ-G4H-28TM group or PSCA-28BBZ-7H-28TM group, and they did not kill the negative cells.

TABLE-US-00014 TABLE 14 Cell killing of PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 in positive/negative cells, respectively IFN-γ (pg/ml) Structure RT4-Luc-GFP PC-3-Luc-GFP PSCA-28BBZ-G4H-28TM-P2A- 45.2911 −0.7548 SIRPγ-28 Control T 25.6632 −8.3602

TABLE-US-00015 TABLE 15 Cell killing of PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 in positive/negative cells, respectively IFN-γ (pg/ml) Structure RT4-Luc-GFP PC-3-Luc-GFP PSCA-28BBZ-7H-28TM-P2A- 46.2139 −2.4081 SIRPγ-28 Control T 25.6623 −8.3602

TABLE-US-00016 TABLE 16 Cell killing of PSCA-28BBZ-G4H-28TM+SIRPγ-28 in positive/negative cells, respectively IFN-γ (pg/ml) Structure RT4-Luc-GFP PC-3-Luc-GFP PSCA-28BBZ-G4H-28TM+SIRPγ-28 69.9491 −9.91984 Control T 25.6632 −18.3602

TABLE-US-00017 TABLE 17 Cell killing of PSCA-28BBZ-7H-28TM+SIRPγ-28 in positive/negative cells, respectively IFN-γ (pg/ml) Structure RT4-Luc-GFP PC-3-Luc-GFP PSCA-28BBZ-7H-28TM+SIRPγ-28 76.6439 −10.0736 Control T 25.6632 −18.3602

TABLE-US-00018 TABLE 18 Cell killing in PSCA-28BBZ-G4H-28TM and PSCA-28BBZ-G4H-28TM+SIRPγ-28 IFN-γ (pg/ml) Structure RT4-Luc-GFP PC-3-Luc-GFP PSCA-28BBZ-G4H-28TM 53.5258 −19.8898 PSCA-28BBZ-G4H-28TM+SIRPγ-28 69.9491 −9.91984 Control T 25.6632 −18.3602

TABLE-US-00019 TABLE 19 Cell killing in PSCA-28BBZ-7H-28TM, PSCA-28BBZ-7H-28TM+SIRPγ-28 IFN-γ (pg/ml) Structure RT4-Luc-GFP PC-3-Luc-GFP PSCA-28BBZ-7H-28TM 52.6503 −12.1743 PSCA-28BBZ-7H-28TM+SIRPγ-28 76.6439 −10.0736 Control T 25.6632 −18.3602

[0179] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the preferred examples, those of ordinary skills in the art should understand that modifications or equivalent substitutions can be made to the technical embodiments of the present application without departing from the spirit and scope of the technical embodiments of the present application and should be included in the scope of the claims of the present application.