CN116903756A

CN116903756A - Bispecific chimeric antigen receptor targeting PSMA antigen and application thereof

Info

Publication number: CN116903756A
Application number: CN202310850291.2A
Authority: CN
Inventors: 刘勋; 许恩顺; 张倩
Original assignee: Nanjing Enrui Kainuo Biotechnology Co ltd
Current assignee: Nanjing Enrui Kainuo Biotechnology Co ltd
Priority date: 2023-07-12
Filing date: 2023-07-12
Publication date: 2023-10-20

Abstract

The invention provides a bispecific chimeric antigen receptor targeting PSMA antigen and application thereof, comprising a dnTGF beta RII domain, a T2A self-cleaving peptide domain, a signal peptide domain, a bispecific PSMA antigen binding domain, a hinge region domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, a P2A self-cleaving peptide domain, and a membrane-bound IL15 domain. Chimeric Antigen Receptor (CAR) technology is a perfect fusion of genetic engineering and immunotherapy, and is one of the most promising research directions in the current tumor treatment field. The chimeric antigen receptor is constructed by utilizing two single domain antibodies combined with PSMA antigen, and the CAR natural killer (anti-PSMA CAR NK) cell targeting PSMA is prepared, so that the anti-PSMA CAR NK cell has a good tumor killing function, and can be used for treating tumors and diseases related to PSMA expression.

Description

Bispecific chimeric antigen receptor targeting PSMA antigen and application thereof

Technical Field

The invention belongs to the technical field of immune cell therapy, and relates to a bispecific chimeric antigen receptor targeting PSMA antigen and application thereof.

Background

Prostate cancer is one of the most common malignant tumors of the male reproductive system. The global new prostate cancer incidence in 2020 is estimated to be 1414259, the proportion of the new cases of the total cancers is 7.3 percent, and the number of the new cases of the cancers is ranked at the 4 th position; the number of cases of prostate cancer death was estimated to be 375304, the proportion of total cases of cancer death was 3.8%, and the number of cases of cancer death was ranked 8. The incidence of prostate cancer tends to rise year by year due to the influence of factors such as aging of the population, changes in dietary structure, and improvement in detection level.

Treatments for castration-resistant prostate cancer (CRPC) include various types of drugs and regimens, which currently improve patient survival and quality of life to some extent. However, survival rates for patients receiving standard treatment remain low, and especially prognosis for castration-resistant prostate cancer is poor. Thus, new therapeutic agents are urgently needed to improve patient survival. Chimeric Antigen Receptor (CAR) engineered immune cells are currently the most promising immunotherapeutic drugs, and have been used to treat relapsed and refractory leukemias and lymphomas, with good therapeutic efficacy. Therefore, there is a need to develop an engineered immune cell drug against prostate cancer.

Disclosure of Invention

The invention provides a bispecific chimeric antigen receptor based on single domain antibody targeting PSMA and application thereof. Chimeric antigen receptor is constructed by utilizing two single domain antibodies combined with PSMA antigen, and CAR natural killer (anti-PSMA CAR NK) cells targeting PSMA are prepared, so that the anti-PSMA CAR NK cells are verified to have good tumor killing function.

The invention adopts the following technical scheme:

a PSMA-targeting bispecific chimeric antigen receptor comprising an extracellular domain, a transmembrane domain (TM), and an intracellular signaling domain, the extracellular domain comprising a PSMA antigen-binding domain, preferably a multispecific PSMA antigen-binding domain, such as a 2-10-specific PSMA antigen-binding domain, further preferably a bispecific PSMA antigen-binding domain.

In the present invention, the bispecific PSMA antigen-binding domain comprises two single domain antibodies that bind to PSMA antigen; the two single domain antibodies that bind PSMA antigen are linked by a GS linker, preferably the two single domain antibodies that bind PSMA antigen are selected from two of P3E1, P2A6, P3E7, further preferably the two single domain antibodies that bind PSMA antigen are a P3E1 and P3E7 combination.

In the present invention, a bispecific Chimeric Antigen Receptor (CAR) targeting PSMA comprises a bispecific PSMA antigen-binding domain comprising two single domain antibodies (sdabs) linked by a Linker (Linker) fragment, wherein each single domain antibody comprises a heavy chain complementarity determining region 1 (HCDR 1), a heavy chain complementarity determining region 2 (HCDR 2), and a heavy chain complementarity determining region 3 (HCDR 3).

Preferably, in the present invention, the amino acid sequence of heavy chain complementarity determining region 1 is selected from the group consisting of SEQ ID NOs: 11. EQ ID NO:17 or SEQ ID NO:23, the amino acid sequence of heavy chain complementarity determining region 2 is selected from the group consisting of SEQ ID NOs: 13. EQ ID NO:19 or SEQ ID NO:25, the amino acid sequence of heavy chain complementarity determining region 3 is selected from the group consisting of SEQ ID NOs: 15. EQ ID NO:21 or SEQ ID NO:27. preferably, the amino acid sequence of heavy chain complementarity determining region 1 is selected from the group consisting of SEQ ID NOs: 11, the amino acid sequence of heavy chain complementarity determining region 2 is selected from the group consisting of SEQ ID NOs: 13, the amino acid sequence of heavy chain complementarity determining region 3 is selected from the group consisting of SEQ ID NOs: 15; alternatively, the amino acid sequence of heavy chain complementarity determining region 1 is selected from the group consisting of EQ ID NO:17, the amino acid sequence of heavy chain complementarity determining region 2 is selected from the group consisting of SEQ ID NOs: 19, the amino acid sequence of heavy chain complementarity determining region 3 is selected from the group consisting of SEQ ID NOs: 21, a step of; alternatively, the amino acid sequence of heavy chain complementarity determining region 1 is selected from the group consisting of SEQ ID NOs: 23, the amino acid sequence of heavy chain complementarity determining region 2 is selected from the group consisting of SEQ ID NOs: 25, the amino acid sequence of heavy chain complementarity determining region 3 is selected from the group consisting of SEQ ID NOs: 27.

preferably, the PSMA antigen-binding domain comprises the amino acid sequence shown as SEQ ID NO. 29, SEQ ID NO. 31 or SEQ ID NO. 33, or a functional variant thereof. The functional variants are selected from the group consisting of: a protein or polypeptide having one or more amino acids substituted, deleted or added in the PSMA antigen-binding domain, and a protein or polypeptide having more than 90% homology, preferably at least 90% to 99% or more sequence homology, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology, to the PSMA antigen-binding domain.

