Anti-PSMA single chain antibodies, related chimeric antigen receptors and their uses

Abstract

Anti-PSMA single-chain antibodies, chimeric antigen receptors related thereto, and uses thereof are provided. The amino acid sequence of the heavy chain of the anti-PSMA single-chain antibody comprises the sequence shown in SEQ ID NO: 1, and the amino acid sequence of the light chain of the anti-PSMA single-chain antibody comprises the sequence shown in SEQ ID NO: 2. The anti-PSMA single-chain antibody is a humanized scFv antibody of PSMA, which is more functional in the human body, has better compatibility, and is less likely to be rejected by the immune system. The chimeric antigen receptor has a better response effect after specific binding to PSMA, allowing CAR-T cells to generate a stronger immune response to tumors, and the chimeric antigen receptor also has better long-term efficacy than other PSMA chimeric antigen receptors.

Description

[Technical Field]

[0001] The present application is in the field of tumor immunotherapy and relates to anti-prostate specific membrane antigen (PSMA) single chain antibodies, related chimeric antigen receptors and their uses. [Background technology]

[0002] With the advancement of tumor immunology theory and clinical technology, chimeric antigen receptor T cell (CAR-T) immunotherapy has become one of the most promising tumor immunotherapies. A chimeric antigen receptor (CAR) typically consists of a tumor-associated antigen (TAA)-binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain. CARs typically contain a single-chain variable fragment (scFv) region of an antibody or binding domain specific for a tumor-associated antigen (TAA), which is linked to the cytoplasmic domain of a T cell signaling molecule via the hinge and transmembrane domain. The most common lymphocyte activation moiety contains a T cell costimulatory domain and a moiety that triggers T cell effector function (e.g., CD3ζ) in tandem.

[0003] CAR-mediated adoptive immunotherapy allows CAR-implanted T cells to directly recognize TAAs on target tumor cells in a non-human leukocyte antigen (HLA)-restricted manner. Currently, CAR-T treatment has achieved consistent efficacy in hematologic tumors, and second-generation CD19 CAR-T cells have demonstrated antitumor efficacy in acute B lymphocytic leukemia, chronic lymphocytic leukemia, and lymphoma with response rates ranging from 50% to 90%, depending on the tumor.

[0004] Prostate-specific membrane antigen (PSMA) is widely expressed in tumors such as prostate cancer, kidney cancer, bladder cancer, glioblastoma, brain tumor, multiple myeloma and B-cell lymphoma, and in the microvessels in tumor microenvironment, but is expressed at low and limited amounts in normal tissues, making it an ideal tumor-associated antigen for immunotherapy.Currently, this cancer antigen is mainly used in the treatment of prostate cancer, which is an epithelial malignant tumor occurring in the prostate gland and is the most common malignant tumor in the male genitourinary system.However, in addition to prostate tumors, PSMA is also highly expressed in the microvessels in the interstitial tissue of most tumors.

[0005] Currently, antibody therapy against PSMA has been carefully developed in immunotherapy for glioblastoma and brain tumors and has achieved preliminary success in clinical trials. Antibodies are present in peripheral blood after administration, rarely accurately penetrate tumor tissue or sites where a small amount of tumor remains, and cannot survive in vivo for long periods. Therefore, chimeric antigen receptor PSMA CAR-T cells have been developed. In addition to possessing the advantages of antibody therapy, due to the properties of T cells themselves, PSMA CAR-T cells can accurately penetrate tumor tissue and exist in vivo with long-term memory, providing a more effective treatment option for patients with recurrent and refractory cancers. Recent clinical reports on the treatment of glioblastoma using PSMA CAR-T cells have preliminary demonstrated the efficacy of CAR-T cells, but long-term observation data are still lacking.

[0006] Currently, CAR-T technology is not very effective in treating solid tumors. The scFv region of various CAR-T cells is derived from mouse-derived antibodies, and such mouse-derived scFvs are easily rejected by the human immune system, which prevents CAR-T cells from persisting in vivo for a long period of time, thereby limiting their therapeutic efficacy. This is one of the reasons why many patients with acute lymphoblastic leukemia relapse after achieving complete remission with CD19 CAR-T cells, making retreatment difficult. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, how to provide humanized PSMA CAR-T cells with good therapeutic efficacy and long duration has become one of the urgent issues in the field of tumor immunotherapy.

