Anti-PSMA antigen binding fragment, antibody and application thereof

Abstract

The invention discloses an anti-PSMA antigen binding fragment, an antibody and application of the anti-PSMA antigen binding fragment and the antibody. CDR1 of a heavy chain variable region of the antigen binding fragment comprises an amino acid sequence as shown in SEQ ID NO.1-2, CDR2 comprises an amino acid sequence as shown in SEQ ID NO.3-4, and CDR3 comprises an amino acid sequence as shown in SEQ ID NO.5-6; cDR1 of a light chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.7-8, CDR2 of the light chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.9-10, and CDR3 of the light chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.11-12. The antigen binding fragment and the antibody have high specificity and affinity, and can effectively recognize and bind PSMA positive cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to an anti-PSMA antigen-binding fragment, an antibody and applications thereof. Background Art

[0002] Prostate cancer is a common type of malignant tumor in men. Early detection and timely treatment play a vital role in improving the survival rate of patients. PSMA, or prostate-specific membrane antigen, is a membrane protein highly expressed on the surface of prostate cancer cells, so it has become a key target for the diagnosis and treatment of prostate cancer. Traditional treatments mainly include surgical resection, radiotherapy, and chemotherapy, but these methods are often accompanied by significant side effects and certain limitations. In recent years, CAR-T therapy, as an emerging immunotherapy, has shown significant effects in the treatment of certain blood tumors by genetically engineering patients' T cells to enable them to specifically recognize and kill tumor cells. However, the application of CAR-T technology to solid tumors, especially prostate cancer, still faces many challenges. These challenges include finding suitable tumor-specific targets and improving the killing efficiency of CAR-T cells on tumor cells.

[0003] Therefore, there is an urgent need to develop an anti-PSMA antigen-binding fragment, antibody and PSMA-targeted CAR-T therapy to improve the treatment effect of prostate cancer and bring new hope and treatment options to prostate cancer patients. Summary of the invention

[0004] In view of the deficiencies in the prior art and actual needs, the present invention provides an anti-PSMA antigen-binding fragment, an antibody and applications thereof. The antigen-binding fragment and the antibody can specifically bind to the PSMA protein and are prepared into chimeric antigen receptors and CAR-T cells, which have obvious cytotoxicity to target cells expressing the PSMA protein.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an anti-PSMA antigen-binding fragment, wherein the CDR1 of the heavy chain variable region of the antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, the CDR2 comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.6;

[0007] The CDR1 of the light chain variable region of the antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.7 or SEQ ID NO.8, CDR2 includes the amino acid sequence shown in SEQ ID NO.9 or SEQ ID NO.10, and CDR3 includes the amino acid sequence shown in SEQ ID NO.11 or SEQ ID NO.12.

[0008] The antigen-binding fragment and antibody of the present invention have high specificity and affinity, and can effectively recognize and bind to PSMA-positive cells. Through genetic engineering technology, the gene sequence encoding the antigen-binding fragment is inserted into T cells to construct T cells that can express specific anti-PSMA CARs. These modified T cells can specifically recognize and kill PSMA-positive tumor cells, thereby showing significant potential in the treatment of prostate cancer and other tumors expressing PSMA. The present invention also provides related preparation methods and applications, providing a new strategy for cancer treatment.

[0009] Preferably, CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.1, CDR2 is the amino acid sequence shown in SEQ ID NO.3, and CDR3 is the amino acid sequence shown in SEQ ID NO.5;

[0010] The CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.7, the CDR2 is the amino acid sequence shown in SEQ ID NO.9, and the CDR3 is the amino acid sequence shown in SEQ ID NO.11.

[0011] Preferably, CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.2, CDR2 is the amino acid sequence shown in SEQ ID NO.4, and CDR3 is the amino acid sequence shown in SEQ ID NO.6;

[0012] The CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.8, the CDR2 is the amino acid sequence shown in SEQ ID NO.10, and the CDR3 is the amino acid sequence shown in SEQ ID NO.12.

[0013] SEQ ID NO.1: NFGMN.

[0014] SEQ ID NO.2: NYWMN.

[0015] SEQ ID NO. 3: WISTYTGEPTYTDDFKG.

[0016] SEQ ID NO.4: MIDPSDSETHYNQMFKD.

[0017] SEQ ID NO.5: NDYPYWYFDV.

[0018] SEQ ID NO.6:RGGYYGYYGMDY.

[0019] SEQ ID NO. 7: KASQSVRNDVG.

[0020] SEQ ID NO. 8: RASQDISNYLN.

[0021] SEQ ID NO.9: FASNRYT.

[0022] SEQ ID NO.10: STSRLHS.

[0023] SEQ ID NO.11:QQDYSSPWT.

[0024] SEQ ID NO.12: QQGHMIPWT.