In the present invention, the extracellular domain further includes dnTGF beta RII domain, T2A self-cleaving peptide domain, and signal peptide domain (SP). Preferably, the signal peptide domain comprises a CD8a signal peptide domain.

In the present invention, a Hinge region (Hinge) domain is provided between the extracellular domains and the transmembrane domains. Preferably, the hinge region domain comprises a CD8a hinge region domain; preferably, the transmembrane domain comprises a CD8a transmembrane domain.

In the present invention, the intracellular signaling domain comprises a costimulatory domain, an intracellular signaling domain, a P2A self-cleaving peptide domain, and a membrane-bound IL15 domain. Preferably, the co-stimulatory domain comprises an OX40 co-stimulatory domain; preferably, the intracellular signaling domain comprises the DAP12 intracellular signaling domain.

Preferably:

the dnTGF beta RII domain comprises an amino acid sequence shown in SEQ ID NO. 1 or a functional variant thereof;

the T2A self-cleaving peptide domain comprises an amino acid sequence shown in SEQ ID NO. 3 or a functional variant thereof;

the CD8 alpha signal peptide domain comprises an amino acid sequence shown in SEQ ID NO. 5 or a functional variant thereof;

the CD8a hinge region domain comprises SEQ ID NO:37, an amino acid fragment shown in seq id no;

the CD8a transmembrane domain comprises SEQ ID NO:39 or a functional variant thereof, comprising a polypeptide from the following proteins: the α, β or ζ chain of T cell receptor, CD28, CD5, CD16, CD22, CD33,4-1bb, nkg2d,2b4, dnam1, igG1 and IgG4;

the OX40 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO. 41 or a functional variant thereof, said co-stimulatory domain functional variant comprising a polypeptide from the group consisting of CD28,4-1BB;

the intracellular signaling domain of DAP12 comprises the amino acid sequence shown in SEQ ID NO. 43 or a functional variant thereof, and the intracellular signaling domain functional variant comprises a polypeptide from the group consisting of DAP10, CD3 ζ;

the P2A self-cleaving peptide domain comprises an amino acid sequence shown in SEQ ID NO. 45 or a functional variant thereof;

the mbIL15 domain comprises SEQ ID NO:47 or a functional variant thereof.

Preferably, the bispecific chimeric antigen receptor of the present invention targeting PSMA based on single domain antibodies comprises dntgfβrili, T2A self-cleaving peptide, CD8a signal peptide, two single domain antibodies that bind PSMA antigen, CD8a hinge region, CD8a transmembrane domain, OX40 co-stimulatory domain, DAP12 intracellular signaling domain, P2A self-cleaving peptide domain, membrane-bound IL15 (mbIL 15) domain; further preferably, the chimeric antigen receptor of the bispecific single domain antibody of the present invention consists of dntgfβrii, T2A self-cleaving peptide, CD8a signal peptide, two single domain antibodies that bind PSMA antigen, CD8a hinge region, CD8a transmembrane domain, OX40 co-stimulatory domain, DAP12 intracellular signaling domain, P2A self-cleaving peptide domain, membrane-bound IL15 (mbIL 15) domain, in series.

The invention discloses a nucleic acid molecule comprising a bispecific chimeric antigen receptor encoding the above-described targeting PSMA, or a vector comprising the nucleic acid molecule; specific vectors and construction methods are conventional techniques, such as vectors selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors.

An immune effector cell comprising the above-described PSMA-targeting bispecific chimeric antigen receptor or a nucleic acid molecule encoding the PSMA-targeting bispecific chimeric antigen receptor or a vector comprising the nucleic acid molecule. Preferably, the immune cells are selected from the group consisting of T lymphocytes, natural Killer (NK) cells, megaphaga (M) cells.

A medicament comprising a bispecific chimeric antigen receptor or immune effector cell targeted to PSMA, and further comprising one or more pharmaceutically acceptable excipients, is conventional.

Preferably, the invention discloses a CAR NK cell, the chimeric antigen receptor of which is the bispecific chimeric antigen receptor targeting PSMA; it is an immunocyte drug, specifically chimeric antigen receptor natural killer cells (anti-PSMA CAR NK) targeting PSMA antigen, for treating castration-resistant prostate cancer.

The invention discloses an application of the PSMA-targeted bispecific chimeric antigen receptor in preparing CAR immune cells, especially CAR NK cells, or in preparing immunotherapeutic drugs; the invention discloses application of the CAR immune cells in preparation of immunotherapeutic drugs. In particular, the present invention discloses the use of the aforementioned CAR, nucleic acid molecule, vector, immune effector cell or drug for treating a disease or cancer associated with expression of PSMA. Preferably, the immunotherapeutic agent is an agent for the treatment of castration-resistant prostate cancer.

The CAR (chimeric antigen receptor) technology is perfect fusion of genetic engineering and immunotherapy, and is one of the most promising research directions in the field of tumor treatment at present. The high cost and long manufacturing cycle of CAR-T therapy remains an obstacle to its widespread use. The invention provides a bispecific chimeric antigen receptor based on single domain antibody targeting PSMA and application thereof, in particular CAR NK cells have a plurality of advantages, wherein one of the most important advantages is safety, unlike T cells, that they do not release a large amount of inflammatory proteins, leading to cytokine storms; another advantage is versatility. The CAR NK cells of the chimeric antigen receptor based on the single domain antibody disclosed by the invention have a good killing effect on prostate cancer.

Drawings

FIG. 1 is a structural (A) and functional schematic (B) diagram of a bispecific chimeric antigen receptor.

FIG. 2 is a map of the pMSCV retroviral vector (PKN 0115) for a bispecific chimeric antigen receptor.

FIG. 3 is a map of the pMSCV retroviral vector (PKN 0117) for a bispecific chimeric antigen receptor.

FIG. 4 is a map of the pMSCV retroviral vector (PKN 0119) for a bispecific chimeric antigen receptor.

FIG. 5 shows the results of the expression detection of PSMA antigen on the surface of prostate cancer cell lines and other tumor cell lines.

Figure 6 is a graph showing the results of CAR positive cell rate detection after CAR transduction into NK cells with retroviral vectors.