[0008] (Abstract) To achieve the above goals, the present application provides an anti-PSMA single-chain antibody (anti-PSMA scFv), a chimeric antigen receptor associated therewith, and uses thereof. The anti-PSMA single-chain antibody has high specificity and affinity and can efficiently bind to PSMA. Through the PSMA chimeric antigen receptor of the anti-PSMA single-chain antibody and the corresponding CAR-T cells, PSMA-positive solid tumors can be eliminated, and the tumor microenvironment can be effectively targeted to eliminate tiny residual tumors without serious adverse effects. Therefore, the safety, efficacy, memory, and long-term maintenance of CAR-T cells can be improved.

[0009] To achieve the above goal, the present application adopts the following solution. [Means for solving the problem]

[0010] In a first aspect of the present application, the anti-PSMA single-chain antibody is a humanized scFv antibody of PSMA. The scFv antibody is specifically engineered against the tumor surface antigen PSMA using humanized codons and a humanized antibody framework, making the engineered scFv antibody more functional in the human body, more compatible with the human body, and less likely to be rejected by the immune system.

[0011] In the present application, the amino acid sequence of the heavy chain (VH) of the anti-PSMA single chain antibody comprises a sequence having 80% or more identity to SEQ ID NO:1, and the amino acid sequence of the light chain (VL) of the anti-PSMA single chain antibody comprises a sequence having 80% or more identity to SEQ ID NO:2.

[0012] SEQ ID NO:1:

[0013] [ka]

[0014] SEQ ID NO:2:

[0015] [ka]

[0016] The anti-PSMA scFv VH and anti-PSMA scFv VL can be connected via a linker, for example, GSTSGSGKPGSSEGSTKG (SEQ ID NO: 12).

[0017] In one particular embodiment, the sequence of the anti-PSMA scFv is SEQ ID NO: 9:

[0018] [ka] is.

[0019] In a second aspect, the present application provides a nucleic acid molecule comprising a nucleic acid sequence encoding the anti-PSMA single chain antibody described in the first aspect.

[0020] Preferably, the nucleotide sequence of the nucleic acid molecule has 80% or greater identity to SEQ ID NO:3.

[0021] SEQ ID NO:3:

[0022] [ka]

[0023] In a third aspect, the present application provides a PSMA chimeric antigen receptor, which comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3ζ signaling domain. The antigen-binding domain comprises the anti-PSMA single-chain antibody described in the first aspect.

[0024] The PSMA chimeric antigen receptor of the present application is obtained by carrying out specific genetic modifications in the costimulatory signaling domain of a humanized chimeric antigen receptor that targets the tumor surface antigen PSMA, and the structure of the PSMA chimeric antigen receptor is shown in Figure 1. The modified chimeric antigen receptor has a better response effect after specifically binding to PSMA, which allows CAR-T cells to generate a stronger immune response to tumors, and the modified chimeric antigen receptor also has better long-term efficacy than other PSMA chimeric antigen receptors.

[0025] Preferably, the transmembrane domain comprises a CD28 transmembrane domain and / or a CD8α transmembrane domain.

[0026] Preferably, the costimulatory signaling domain comprises a CD28 signaling domain and a CD27 signaling domain, or comprises a CD28 signaling domain and an IL-15Ra signaling domain.

[0027] In one specific embodiment, the PSMA CAR comprises a CD28 transmembrane domain, a CD28 signaling domain, and a CD27 signaling domain. In this embodiment, the PSMA CAR preferably comprises an amino acid sequence having 90% or greater identity to SEQ ID NO:4.

[0028] In one specific embodiment, the PSMA CAR comprises a CD28 transmembrane domain, a CD28 signaling domain, and an IL-15Ra signaling domain. In this embodiment, the PSMA CAR preferably comprises an amino acid sequence having 90% or greater identity to SEQ ID NO:5.