[0025] In a second aspect, the present invention provides an anti-PSMA antibody, wherein the anti-PSMA antibody comprises the antigen-binding fragment according to the first aspect.

[0026] Preferably, the amino acid sequence of the heavy chain variable region of the anti-PSMA antibody is shown as SEQ ID NO.13-SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.16-SEQ ID NO.18.

[0027] SEQ ID NO.13 (27D11-VH):

[0028] QILLVQSGPELKKPGETVKISCKASGYTFANFGMNWVKQAPGEGLKWMGWISTYTGEPTYTDDFKGRFAFSLETSASTAYLQINNLRNEDAATYFCARNDYPYWYFDVWGAGTTVTVSS.

[0029] SEQ ID NO.14 (12B1-VH):

[0030] QVQLQQSGAELVRPGASVKLSCKVSDYTFTNYWMNWVKQRPGQGLEWIGMIDPSDSETHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAPRGGYYGYYGMDYWGQGTSVTVSS.

[0031] SEQ ID NO.15 (Hu27D11-VH1):

[0032] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQRLEWMGWIYPGNVNTKFNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARGAGTFAYWGQGTLVTVSA.

[0033] SEQ ID NO.16 (27D11-VL):

[0034] DIQMTQTPKFLLVSAGDRVTLTCKASQSVRNDVGWYQQRPGQSPKLLINFASNRYTGVPDRFTGSGYGTDFTFTINTVRAEDLAVYFCQQDYSSPWTFGGGTKLEIK.

[0035] SEQ ID NO.17(12B1-VL):

[0036] VIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPNGTVKLLIHSTSRLHSGVPSRFSGSGSGTDYSLTITNLEQEDVATYFCQQGHMIPWTFGGGNKLEIK.

[0037] SEQ ID NO.18 (Hu27D11-VL1):

[0038] DIQMTQSPSSVSASVGDRVTITCKAGQDVGTAVGWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSYPWTFGGGT KLEIK.

[0039] Preferably, the anti-PSMA antibody further comprises a constant region.

[0040] At the same time, the present invention also provides a method for preparing the anti-PSMA antibody as described in the second aspect, which specifically comprises the following steps:

[0041] (1) Immunizing BALB / c mice with PSMA antigen for hybridoma preparation, selecting 5 healthy female BALB / c mice aged 7 to 8 weeks for immunization, and testing the serum titer of the immunized mice after a certain immunization period, selecting mouse spleen cells with serum titers that meet the requirements of the fusion experiment, and fusing them with myeloma cells SP2 / 0 at an appropriate ratio under the action of a fusion agent to prepare hybridoma monoclonal cells;

[0042] (2) Cultivating hybridoma cells using selective culture medium R1640-HAT, and after 7 to 10 days, switching to HT culture medium for 3 to 4 days, and detecting hybridoma supernatant samples using ELISA on the 10th to 14th day to obtain positive clones;

[0043] (3) Perform flow cytometry screening experiments, subclone the mother clones that bind to PSMA, and sequence to obtain clones with the correct sequence.

[0044] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-PSMA antigen-binding fragment of the first aspect or the anti-PSMA antibody of the second aspect.

[0045] In a fourth aspect, the present invention provides an expression vector, wherein the expression vector comprises the nucleic acid molecule described in the third aspect.

[0046] In a fifth aspect, the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the anti-PSMA antibody according to the second aspect.

[0047] Preferably, the chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane domain and a signaling domain.

[0048] Preferably, the signal peptide comprises a CD8α signal peptide and / or an IgGκ light chain signal peptide, preferably an IgGκ light chain signal peptide.

[0049] Preferably, the hinge region comprises any one of CD8α, CD28, human IgG1, IgG2, IgG4 or IgA, preferably CD8α.

[0050] Preferably, the transmembrane domain comprises CD8α and / or CD28, preferably CD8α.

[0051] Preferably, the signaling domain comprises a CD3zeta signaling domain.

[0052] Preferably, the signal transduction domain further comprises a co-stimulatory domain, and the co-stimulatory domain comprises any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.

[0053] In the present invention, the PSMA-targeted chimeric antigen receptor comprises an IgGκ light chain signal peptide, an anti-PSMA antibody (scFv), a CD8α hinge region, a CD8α transmembrane region, 4-1BB and CD3ζ.

[0054] In the present invention, the chimeric antigen receptor includes an IgGκ light chain signal peptide sequence, an antibody sequence (scFv) that specifically binds to the PSMA antigen, a hinge region of CD8a, a transmembrane region sequence, a 4-1BB co-stimulatory domain sequence, and a CD3ζ signaling domain sequence.