FIG. 7 shows the results of dnTGF beta II positive cell rate assays after CAR transduction into NK cells with retroviral vectors, UNK, PKN0115; PKN0117, PKN0119.

FIG. 8 shows the killing result of anti-PSMA CAR NK cells on PSMA target cells.

FIG. 9 shows the killing result of anti-PSMA CAR NK cells on PSMA target cells.

FIG. 10 shows the killing result of anti-PSMA CAR NK cells on PSMA target cells.

FIG. 11 shows the killing result of anti-PSMA CAR NK cells on PSMA target cells.

FIG. 12 shows the results of two single domain antibodies binding to different epitopes of PSMA protein.

FIG. 13 shows the results of two single domain antibodies binding to different epitopes of PSMA protein.

FIG. 14 shows the affinity assay results for single domain antibodies.

FIG. 15 shows the affinity detection results of single domain antibodies.

Detailed Description

The inventive subject matter of the present invention is the disclosure of novel Chimeric Antigen Receptor (CAR) structures, particularly bispecific chimeric antigen receptors targeting PSMA, particularly bispecific PSMA antigen-binding domains; the related reagent is the existing product, the specific preparation operation and performance test are conventional technologies, such as plasmid preparation, virus infection, cell experiment and other specific experimental methods, which are all conventional technologies in the field.

The structure and the function of the Chimeric Antigen Receptor (CAR) are shown as A in figure 1 and B in figure 1, and the chimeric antigen receptor of the bispecific single domain antibody is formed by sequentially connecting dnTGF beta RII, T2A self-shearing peptide, CD8 alpha signal peptide, two single domain antibodies combined with PSMA antigen, CD8 alpha hinge region, CD8 alpha transmembrane domain, OX40 co-stimulatory domain, DAP12 intracellular signaling domain, P2A self-shearing peptide domain and membrane-binding IL15 (mbiL 15) domain in series. Wherein the two single domain antibodies that bind PSMA antigen are linked by a GS linker, preferably the two single domain antibodies that bind PSMA antigen are selected from two of P3E1, P2A6, P3E7, further preferably the two single domain antibodies that bind PSMA antigen are a P3E1, P2A6 combination or a P3E1, P3E7 combination.

Wherein, the amino acid sequence of the dnTGF beta RII is SEQ ID NO:1, a step of; the amino acid sequence of the T2A self-cleaving peptide is SEQ ID NO:3, a step of; the amino acid sequence of the CD8a signal peptide is SEQ ID NO:5, a step of; the amino acid sequence of the P3E1 HCDR1 is SEQ ID NO:11 The amino acid sequence of the P3E1 HCDR2 is SEQ ID NO:13 The amino acid sequence of the P3E1 HCDR3 is SEQ ID NO:15; the amino acid sequence of P2A6 HCDR1 is SEQ ID NO:17 The amino acid sequence of P2A6 HCDR2 is SEQ ID NO:19 The amino acid sequence of P2A6 HCDR3 is SEQ ID NO:21, a step of; the amino acid sequence of the P3E7 HCDR1 is SEQ ID NO:23 The amino acid sequence of the P3E7 HCDR2 is SEQ ID NO:25 The amino acid sequence of the P3E7 HCDR3 is SEQ ID NO:27; the amino acid sequence of the P3E1 SdAb is SEQ ID NO:29; the amino acid sequence of the P2A6 SdAb is SEQ ID NO:31; the amino acid sequence of the P3E7 SdAb is SEQ ID NO:33; the amino acid sequence of the GS linker is SEQ ID NO:35; the amino acid sequence of the CD8a hinge region is SEQ ID NO:37, respectively; the amino acid sequence of the CD8a transmembrane domain is SEQ ID NO:39; the amino acid sequence of OX40 co-stimulatory domain is SEQ ID NO:41; the amino acid sequence of the intracellular signaling domain of DAP12 is SEQ ID NO: 43. The amino acid sequence of the P2A self-cleaving peptide is SEQ ID NO:45; the amino acid sequence of the mbIL15 domain is SEQ ID NO:47.

preferably, the nucleic acid molecule encoding the amino acid sequence is SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO: 36. SEQ ID NO: 38. SEQ ID NO: 40. SEQ ID NO: 42. SEQ ID NO: 44. SEQ ID NO: 46. SEQ ID NO:48. conventional enzymes of nucleic acid molecules cut into conventional plasmids to construct vectors comprising the nucleic acid molecules.

EXAMPLE bispecific chimeric antigen receptor vector construction

The method comprises the steps of artificially synthesizing a nucleotide sequence (DNA fragment) for encoding a Chimeric Antigen Receptor (CAR) by Beijing qing biological science and technology, enzyme-cutting a pMSCV retrovirus vector plasmid by NcoI and SalI, running the plasmid fragment on agarose gel, then recovering the enzyme-cut pMSCV DNA fragment, and using recombinase to carry out homologous recombination on the DNA fragment of the CAR and the pMSCV DNA fragment to construct the pMSCV retrovirus vector plasmid containing the CAR, wherein the specific operation is a conventional technology, and the technical effect of the invention is not influenced.

A pMSCV retroviral vector comprising a heavy chain (VH) comprising a single chain antibody (SEQ ID NO:8 for the nucleotide sequence, SEQ ID NO:7 for the encoded amino acid sequence) and a light chain (VL) (SEQ ID NO:10 for the nucleotide sequence, SEQ ID NO: 9) with the complementarity determining region selected from the J591 clone was designated PKN 0115. The J591 antibody sequence was derived from WO2002098897A2 and the vector map is shown in FIG. 2 for comparison. The heavy chain (VH) and the light chain (VL) of the single chain antibody are connected by a GS linker (the nucleotide sequence is SEQ ID NO: 36); further comprises dnTGF beta RII (nucleotide sequence SEQ ID NO: 2), T2A self-cleaving peptide (nucleotide sequence SEQ ID NO: 4), CD8a signal peptide (nucleotide sequence SEQ ID NO: 6), CD8a hinge region (nucleotide sequence SEQ ID NO: 38), CD8a transmembrane region (nucleotide sequence SEQ ID NO: 40), OX40 co-stimulatory factor (nucleotide sequence SEQ ID NO: 42), DAP12 intracellular signaling domain (nucleotide sequence SEQ ID NO: 44), P2A self-cleaving peptide (nucleotide sequence SEQ ID NO: 46), mbiL15 domain (nucleotide sequence SEQ ID NO: 48).