[0029] Preferably, the PSMA chimeric antigen receptor further comprises a suicide-inducing fusion domain.

[0030] Preferably, the suicide-inducing fusion domain comprises a caspase 9 domain fused to FK506 binding protein (FKBP) (such a fused caspase 9 is abbreviated as FKBP.Casp9).

[0031] Preferably, the amino acid sequence of the FKBP.Casp9 domain has 90% or greater identity to SEQ ID NO:6.

[0032] Preferably, the PSMA chimeric antigen receptor further comprises a signal peptide and / or a 2A sequence.

[0033] Preferably, the signal peptide comprises a secretory signal peptide.

[0034] Preferably, the secretory signal peptide is the signal peptide of the CD8α gene, and the amino acid sequence of the secretory signal peptide is: MALPVTALLLPLALLLHAARP (SEQ ID NO: 10). Alternatively, the secretory signal peptide may be the signal peptide of the GM-CSFR gene, and the amino acid sequence of the secretory signal peptide is: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 11). Those skilled in the art can select a secretory signal peptide according to the actual situation, and the secretory signal peptide is not specifically limited herein. The presence of a secretory signal peptide does not affect the performance of the chimeric antigen receptor of the present application.

[0035] SEQ ID NO:4:

[0036] [ka] (CD28 hinge domain + CD28 transmembrane domain + CD28 signaling domain + linker + CD27 signaling domain).

[0037] SEQ ID NO:5:

[0038] [ka] (CD28 hinge domain + CD28 transmembrane domain + CD28 signaling domain + linker + IL-15Ra signaling domain).

[0039] SEQ ID NO:6:

[0040] [ka] (FKBP+linker+Casp9).

[0041] According to the present application, the signal peptide in the PSMA chimeric antigen receptor is a signal peptide that can direct transmembrane translocation of the chimeric antigen receptor, and those skilled in the art can select a signal peptide from conventional secretory protein genes as needed.

[0042] In this application, the suicide-inducing fusion domain is connected in tandem to the CD3ζ signaling domain via the 2A sequence. The 2A sequence can cleave the protein expressed by the suicide-inducing fusion domain and the protein of the PSMA chimeric antigen receptor, allowing the chimeric antigen receptor to function. When an activator is injected, the suicide-inducing fusion domain is activated, causing the chimeric antigen receptor to be inactivated.

[0043] In the present application, the chimeric antigen receptor further comprises a linker, and the amino acid sequence of the linker is GSTSGSGKPGSSEGSTKG (SEQ ID NO: 12) or a repeating combination of multiple GGGGS (SEQ ID NO: 13), for example, GGGGSGGGGS (SEQ ID NO: 14) or GGGGSGGGGGSGGGGS (SEQ ID NO: 15). Those skilled in the art can select a linker according to the actual situation, and the linker is not specifically limited herein. The presence of the linker does not affect the performance of the chimeric antigen receptor of the present disclosure.

[0044] Preferably, the PSMA chimeric antigen receptor comprises a secretory signal peptide, an antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, a CD3ζ signaling domain, a 2A sequence, and a suicide-inducing fusion domain.

[0045] In a preferred solution, the PSMA chimeric antigen receptor is composed of a tandemly connected secretory signal peptide (secretory signal), an anti-PSMA single-chain antibody (PSM scFv), a CD8α and / or CD28 transmembrane domain, a CD28 and CD27 signaling domain, a CD3ζ signaling domain, a 2A sequence, and a caspase 9 domain (FKBP.Casp9), with a specific configuration as follows: secretory signal-PSM scFv-CD28-CD27-CD3ζ-2A-FKBP.Casp9, where CD28 represents the CD28 extracellular signaling structure, the CD28 transmembrane domain, and its intracellular signaling domain. Alternatively, the PSMA chimeric antigen receptor is composed of a tandemly connected secretory signal peptide, an anti-PSMA single-chain antibody (PSM scFv), a CD8α and / or CD28 transmembrane domain, a CD28 and IL-15Ra signaling domain, a CD3ζ signaling domain, a 2A sequence, and a caspase 9 domain (FKBP.Casp9), with the specific configuration being as follows: secretory signal-PSMA scFv-CD28-IL-15Ra-CD3ζ-2A-FKBP.Casp9, where CD28 represents the CD28 transmembrane domain and its intracellular signaling domain.