[0055] Among them, the amino acid sequence of the IgGκ light chain signal peptide (SEQ ID NO.19) is:

[0056] MDMRVPAQLLGLLLLWLRGARC;

[0057] The amino acid sequence of the hinge region of CD8α (SEQ ID NO.20) is:

[0058] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

[0059] The amino acid sequence of the CD8α transmembrane region (TM) (SEQ ID NO.21) is:

[0060] IYIWAPLAGTCGVLLLSLVITLYC;

[0061] The amino acid sequence of the 4-1BB intracellular costimulatory domain (ICD) (SEQ ID NO.22) is:

[0062] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

[0063] The amino acid sequence of the CD3ζ signaling domain (SEQ ID NO.23) is:

[0064] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR.

[0065] Preferably, the amino acid sequence of the chimeric antigen receptor includes the sequence shown in SEQ ID NO.24-SEQ ID NO.26.

[0066] SEQ ID NO.24 (Amino acid sequence of 27D11 CAR):

[0067] MDMRVPAQLLGLLLLWLRGARCDIQMTQTPKFLLVSAGDRVTLTCKASQSVRNDVGWYQQRPGQSPKLLINFASNRYTGVPDRFTGSGYGTDFTFTINTVRAEDLAVYFCQQDYSSPWTFGGGTKLEIKGGGGSGGGGSGGGGSQILLVQSGPELKKPGETVKISCKASGYTFANFGMNWVKQAPGEGLKWMGWISTYTGEPTYTDDFKGRFAFSLETSASTAYLQINNLRNEDAATYFCARNDYPYWYFDVWGAGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0068] SEQ ID NO.25 (Amino acid sequence of 12B1 CAR):

[0069] MDMRVPAQLLGLLLLWLRGARCDVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLMISRVEAEDLGVYYCFQSNYPPLYTFGGGTKLEIKGGGGSGGGGSGGGGSDVKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPEKRLEWVATISSGSSFTYYPDSVKGRFTISRDNAKNTLHLQMSSLKSEDTAMYYCTRDFFGSTFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0070] SEQ ID NO.26 (Amino acid sequence of humanized 27D11):

[0071] MALPPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQSVRNDVGWYQQKPGKAPKLLIYFASNRYTGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQ GTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCKASGYTFANFGMNWVRQAPGKGLEWMGWISTYTGEPTYTDSFKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARND YPYWYFDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0072] In a sixth aspect, the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the chimeric antigen receptor described in the fifth aspect.

[0073] In a seventh aspect, the present invention provides a pharmaceutical composition, comprising the anti-PSMA antibody according to the second aspect.

[0074] Preferably, the pharmaceutical composition further comprises an anti-tumor drug.

[0075] In the present invention, the pharmaceutical composition can also be used in combination with other anti-tumor drugs, including simultaneous administration, separate administration or sequential administration.

[0076] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.

[0077] In an eighth aspect, the present invention provides use of the anti-PSMA antigen-binding fragment of the first aspect, the anti-PSMA antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the chimeric antigen receptor of the fifth aspect, the host cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect in the preparation of cancer detection reagents and / or cancer therapeutic drugs.

[0078] Preferably, the cancer comprises a PSMA-positive cancer.

[0079] Preferably, the cancer includes any one of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer or gallbladder cancer.

[0080] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0081] Compared with the prior art, the present invention has the following beneficial effects:

[0082] (1) The antigen-binding fragments and anti-PSMA antibodies provided by the present invention can specifically bind to PSMA proteins from multiple species (including humans, mice, and cynomolgus monkeys), providing more options for the selection of subsequent animal models. The anti-PSMA antibodies of the present invention can specifically bind to PSMA but not to other non-target proteins, and have obvious specificity;

[0083] (2) The present invention provides two anti-PSMA chimeric antigen receptors (CARs) and one humanized anti-PSMA chimeric antigen receptor. After these chimeric antigen receptors are transferred into T cells via lentiviral vectors, CAR-T cells expressing anti-PSMA CAR are obtained. The CAR-T cells have obvious cytotoxicity to cells stably expressing PSMA protein. Therefore, they have obvious therapeutic value for PSMA-positive cancers, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 It is a curve chart of the test results of the binding ability between the antibody and the cell line;

[0085] Figure 2 It is the schematic diagram of PSMACAR structure;

[0086] Figure 3 This is a map of the PSMACAR vector;

[0087] Figure 4 This is a graph showing the results of humanized 27D11 (Hu27D11) CAR binding to PSMA. DETAILED DESCRIPTION

[0088] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0089] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0090] Example 1

[0091] In this example, hybridoma monoclonal cells were prepared by DNA immunization of BALB / c mice, and the hybridoma cells were cultured using the selective culture medium R1640-HAT, and then replaced with HT culture medium for culture, and then the hybridoma supernatant samples were detected by ELISA to obtain positive clones;

[0092] Subsequently, flow cytometry screening experiments were performed to obtain subclones, and sequencing was performed to obtain two anti-PSMA antibody correct sequence clones 12B1 and 27D11. After sequencing and identification, it was found that the anti-PSMA antibody sequence was as follows:

[0093] 12B1-VL (SEQ ID NO.17):

[0094] VIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPNGTVKLLIHS TSRLHSGVPSRFSGSGSGTDYSLTITNLEQEDVATYFCQQGHMIPWTFGGGNK LEIK.