A pMSCV retroviral vector comprising a single domain antibody selected from the group consisting of P3E1 (SEQ ID NO: 30) and P2A6 (SEQ ID NO: 32) was designated PKN0117 and the vector map is shown in FIG. 3. The two single domain antibodies are linked by a GS linker (SEQ ID NO: 36); further comprises dnTGF beta RII (nucleotide sequence SEQ ID NO: 2), T2A self-cleaving peptide (nucleotide sequence SEQ ID NO: 4), CD8a signal peptide (nucleotide sequence SEQ ID NO: 6), CD8a hinge region (nucleotide sequence SEQ ID NO: 38), CD8a transmembrane region (nucleotide sequence SEQ ID NO: 40), OX40 co-stimulatory factor (nucleotide sequence SEQ ID NO: 42), DAP12 intracellular signaling domain (nucleotide sequence SEQ ID NO: 44), P2A self-cleaving peptide (nucleotide sequence SEQ ID NO: 46), mbiL15 domain (nucleotide sequence SEQ ID NO: 48).

A pMSCV retroviral vector comprising a single domain antibody selected from the group consisting of P3E1 (SEQ ID NO: 30) and P3E7 (SEQ ID NO: 34) was designated PKN0119 and the vector map is shown in FIG. 4. The two single domain antibodies are linked by a GS linker (SEQ ID NO: 36); further comprises dnTGF beta RII (nucleotide sequence SEQ ID NO: 2), T2A self-cleaving peptide (nucleotide sequence SEQ ID NO: 4), CD8a signal peptide (nucleotide sequence SEQ ID NO: 6), CD8a hinge region (nucleotide sequence SEQ ID NO: 38), CD8a transmembrane region (nucleotide sequence SEQ ID NO: 40), OX40 co-stimulatory factor (nucleotide sequence SEQ ID NO: 42), DAP12 intracellular signaling domain (nucleotide sequence SEQ ID NO: 44), P2A self-cleaving peptide (nucleotide sequence SEQ ID NO: 46), mbiL15 domain (nucleotide sequence SEQ ID NO: 48).

EXAMPLE two expression levels of PSMA on target cell surfaces

LNCAP (Punuxel, CL-0143), 22RV1 (Punuxel, CL-0004), raji-ffluc (Fu He, FH 0142), KG1a (ATCC, CCL-246.1) cell lines were purchased and the expression level of PSMA antigen on the cell surface was examined by flow cytometry (Backman, cytoflex). The experimental procedure is briefly as follows:

1) LNCAP,22RV1,KG1a and Raji cell lines were cultured separately in 1640 (Gbico, 22400105) containing 10% FBS (excelbio, FSP 50);

2) Taking 5E+05 cells into a 96-well V bottom plate respectively, and centrifuging 400g for 5 minutes;

3) The supernatant was discarded, and 200ul of 1XDPBS (Cytics, SH 30028.02) was added separately and washed by centrifugation 2 times (400 g for 5 minutes);

4) Cells were resuspended with 200ul 1X DPBS and divided into two portions, 100ul each, control and experimental groups, respectively;

5) Then, in the experimental group cells, 1:50 dilution of PE-anti-PSMA antibody (Biolegend, 342504), incubated at room temperature for 15 min in the absence of light;

6) After the incubation, 400g is centrifuged for 5 minutes, the supernatant is discarded, and 200ul of 1XDPBS is added for centrifugal cleaning for 1 time (400 g is centrifuged for 5 minutes);

7) The supernatant was discarded and 100ul of 1XDPBS was added per well to resuspend the cells;

8) PSMA expression was measured using a flow cytometer, and the PSMA positive cell ratios of the LNCAP,22RV1,KG1a and Raji cell lines were analyzed to be 99.8%,99.9%,0.37% and 0.093% with the results shown in fig. 5. LNCAP and 22RV1 cells were indicated as positive PSMA-expressing cells, KG1a and Raji cells as negative PSMA-expressing cells.

EXAMPLE three retrovirus preparation

The retroviral vectors of PKN0115, PKN0117 and PKN0119 were co-transfected with 293T cells simultaneously with the retroviral helper packaging plasmid, respectively, to prepare retroviruses. The experimental procedure was briefly described as follows:

(1) Resuscitating 293T cells, adding 5E+06 cells into T75 cell bottle, culturing in DMEM medium containing 10% FBS, and placing in CO ₂ Culturing in a cell incubator;

(2) When the cell fusion degree reaches about 80%, changing a fresh cell culture medium (DMEM medium containing 10% FBS);

(3) Taking 500ul opti-DMEM medium, adding 10ug pMSCV plasmid (PKN 0115, PKN0117 or PKN 0119), 10ug pUMVC plasmid and 5ug pRD114 plasmid and mixing well;

(4) Another 500ul of opti-DMEM medium was added with 25ug of Polyetherimide (PEI) solution and mixed well;

(5) Adding the PEI solution into the plasmid solution in the step (3), uniformly mixing, standing at room temperature for 20min, and adding into 293T cells; after incubation for 8 hours, the medium was aspirated and 15ml of fresh cell medium was added;

(6) Culture supernatant was collected 48 hours after transfection, centrifuged at 400g for 5 minutes, and the supernatant was kept in a-80℃refrigerator for NK cell transduction, which was PKN0115 retrovirus supernatant, PKN0117 retrovirus supernatant, and PKN0119 retrovirus supernatant, respectively.