[0046] Preferably, the amino acid sequence of the CD3ζ signaling domain comprises the sequence shown in SEQ ID NO:7.

[0047] Preferably, the amino acid sequence of the 2A sequence comprises the sequence shown in SEQ ID NO:8.

[0048] SEQ ID NO:7:

[0049] [ka]

[0050] SEQ ID NO:8:

[0051] [ka]

[0052] In the present application, the chimeric antigen receptor further comprises a promoter, which is either EF1α or one of the highly expressed promoters.

[0053] In a fourth aspect, the present application provides a nucleic acid molecule that encodes a PSMA chimeric antigen receptor described in the third aspect.

[0054] In a fifth aspect, the present application provides a viral vector, the viral vector comprising a nucleic acid molecule encoding the PSMA chimeric antigen receptor described in the third aspect.

[0055] The viral vector comprises at least one copy of the nucleic acid molecule described in the fourth aspect.

[0056] Preferably, the viral vector comprises a lentiviral vector or a retroviral vector, preferably a lentiviral vector.

[0057] In this application, viral vectors can effectively modify immune cells to prepare target cells.

[0058] In a sixth aspect, the present application provides a recombinant virus obtained by co-transduction of a viral vector described in the fifth aspect and a packaging helper plasmid into a mammalian cell.

[0059] Preferably, the packaging helper plasmids include pNHP and pHEF-VSVG.

[0060] Preferably, the mammalian cells include any one of 293 cells, 293T cells, or TE671 cells.

[0061] In a seventh aspect, the present application provides a chimeric antigen receptor cell, which expresses the PSMA chimeric antigen receptor described in the third aspect.

[0062] In the present application, the mechanism of action of chimeric antigen receptor cells is shown in Figure 1, which shows that chimeric antigen receptor cells have a high target killing effect, release low amounts of immune factors, and have low toxicity and high immune killing response characteristics.

[0063] Preferably, the chimeric antigen receptor cells are prepared by transduction of immune cells with a nucleic acid molecule encoding the PSMA chimeric antigen receptor described in the third aspect.

[0064] Preferably, the mode of transduction comprises any one of transduction by a viral vector, transduction by a eukaryotic expression plasmid or transduction by mRNA, preferably transduction by a viral vector.

[0065] Preferably, the immune cells include T cells.

[0066] In an eighth aspect, the present application provides a composition comprising any one or a combination of at least two of the anti-PSMA single chain antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the PSMA chimeric antigen receptor described in the third aspect, the viral vector described in the fifth aspect, the recombinant virus described in the sixth aspect, or the chimeric antigen receptor cell described in the seventh aspect.

[0067] In a ninth aspect, the present application provides the use of any one or a combination of at least two of the anti-PSMA single chain antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the PSMA chimeric antigen receptor described in the third aspect, the viral vector described in the fifth aspect, the recombinant virus described in the sixth aspect, the chimeric antigen receptor cell described in the seventh aspect or the composition described in the eighth aspect in the preparation of a medicament for treating a tumor.

[0068] Preferably, the tumor comprises a tumor that expresses a PSMA-specific antigen.

[0069] Preferably, the tumor comprises prostate cancer, lymphoma, kidney cancer, bladder cancer, colon cancer, neuroblastoma or brain cancer.

[0070] Compared with existing technologies, the present application has the beneficial effects described below.

[0071] (1) The anti-PSMA single-chain antibody of the present application is a humanized scFv antibody of PSMA. The scFv antibody is specifically engineered against the tumor surface antigen PSMA using humanized codons and a humanized antibody framework, making the engineered scFv antibody more functional in the human body, more compatible with the human body, and less likely to be rejected by the immune system.

[0072] (2) The chimeric antigen receptor of the present application is obtained by carrying out specific genetic modifications in the costimulatory signaling domain of a humanized chimeric antigen receptor that targets the tumor surface antigen PSMA. The modified chimeric antigen receptor has a better response effect after specifically binding to PSMA, thereby enabling CAR-T cells to generate a stronger immune response to tumors, and the modified chimeric antigen receptor also has better long-term efficacy than other PSMA chimeric antigen receptors.