EXAMPLE four retrovirus transduced NK cells to prepare Anti-PSMA CAR NK cells

NK cell sorting and activation As a conventional technique, the present invention sorts and activates NK cells from human Cord Blood Mononuclear Cells (CBMC) using methoprene NK Cell Isolation Kit (Miltenyi Biotec, 130-092-657). The experimental procedure is briefly as follows:

fresh MACS buffer (1 XDPBS solution with 2mM EDTA and 0.5% FBS) was prepared and placed in pre-chill at 4 ℃. 25M human umbilical Cord Blood Mononuclear Cells (CBMC) were thawed in a water bath at 37 ℃. 10ul of CBMC was taken in a biosafety cabinet and counted with a cytometer. CBMC was transferred to a 15ml centrifuge tube, then 6ml of pre-chilled 1X dpbs was added and 400g centrifuged for 5 minutes. After centrifugation, the supernatant was discarded and the CBMC cells (40 ul/1E+07 cells) were resuspended by addition of MACS buffer. NK cell Biotin-Antibody Cocktail (10 ul/1E+07 cells) was added, and after mixing well, incubated for 5 minutes in a 4℃refrigerator. MACS buffer (30 ul/1E+07 cells) and NK Cell Microbeads Cocktail (20 ul/1E+07 cells) were added, mixed well and incubated in a refrigerator at 4℃for 10 minutes. LS columns were rinsed with 3ml MACS buffer (Miltenyi Biotec, 130-042-401), then the cell suspension was added to the LS columns, immediately followed by 3ml MACS buffer, after the cell suspension had flowed completely into a 15ml centrifuge tube, 3ml MACS buffer was added, and all cells were collected into a 15ml centrifuge tube. 400g of the cell suspension was centrifuged for 10 minutes. After resuspension of cells with the eketaxel NK medium containing 10% FBS, 10ul cell counts were taken and K562 feeder cells were added to NK cells at a ratio of NK: K562 feeder=1:1 for activation culture.

On day 6 of NK cell activation culture, PKN0117 or PKN0119 retrovirus was added to prepare and obtain PKN0117, PKN0117 and PKN0119 anti-PSMA CAR NK cells, respectively. The experimental procedure is briefly as follows:

(1) Taking 5E+05 activated NK cells into a 12-well cell culture plate, adding PKN0115 retrovirus supernatant with a virus infection complex number MOI (Multiplicity Of Infection) =1, then adding a vector fusion-1 (Miltenyi Biotec, 130-111-163) transduction-aiding reagent with a concentration of 10 [ mu ] g/mL, wherein the cell culture medium is Ikesaine NK cell culture medium (Ikesaine, NE 000-N012) containing 5% FBS and 200IU IL2, and the cell density is 5E+05 cells/mL;

(2) 1000g of cells were centrifuged for 30min and then placed in CO ₂ Culturing in a cell incubator, and preparing and obtaining PKN0115 anti-PSMA CAR NK cells (PKN 0115 CAR NK).

The same method is used for preparing and obtaining PKN0117 anti-PSMA CAR NK cells (PKN 0117 CAR NK) or PKN0119 anti-PSMA CAR NK cells (PKN 0119 CAR NK) by replacing PKN0115 retrovirus supernatant obtained in the step (1) with PKN0117 retrovirus supernatant or PKN0119 retrovirus supernatant.

Example five Anti-PSMA CAR NK cell CAR Positive Rate

Anti-PSMA CAR NK cells were cultured in the Ikesai NK cell medium containing 5% FBS and 200IU IL2, and the positive rate of CAR was detected at 10, 13 and 18 days of culture, and the virus transduction efficiency was verified. The experimental procedure is briefly as follows:

(1) Non-transduced virus NK cells (UNK), transduced virus CAR NK cells (KN 0115, PKN0117 or PKN 0119), 5e+05 cells each were taken into 96-well V-bottom plates, and centrifuged at 400g for 5 min;

(2) The supernatant was discarded, and 200ul of 1XDPBS was added to each for 2 times of centrifugation (400 g for 5 minutes); then, FITC-PSMA protein (ACROBiosystems, PSA-HF 244) was added at a 1:50 dilution, 100 ul/well. Incubating for 15 minutes at normal temperature in a dark place;

(3) After the incubation, 400g was centrifuged for 5 minutes. The supernatant was discarded and 200ul of 1XDPBS was added for 1 centrifugation (400 g for 5 minutes); the supernatant was discarded and 100ul of 1XDPBS was added to resuspend cells per well. Analysis of UNK, PKN0117 and PKN0119 cell CAR positive cell results using flow cytometry detection are shown in table 1 and fig. 6, which can demonstrate that the CAR expressed by the engineered NK cells is the CAR designed as described above.

EXAMPLE six Anti-PSMA CAR NK cell Dn TGF beta RII expression levels

On day 10 of Anti-PSMA CAR NK cell culture, the expression level of dnTGF-. Beta.RII was examined. The experimental procedure is briefly as follows:

(1) Non-transduced virus NK cells (UNK), transduced virus CAR NK cells (PKN 0115, PKN0117 or PKN 0119) were each taken 5e+05 cells into 96 well V-bottom plates, and centrifuged at 400g for 5 min. The supernatant was discarded, and 200ul of 1XDPBS was added to each for 2 times of centrifugation (400 g for 5 minutes);

(2) Then, 100 ul/well of PE-anti-TGF-beta RII antibody (Biolegend, 399704) diluted 1:50 was added to each. Incubate at room temperature for 15 minutes in the dark. After the incubation, 400g was centrifuged for 5 minutes. The supernatant was discarded and 200ul of 1XDPBS was added for 1 centrifugation (400 g for 5 minutes);

(3) The supernatant was discarded and 100ul of 1XDPBS was added to resuspend cells per well. The expression of dnTGF-beta RII was examined using a flow cytometer, and the results showed that the proportion of dnTGF-beta RII positive cells in UNK, PKN0115, PKN0117, and PKN0119 cells was 0.19%, 98.1%, 97.8%, and 98.8%, respectively, as shown in FIG. 7.

Example seven in vitro killing function of Anti-PSMA CAR NK cells

LNCAP,22RV1 and KG1a cells were transduced by lentivirus to obtain target cells (ffluc, firefly luciferase) stably expressing firefly luciferase. Anti-PSMA CAR NK cells were incubated with target cells (LNCAP-ffluc, 22RV1-ffluc, raji-ffluc, and KG1 a-ffluc) and the tumor killing function of Anti-PSMA CAR NK cells was examined. The experimental procedure is briefly as follows:

(1) PSMA positive target cells (LNCAP-ffluc, 22RV 1-ffluc) and PSMA negative target cells (Raji-ffluc, KG1 a-ffluc) were counted and 20000 cells/well were added to 96 Kong Baiban in a volume of 100ul. The culture medium is 1640 containing 10% FBS;

(2) Counting UNK, PKN0115 CAR NK, PKN0117 CAR NK and PKN0119 CAR NK cells according to the CAR positive rate and target ratio (E: T ratio) of 6: 1. 2: 1. 0.67:1 cells were added in medium 1640 with 10% FBS, 100 ul/well;

(3) Put into CO ₂ The cell culture incubator was incubated for 4 hours. After the incubation, 50ul of firefly luciferase substrate ONE-Glo (Promega, E6110) reagent was added to each well, mixed by shaking, and after 5 minutes of standing, the fluorescence value was detected by an enzyme-labeled instrument (TECAN, SAPRK).

The test results show that compared with UNK cells, the Anti-PSMA CAR NK cells of PKN0115, PKN0117 and PKN0117 can effectively kill PSMA expression positive target cells (LNCAP-ffluc and 22RV 1-ffluc) when the effective target ratio is 6:1, 2:1 and 0.67:1, and the killing effect of PKN0117 is more obvious than that of PKN0115 and PKN0117 (figures 8 and 9), and the statistical difference is obvious. PKN0115, PKN0117 and PKN0119 did not significantly kill PSMA-expressing negative target cells (Raji-ffluc and KG1 a-ffluc) (FIGS. 10 and 11).

Example eight Single Domain antibody epitope Competition detection

In 293 cells, the human IgG-tagged P3E1, P3E7 and P2A6 antibodies were expressed, antibody binding epitopes were detected using a BLI (Gator) instrument, his-targeting probes were added on the instrument, PSMA protein was then added, P3E1 antibody was added after washing, washing after 30min of binding, and P3E7 and P2A6 antibodies were then added, respectively, with channels CH3 and CH2.CH4 and CH5 are experimental control groups. The experimental procedure is briefly as follows:

(1) Depending on the molecular weight of the P3E1, P3E7 and P2A6 antibodies, the antibodies were diluted to 50nM with 0.05% PBST, a final volume of 2ml;

(2) The antigen was diluted to 500nM with 0.05% PBST based on the molecular weight of the human PSMA antigen, and then diluted in a double-fold ratio gradient with 500nM as the starting concentration. Adding the diluted antigen antibody into a 96-well sample plate;

(3) 0.05% PBST,250 ul/well, for a total of 8 wells were added to the MAX plate, and then the corresponding biosensor was placed into the MAX plate with 0.05% PBST;

(4) Placing the sample plate which is already loaded and the MAX plate which is already placed in the biosensor into a sample bin of a biological film layer interference technology analyzer (Gator);

(5) And starting to run the instrument program, and after the program is run, deriving a combination curve chart.

Analysis of the results by instrument data showed that P3E1 and P2A6 bound to the same epitope of PSMA protein and P3E1 and P3E7 bound to different epitopes of PSMA protein, as shown in FIGS. 12 and 13.

Example nine Single Domain antibody affinity detection

P3E1 and P3E7 antibodies were expressed artificially at the baiying biotechnology company, and then the affinity of the antibodies was analyzed by a Biacore instrument. The experimental procedure is briefly as follows:

(1) P3E1 and P3E7 antibodies were diluted to 0.5. Mu.g/mL and 10. Mu.g/mL, respectively, with 1 XHBS-EP running buffer (0.1M HEPES:1.5M NaCl,30 mM EDTA,0.05% Tween-20, pH 7.4);

(2) Capture about 120/2400 RU, 10. Mu.L/min on a Biacore 8K instrument;

(3) The human PSMA antigen was diluted in double ratio with running buffer. The diluted PSMA antigen was sequentially injected into the experimental channel and the reference channel at a flow rate of 30. Mu.L/min, and was combined and dissociated for the corresponding time. The binding dissociation steps were all performed in running buffer. The specific parameters are shown in table 2;

(4) Affinity values of ka, KD, etc. for the P3E1 and P3E7 antibodies were calculated using Biacore 8K analysis software Biacore Insight Evaluation Software, as shown in table 3 and fig. 14 and 15.

The nucleotide sequence and the coded amino acid sequence are shown as follows.

dnTGF beta RII amino acid sequence (SEQ ID NO: 1):

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQ

dnTGF beta RII nucleotide sequence (SEQ ID NO: 2):

ATGGGAAGAGGTTTACTGAGAGGACTGTGGCCTTTACACATCGTGCTGTGGACTCGTATCGCCAGCACCATCCCCCCCCATGTCCAAAAGAGCGTGAACAACGACATGATCGTGACCGACAACAATGGCGCCGTGAAGTTCCCCCAGCTGTGCAAGTTCTGCGACGTGAGGTTCAGCACTTGTGACAACCAGAAGAGCTGCATGAGCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGAGGAAGAACGACGAGAACATCACTTTAGAGACAGTGTGCCACGACCCCAAGCTGCCCTACCACGACTTCATTTTAGAAGATGCCGCCAGCCCCAAGTGCATCATGAAGGAGAAGAAGAAGCCCGGCGAGACCTTCTTCATGTGTTCTTGTTCGTCTGATGAGTGCAACGATAACATCATCTTCAGCGAGGAGTACAACACCAGCAACCCCGATTTACTGCTGGTGATCTTCCAAGTTACCGGCATTTCTTTACTGCCTCCGTTGGGCGTGGCTATCAGCGTGATCATCATCTTCTACTGCTATCGTGTTAATCGTCAA

T2A self-cleaving peptide amino acid sequence (SEQ ID NO: 3):

GSGEGRGSLLTCGDVEENPGP

T2A self-cleaving peptide nucleotide sequence (SEQ ID NO: 4):

GGATCCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

CD8 alpha Signal peptide amino acid sequence (SEQ ID NO: 5):

ALPVTALLLPLALLLHAARP

CD8 alpha Signal peptide nucleotide sequence (SEQ ID NO: 6):

GCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

j591 VH amino acid sequence (SEQ ID NO: 7):

EVQLVQSGAEVKKPGASVKISCKTSGYTFTEYTIHWVKQASGKGLEWIGNINPNNGGTTYNQKFEDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSS

j591 VH nucleotide sequence (SEQ ID NO: 8):

GAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGACCTCCGGCTACACCTTCACCGAGTACACCATCCACTGGGTGAAACAGGCCTCCGGCAAGGGCCTGGAATGGATCGGCAACATCAACCCTAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACCGGGCCACCCTGACCGTGGACAAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGCGGTCTGAGGACACCGCCGTGTACTACTGCGCCGCTGGCTGGAACTTCGACTACTGGGGCCAGGGCACCACAGTGACAGTCTCGAGC

j591 VL amino acid sequence (SEQ ID NO: 9):