[0073] (3) The chimeric antigen receptor T cells of the present application have greater safety and durability than other PSMA chimeric antigen receptor T cells. Even if an excessively strong immune response occurs in a patient and an immune factor storm occurs, the chimeric antigen receptor T cells can be eliminated by drug-induced CAR-T cell apoptosis due to the apoptosis induction mechanism. After the chimeric antigen receptor T cells of the present application are infused, the presence of CAR-T cells can be monitored in vivo for a long period of time, demonstrating that the chimeric antigen receptor T cells have long-term efficacy in patients and can achieve long-term remission.

[0074] (4) The humanized antibody-related preparations in this application can act against all PSMA-positive diseases and have been applied to patients with tumors that express the tumor-specific target PSMA, with few clinical side effects, high safety, and effective removal of chemotherapy-resistant minimal residual tumors. In addition, PSMA CAR-T cells can also be used in combination with other targeted CAR-T cells in patients with brain tumors, and the presence of PSMA CAR-T cells can be monitored in vivo in patients over a long period of time, contributing to the maintenance of long-term remission. [Brief explanation of the drawings]

[0075] [Figure 1] FIG. 1 is a schematic diagram showing the structure of a chimeric antigen receptor and the mechanism of action of chimeric antigen receptor T cells. [Figure 2] FIG. 1 shows in vitro killing of PSMA CAR-T cells against PSMA-positive tumor cell lines. [Figure 3A] FIG. 1 is a schematic diagram showing the process of treating brain glioma with PSMA CAR-T cells. [Figure 3B] 1 is an image (20x magnification) showing the results of immunohistochemical staining of a tumor section from a patient with brain glioma. [Figure 4] 1 is a curve graph of CAR copy number detected in vivo after infusion of PSMA CAR-T cells in Example 8. [Figure 5] MRI images of changes in brain tumor lesions before and after PSMA CAR-T cell infusion. DETAILED DESCRIPTION OF THE INVENTION

[0076] In order to further describe the technical means adopted in the present application and the effects achieved, the present application is further described below in conjunction with examples and drawings. It is understood that the specific examples described below are intended to illustrate the present application, and do not limit the present application.

[0077] Experiments for which no specific techniques or conditions are specified in the examples are carried out according to techniques or conditions described in the literature in the art or according to product specifications. Reagents or equipment used herein that are not specified by the manufacturer are conventional products that are commercially available in accordance with regular procedures. [Example]

[0078] [Example 1] This example provides a humanized scFv antibody to PSMA, which has binding activity to the PSMA antigen.

[0079] The nucleic acid sequence of the humanized scFv antibody to PSMA is shown in SEQ ID NO:3.

[0080] [Example 2] This example provides a chimeric antigen receptor (CAR), which is composed of a tandemly connected secretory signal peptide (MALPVTALLLPLALLLHAARP (SEQ ID NO: 10)), an anti-PSMA single-chain antibody (SEQ ID NO: 9), a CD28 transmembrane domain, CD28 and CD27 signaling domains (SEQ ID NO: 4), a CD3ζ signaling domain (SEQ ID NO: 7), a 2A sequence (SEQ ID NO: 8), and a caspase-9 domain (SEQ ID NO: 6). The specific configuration is as follows: secretory signal PSMA scFv-CD28-CD27-CD3ζ-2A-FKBP.Casp9, where CD28 represents the CD28 transmembrane domain and its intracellular signaling domain. The CAR was named chimeric antigen receptor 12313.

[0081] [Example 3] This example provides a lentiviral vector encoding the chimeric antigen receptor of Example 2.

[0082] The backbone vector of the lentiviral vector is pTYF. For details, see Chang, L.-J. and Zaiss, A.-K. (2001) Methods for the preparation and use of lentivirus vectors. Methods in Molecular Medicine, Gene Therapy Protocols, 2nd ed., pp. 303-318, Jeffrey Morgan (ed.), Humana Press, Inc.; Cui, Y. and Chang, L.-J. (2003) Detection and selection of lentiviral vector transduced cells. Methods in Molecular Biology, Vol. 229: Lentivirus Gene Engineering Protocols, pp. 69-85, Maurizio Federico (ed.), Humana Press, Inc.; Oka, M., Chang, L.-J., Costantini, F., and Terada, N. (2005) Lentivirus-mediated gene transfer in embryonic stem cells. Series: "Methods in Molecular See Biology”Embryonic Stem Cells 2.

[0083] [Example 4] This example provides a recombinant lentiviral vector, which can be obtained by co-transducing mammalian cells with the lentiviral vector described in Example 3 or a control viral protein (EBV LMP 2) antibody CAR lentiviral vector that does not associate with target cells and a packaging helper plasmid, by the following steps: (1) 293T cells were cultured for 18 hours.

[0084] (2) Fresh DMEM (purchased from Thermo Fisher) was added.

[0085] (3) The following reagents were placed in a sterile centrifuge tube: each well was filled with DMEM, packaging helper plasmids (pNHP and pHEF-VSV-G), and pTYF CAR DNA vector (for specific procedures, see Chang, L.-J. and Zaiss, A.-K. (2001) Methods for the preparation and use of lentivirus vectors. Methods in Molecular Medicine, Gene Therapy Protocols, 2nd Edition, pp. 303-318, Jeffrey Morgan (ed.), Humana Press, Inc.), and then the tube was vortexed and shaken.

[0086] (4) Superfect (purchased from Qiagene) was placed in a centrifuge tube and placed at 25°C for 8 minutes.

[0087] (5) The DNA-Superfect mixture in the centrifuge tube was added dropwise to the cultured cells and vortexed.

[0088] (6) The system was incubated for 5 hours at 37°C in a CO2 incubator.

[0089] (7) The solution in the culture medium was aspirated and removed, the culture medium was washed with AIM-V (BRL), and fresh AIM-V was added to continue the incubation.

[0090] (8) The cells were returned to a CO2 incubator and cultured overnight. The transduction efficiency was observed the next day.

[0091] [Example 5] In this example, lentivirus purification and concentration were carried out.

[0092] 1. Viral Vector Purification Cell debris was removed using centrifugation (1000 xg) to obtain the viral supernatant, which was filtered through a low protein binding filter, and the virus was aliquoted and stored at -80°C.

[0093] 2. Lentiviral vector concentration using a centrifugal filter (1) In a biosafety cabinet, the concentration tubes were sterilized and washed twice under sterile conditions.

[0094] (2) The viral vector supernatant was placed in each centrifugal filter tube and centrifuged until the viral volume was reduced to 1 / 30.

[0095] (3) The filter tube was shaken and centrifuged, the concentrated virus was collected in a collection cup, and the viral vectors from all the tubes were pooled into one centrifuge tube.

[0096] [Example 6] This example provided two types of chimeric antigen receptor T cells. The chimeric antigen receptor T cells expressed a chimeric antigen receptor targeting PSMA (12313) in Example 2 and a control chimeric antigen receptor targeting LMP2 in Example 4. The preparation method was as follows: The activated T cells were suspended in culture medium and 10 μg / mL polybrene (purchased from Sigma) was added. The culture medium was AIM-V containing cell culture factors IL-2, IL-7, and IL-15 (purchased from Peprotech). The lentivirus concentrated in Example 5 was added to each suspension. The cells were centrifuged at 100 g for 100 minutes at 25°C and cultured for 24 hours at 37°C. Culture medium was added. After 4 days of culture, the cells were collected and counted. After 2 days of culture, the cells were successfully detected and transferred to patients.

[0097] [Example 7] This example performed an in vitro killing assay of target cell-associated CAR-T cells (12313) and target cell-unassociated CAR-T cells (LMP2).

[0098] (1) Green fluorescent protein was transferred into two PSMA-positive tumor cell lines, namely, the prostate tumor cell line LNCap2 and the multiple myeloma cell line Molp2, using a lentiviral vector for stable expression.