DIVMTQSPSSLSASVGDRVTITCKASQDCGTAVDWYQQKPGKAPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSLQPEDFADYFCQQYNSYPLTFGGGTKLEIK

j591 VL nucleotide sequence (SEQ ID NO: 10):

GACATCGTGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCAAGATTGTGGCACCGCCGTGGACTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGGGCCTCCACCAGACACACCGGCGTGCCTGACAGATTCACCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCGACTACTTCTGCCAGCAGTACAACTCCTACCCTCTGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAA

P3E1 HCDR1 amino acid sequence (SEQ ID NO: 11):

GFTLDYYA

P3E1 HCDR1 nucleotide sequence (SEQ ID NO: 12):

GGCTTCACACTGGACTATTACGCT

P3E1 HCDR2 amino acid sequence (SEQ ID NO: 13):

ISSSGGTT

P3E1 HCDR2 nucleotide sequence (SEQ ID NO: 14):

ATCAGCAGCAGCGGCGGGACAACC

P3E1 HCDR3 amino acid sequence (SEQ ID NO: 15):

AAVYRHYYSDLGGPPRLEYEYDY

P3E1 HCDR3 nucleotide sequence (SEQ ID NO: 16):

GCCGCTGTGTACAGACACTACTACAGCGACCTGGGCGGCCCCCCTAGACTGGAGTACGAATATGACTAT

P2A6 HCDR1 amino acid sequence (SEQ ID NO: 17):

GFALDDYA

P2A6 HCDR1 nucleotide sequence (SEQ ID NO: 18):

GGCTTCGCTCTGGACGACTACGCC

P2A6 HCDR2 amino acid sequence (SEQ ID NO: 19):

ISSSGSST

P2A6 HCDR2 nucleotide sequence (SEQ ID NO: 20):

ATCAGCAGCAGCGGCAGCAGCACC

P2A6 HCDR3 amino acid sequence (SEQ ID NO: 21):

AARYSAGWGSHMTFLKRYEYDY

P2A6 HCDR3 nucleotide sequence (SEQ ID NO: 22):

GCTGCTCGGTACAGCGCCGGCTGGGGCAGCCACATGACCTTCCTGAAGAGATACGAATACGATTAC

P3E7 HCDR1 amino acid sequence (SEQ ID NO: 23):

GFTGDYYF

P3E7 HCDR1 nucleotide sequence (SEQ ID NO: 24):

GGCTTCACCGGCGACTACTACTTC

P3E7 HCDR2 amino acid sequence (SEQ ID NO: 25):

INYSGSST

P3E7 HCDR2 nucleotide sequence (SEQ ID NO: 26):

ATCAACTACAGCGGCAGCAGCACC

P3E7 HCDR3 amino acid sequence (SEQ ID NO: 27):

AASNLIATMTSYECSESSDS

P3E7 HCDR3 nucleotide sequence (SEQ ID NO: 28):

GCTGCTAGCAACCTGATCGCCACCATGACAAGCTACGAGTGCAGCGAGAGCAGCGACAGC

P3E1 SdAb amino acid sequence (SEQ ID NO: 29):

QLQLVETGGGLVQAGGSLRLSCAASGFTLDYYAIGWFRQALGKEREGVSSISSSGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVYRHYYSDLGGPPRLEYEYDYWGQGTQVTVSS

P3E1 SdAb nucleotide sequence (SEQ ID NO: 30):

CAACTGCAACTCGTCGAAACCGGCGGGGGCCTGGTGCAAGCCGGGGGCAGCCTGAGACTGAGCTGCGCCGCTTCCGGCTTCACACTGGACTATTACGCTATCGGCTGGTTTAGACAAGCCCTGGGGAAGGAAAGAGAGGGCGTGAGCTCCATCAGCAGCAGCGGCGGGACAACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATTAGCCGGGACAATGCCAAGAACACCGTCTACCTGCAAATGAATAGCCTGAAGCCTGAGGACACCGCTGTGTATTACTGCGCCGCTGTGTACAGACACTACTACAGCGACCTGGGCGGCCCCCCTAGACTGGAGTACGAATATGACTATTGGGGGCAAGGGACCCAAGTGACCGTCAGCTCC

P2A6 SdAb amino acid sequence (SEQ ID NO: 31):

QVQLVESGGGLVQPGGSLRLSCAASGFALDDYAIGWFRQAPGKEREGVSCISSSGSSTNYTDSVKGRFTISRDNAKNTVYLQVNSLKPEDTAVYYCAARYSAGWGSHMTFLKRYEYDYWGQGTQVTVSS

P2A6 SdAb nucleotide sequence (SEQ ID NO: 32):

CAAGTGCAACTCGTGGAATCCGGGGGGGGGCTCGTGCAGCCTGGCGGCTCCCTGAGACTGTCCTGTGCCGCCTCCGGCTTCGCTCTGGACGACTACGCCATCGGCTGGTTCAGACAAGCCCCCGGCAAAGAGAGAGAAGGCGTGAGCTGCATCAGCAGCAGCGGCAGCAGCACCAACTACACAGACAGCGTCAAGGGCCGGTTCACCATCAGCCGGGACAACGCTAAGAACACAGTCTATCTGCAAGTGAACAGCCTGAAGCCCGAGGATACCGCCGTGTACTACTGCGCTGCTCGGTACAGCGCCGGCTGGGGCAGCCACATGACCTTCCTGAAGAGATACGAATACGATTACTGGGGCCAAGGCACCCAAGTGACAGTCAGCTCC

P3E7 SdAb amino acid sequence (SEQ ID NO: 33):

QVQLVESGGGLVQPGGSLRLSCAASGFTGDYYFIGWFRQAPGKEREGVSCINYSGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASNLIATMTSYECSESSDSWGQGTQVTVSS

P3E7 SdAb nucleotide sequence (SEQ ID NO: 34):