[0099] (2) LMP2 CAR-T cells were used as a negative control, and the 12313 PSMA CAR-T cells in Example 6 were used as the experimental group. The two types of CAR-T cells were co-cultured with the two tumor types in step (1) for 1 to 8 days in a 37°C, 5% CO2 incubator. During the culture process, tumor cell killing was observed and recorded daily using a fluorescence microscope. The results are shown in Figure 2. As can be seen, the killing effect of the PSMA CAR-T (12313) group was significantly superior to that of the control group (LMP2).

[0100] [Example 8] This example used PSMA CAR-T cells (12313 CAR-T) to treat brain glioma.

[0101] (1) The overall treatment flow was shown in Figure 3A, which was adopted for one patient with intractable brain glioma.

[0102] (2) Unstained tumor sections from the patient were confirmed to have positive PSMA expression using immunohistochemical staining, as shown in Figure 3B.

[0103] (3) The patient's white blood cell concentrate was collected. Peripheral mononuclear lymphocytes in the white blood cell concentrate were separated using density gradient centrifugation with Ficoll. T cells were selected using CD3 magnetic beads and activated with anti-CD28 antibody (purchased from BD Biosciences). PSMA CAR-T cells were injected at a dose of 2 × 10 CAR-T cells per kilogram of body weight. 6 It was prepared individually.

[0104] (4) Before infusion, patients were conditioned with low-dose chemotherapy. The conditioning regimen consisted of cyclophosphamide (250 mg / m 2) for 3 days and fludarabine (25 mg / m 2 The CAR-T cell infusion was performed 24 hours after the conditioning treatment, for a total of 4 days of conditioning.

[0105] (5) CAR-T cells were infused intravenously.

[0106] (6) After infusion, patients were monitored by clinicians and evaluated for cytotoxic responses. The clinical cytotoxic response was cytokine release syndrome (CRS). The results show that no CRS responses were observed in patients.

[0107] (7) The patient's tumor lesion was evaluated by MRI before and after injection, and the patient was assessed to be in stable condition.

[0108] (8) After the infusion, small amounts of peripheral blood were collected periodically from the patient, and mononuclear lymphocytes were isolated from the peripheral blood, followed by extraction of cellular chromosomal DNA (gDNA). The CAR copy number in the peripheral blood was then quantified by qPCR using specific primers. (For specific procedures, see Chang, L.-J. and Zaiss, A.-K. (2001) Methods for the preparation and use of lentivirus vectors. Methods in Molecular Medicine, Gene Therapy Protocols, 2nd ed., pp. 303-318, Jeffrey Morgan (ed.), Humana Press, Inc.) Figure 4 shows a curve graph of the change in PSMA CAR copy number in a patient with glioblastoma obtained after the CAR copy number was detected in blood collected from a patient infused with CAR-T cells. Figure 5 shows MRI images of the brain of a patient with cerebral glioma before and after CAR-T cell infusion, when the patient was clinically infused with PSMA chimeric antigen receptor T cells for the treatment of cerebral glioma. The results show that tumor images were significant 5 days (-5) before CAR-T cell injection, and 12 days after CAR-T cell injection, the tumor enlarged with pseudoprogression due to CAR-T cell infiltration, and the tumor shrank 55 days after CAR-T cell injection.

[0109] In summary, the PSMA chimeric antigen receptor of the present application has better response efficacy and long-term efficacy. When applied to patients with glioblastoma that expresses the tumor-specific target PSMA, the PSMA chimeric antigen receptor has few clinical side effects and high safety, and can effectively eliminate minimal residual tumors that are insensitive to chemotherapy. In addition, PSMA CAR-T cells can also be used in combination with other targeted CAR-T cells in patients with brain glioma, and the presence of PSMA CAR-T cells can be monitored in vivo in patients over a long period of time, contributing to maintaining long-term remission.

[0110] The applicant states that the detailed methods of the present application are described through the above-described embodiments, but the present application is not limited to the detailed methods described above, which means that the implementation of the present application does not necessarily depend on the detailed methods described above. Any improvements made to the present application, equivalent replacement of raw materials of the products of the present application, addition of adjuvant components, selection of specific modes, etc., are all within the scope of protection and disclosure of the present application, as will be apparent to those skilled in the art.