CAAGTGCAGCTGGTCGAGTCCGGGGGGGGCCTGGTGCAACCCGGCGGCTCCCTGCGGCTGAGCTGCGCCGCTTCCGGCTTCACCGGCGACTACTACTTCATCGGCTGGTTCAGACAAGCCCCCGGGAAGGAGCGGGAGGGCGTGAGCTGCATCAACTACAGCGGCAGCAGCACCTATTATGCCGATAGCGTGAAAGGCAGATTCACCATCAGCAGAGATAACGCCAAGAATACAGTGTACCTGCAGATGAATTCCCTGAAACCCGAGGACACCGCCGTGTACTACTGCGCTGCTAGCAACCTGATCGCCACCATGACAAGCTACGAGTGCAGCGAGAGCAGCGACAGCTGGGGGCAAGGCACCCAAGTGACAGTCAGCAGC

GS linker amino acid sequence (SEQ ID NO: 35):

GGGGSGGGGSGGGGS

GS linker nucleotide sequence (SEQ ID NO: 36):

GGAGGAGGCGGCAGTGGCGGCGGCGGGTCCGGCGGGGGCGGCAGC

CD8 alpha hinge region amino acid sequence (SEQ ID NO: 37):

AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 alpha hinge region nucleotide sequence (SEQ ID NO: 38):

GCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

CD8 alpha transmembrane domain amino acid sequence (SEQ ID NO: 39):

IYIWAPLAGTCGVLLLSLVITLYC

CD8 alpha transmembrane domain nucleotide sequence (SEQ ID NO: 40):

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

OX40 co-stimulatory domain amino acid sequence (SEQ ID NO: 41):

ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

OX40 co-stimulatory domain nucleotide sequence (SEQ ID NO: 42):

GCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATC

DAP12 intracellular signaling domain amino acid sequence (SEQ ID NO: 43):

YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

DAP12 intracellular signaling domain nucleotide sequence (SEQ ID NO: 44):

TACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAA

P2A self-cleaving peptide amino acid sequence (SEQ ID NO: 45):

ATNFSLLKQAGDVEENPGP

P2A self-cleaving peptide nucleotide sequence (SEQ ID NO: 46):

GCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT

mbIL15 domain amino acid sequence (SEQ ID NO: 47):

MYRMQLLSCIALSLALVTNSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

mbIL15 domain nucleotide sequence (SEQ ID NO: 48):

ATGTACCGGATGCAGCTGCTGTCTTGCATTGCTCTGAGCCTTGCCCTGGTGACAAACTCTAATTGGGTGAACGTGATCTCCGACCTGAAGAAGATCGAAGATCTGATCCAGTCAATGCACATCGACGCCACCCTGTATACCGAGAGCGACGTGCACCCATCTTGCAAAGTGACCGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTGATCAGCCTCGAGTCTGGCGACGCCAGCATCCATGACACCGTGGAGAACCTGATCATCCTGGCAAATAACTCCCTGTCTTCCAACGGCAATGTGACGGAATCCGGCTGTAAGGAATGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTTGTGCACATCGTGCAGATGTTTATTAATACCTCCGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC。

Claims

1. a PSMA-targeting bispecific chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a PSMA antigen-binding domain, wherein the PSMA antigen-binding domain is a multispecific PSMA antigen-binding domain.

2. The PSMA-targeting bispecific chimeric antigen receptor according to claim 1, wherein the PSMA antigen-binding domain comprises two single domain antibodies linked by a linker; each of the single domain antibodies comprises a heavy chain complementarity determining region 1, a heavy chain complementarity determining region 2, and a heavy chain complementarity determining region 3.

3. The PSMA-targeted bispecific chimeric antigen receptor according to claim 2, wherein the amino acid sequence of heavy chain complementarity determining region 1 is selected from the group consisting of SEQ ID NO: 11. SEQ ID NO:17 or SEQ ID NO:23, the amino acid sequence of heavy chain complementarity determining region 2 is selected from the group consisting of SEQ ID NOs: 13. SEQ ID NO:19 or SEQ ID NO:25, the amino acid sequence of heavy chain complementarity determining region 3 is selected from the group consisting of SEQ ID NOs: 15. SEQ ID NO:21 or SEQ ID NO:27.

4. the bispecific chimeric antigen receptor targeting PSMA according to claim 3, wherein the PSMA antigen-binding domain comprises the amino acid sequence shown in SEQ ID No. 29, SEQ ID No. 31 or SEQ ID No. 33, or a functional variant thereof.

5. The PSMA-targeted bispecific chimeric antigen receptor of claim 1, wherein the extracellular domain further comprises a dntgfβrii domain, a T2A self-cleaving peptide domain, a signal peptide domain; a hinge region domain is arranged between the extracellular domain and the transmembrane domain; the intracellular signaling domain includes a costimulatory domain, an intracellular signaling domain, a P2A self-cleaving peptide domain, and a membrane-bound IL15 domain.

6. The PSMA-targeting bispecific chimeric antigen receptor according to claim 5, wherein the dntgfβrii domain comprises the amino acid sequence shown in SEQ ID No. 1 or a functional variant thereof; the T2A self-cleaving peptide domain comprises an amino acid sequence shown in SEQ ID NO. 3 or a functional variant thereof; the signal peptide domain comprises SEQ ID NO:5 or a functional variant thereof; the hinge region domain comprises SEQ ID NO:37, an amino acid fragment shown in seq id no; the transmembrane domain comprises SEQ ID NO:39 or a functional variant thereof; the co-stimulatory domain comprises SEQ ID NO:41 or a functional variant thereof; the intracellular signaling domain comprises SEQ ID NO:43 or a functional variant thereof; the P2A self-cleaving peptide domain comprises SEQ ID NO:45 or a functional variant thereof; the membrane-bound IL15 domain comprises SEQ ID NO:47 or a functional variant thereof.

7. A nucleic acid molecule encoding the PSMA-targeted bispecific chimeric antigen receptor of claim 1, or a vector comprising the nucleic acid molecule.

8. An immune effector cell comprising the PSMA-targeting bispecific chimeric antigen receptor of claim 1 or the nucleic acid molecule encoding the PSMA-targeting bispecific chimeric antigen receptor of claim 1 of claim 7 or a vector comprising the nucleic acid molecule.

9. A medicament comprising the PSMA-targeted bispecific chimeric antigen receptor of claim 1 or the immune effector cell of claim 8.

10. The use of a bispecific chimeric antigen receptor targeted to PSMA of claim 1 for the preparation of immune effector cells, or for the preparation of immunotherapeutic drugs; use of an immune effector cell according to claim 8 or a medicament according to claim 9 for the preparation of an immunotherapeutic medicament.