Claims

1. 1. An anti-prostate specific membrane antigen (anti-PSMA) single chain antibody, A heavy chain comprising an amino acid sequence having 80% or more identity to SEQ ID NO:1; and A light chain comprising an amino acid sequence having 80% or more identity with SEQ ID NO:

2.

1. An anti-prostate specific membrane antigen (anti-PSMA) single chain antibody comprising:

2. A nucleic acid molecule comprising a nucleic acid sequence encoding the anti-PSMA single chain antibody of claim 1.

3. A PSMA chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and a CD3ζ signaling domain, wherein the antigen-binding domain comprises the anti-PSMA single-chain antibody of claim 1.

4. the transmembrane domain comprises a CD28 transmembrane domain and / or a CD8α transmembrane domain; Preferably, the costimulatory signaling domain comprises a CD28 signaling domain and a CD27 signaling domain, or a CD28 signaling domain and an IL-15Ra signaling domain; Preferably, the PSMA chimeric antigen receptor comprises a CD28 transmembrane domain, a CD28 signaling domain, and a CD27 signaling domain; Preferably, the PSMA chimeric antigen receptor comprises a CD28 transmembrane domain, a CD28 signaling domain, and an IL-15Ra signaling domain; Preferably, the PSMA chimeric antigen receptor further comprises a suicide-inducing fusion domain; Preferably, the suicide-inducing fusion domain comprises a caspase-9 domain fused to FK506 binding protein (FKBP); Preferably, the PSMA chimeric antigen receptor further comprises a signal peptide and / or a 2A sequence; Preferably, the signal peptide comprises a secretory signal peptide, Preferably, the PSMA chimeric antigen receptor comprises a secretory signal peptide, an antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, a CD3ζ signaling domain, a 2A sequence, and a suicide-inducing fusion domain. The PSMA chimeric antigen receptor of claim 3.

5. A viral vector comprising a nucleic acid molecule encoding the PSMA chimeric antigen receptor of claim 3, Preferably, the viral vector comprises a lentiviral vector or a retroviral vector, preferably a lentiviral vector. Viral vector.

6. A recombinant virus obtained by cotransfecting a mammalian cell with the viral vector of claim 5 and a packaging helper plasmid, Preferably, the packaging helper plasmid comprises pNHP and pHEF-VSVG; Preferably, the mammalian cells include any one of 293 cells, 293T cells, or TE671 cells. Recombinant viruses.

7. A chimeric antigen receptor cell expressing the PSMA chimeric antigen receptor of claim 3, Preferably, the chimeric antigen receptor is prepared by transducing immune cells with a nucleic acid molecule encoding the PSMA chimeric antigen receptor of claim 3, Preferably, the mode of transduction comprises any one of transduction by a viral vector, transduction by a eukaryotic expression plasmid, or transduction by mRNA, preferably transduction by a viral vector; Preferably, the immune cells comprise T cells. Chimeric antigen receptor cells.

8. A composition comprising any one or a combination of at least two of the anti-PSMA single-chain antibody of claim 1, the nucleic acid molecule of claim 2, the PSMA chimeric antigen receptor of claim 3, the viral vector of claim 5, the recombinant virus of claim 6, or the chimeric antigen receptor cell of claim 7.

9. Use of any one or a combination of at least two of the anti-PSMA single-chain antibody of claim 1, the nucleic acid molecule of claim 2, the PSMA chimeric antigen receptor of claim 3, the viral vector of claim 5, the recombinant virus of claim 6, the chimeric antigen receptor cell of claim 7, or the composition of claim 8 in the preparation of a drug for treating a tumor.

10. the tumor comprises a tumor that expresses a PSMA-specific antigen; Preferably, the tumors include PSMA-specific antigen-expressing hematological tumors and PSMA-specific antigen-expressing solid tumors; Preferably, the tumor comprises prostate cancer, lymphoma, multiple myeloma, kidney cancer, bladder cancer, colon cancer, neuroblastoma, or brain tumor; 10. The use according to claim 9.

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