[0095] 12B1-VH (SEQ ID NO.14):

[0096] QVQLQQSGAELVRPGASVKLSCKVSDYTFTNYWMNWVKQRPGQGLE WIGMIDPSDSETHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAP RGGYYGYYGMDYWGQGTSVTVSS.

[0097] 27D11-VL (SEQ ID NO. 16):

[0098] DIQMTQTPKFLLVSAGDRVTLTCKASQSVRNDVGWYQQRPGQSPKLLIN FASNRYTGVPDRFTGSGYGTDFTFTINTVRAEDLAVYFCQQDYSSPWTFGGG TKLEIK.

[0099] 27D11-VH (SEQ ID NO.13):

[0100] QILLVQSGPELKKPGETVKISCKASGYTFANFGMNWVKQAPGEGLKWM GWISTYTGEPTYTDDFKGRFAFSLETSASTAYLQINNLRNEDAATYFCARNDY PYWYFDVWGAGTTVTVSS.

[0101] Example 2

[0102] Antibody affinity testing.

[0103] 1. Antigen coating: PSMA antigen is coated on a 96-well plate. The coating process must be carried out at room temperature to ensure the activity of the antigen.

[0104] 2. Antibody incubation: Incubate the coated antigen with different concentrations of anti-PSMA antibodies at 37°C to simulate the environment in the human body.

[0105] 3. Binding detection: Add biotin-labeled PSMA antigen, followed by streptavidin-labeled HRP. The binding detection process must be performed at room temperature to ensure enzyme activity.

[0106] 4. Color development reaction: Add TMB substrate and perform color development reaction according to the color change. The color development reaction must be carried out under light-proof conditions to prevent substrate decomposition.

[0107] 5. Data Analysis: OD Measurement by Microplate Reader 450 The affinity constant of the antibody was calculated using the Scatchard analysis method.

[0108] The binding and dissociation data were processed using ForteBIO analysis software to calculate the affinity constant (KD value) of the antibody. The results are shown in Table 1.

[0109] Table 1

[0110]

[0111] Affinity test results of the two antibodies (12B1 and 27D11): The binding rate constants were comparable to those of the control antibody. Both antibodies (12B1 and 27D11) showed relatively high affinity. The affinity constant KD value of the 12B1 antibody was 3.00E-10M, and the affinity constant KD value of the 27D11 antibody was 1.59E-10M.

[0112] Example 3

[0113] Binding to PSMA on LNCaP cells (human prostate cancer cells).

[0114] The binding of the antibodies of the invention to human PSMA can be measured in a flow cytometry assay. 5 LNCaP cells (human prostate cancer cells) were incubated in PBS + 5% BSA at 4°C for 30 min. They were incubated with the experimental antibody + 5% BSA PBS at a minimum concentration of 1 μg / mL (10-fold gradient dilution) to 0.01 μg / mL for 60 min at 4°C, washed by centrifugation, and incubated with the secondary antibody (FITC-labeled, 1:200, sigma, F9512) in PBS + 5% BSA on ice for 30 min (protected from light). The cells were washed 3 times and analyzed by flow cytometry. Results Figure 1 Shown: EC of 2 chimeric antibodies 50 They are 27D11 (0.2482 μg / mL) and 12B1 (2.476 μg / mL) respectively.

[0115] Example 4

[0116] In this example, an anti-PSMA chimeric antigen receptor and its expression vector were constructed.

[0117] (1) Sequence design

[0118] The chimeric antigen receptor includes an IgGκ light chain signal peptide sequence (Leader), an antibody sequence (scFv) that specifically binds to the PSMA antigen, a hinge region (Hinge) and a transmembrane region sequence (Transmembrane) of CD8a, a 4-1BB costimulatory domain sequence, and a CD3ζ signaling domain sequence;

[0119] Specific structure such as Figure 2 As shown; wherein the amino acid sequence of each part is shown in Table 2 below.

[0120] Table 2

[0121]

[0122] (2) Construction of anti-PSMA chimeric antigen receptor expression vector

[0123] First, the CAR sequence was fully synthesized, and the fully synthesized CAR and empty vector were double-digested with EcoRI and BamHI. After digestion in a 37°C water bath for 30 minutes, DNA was electrophoresed using 1.5% agarose gel, and then purified and recovered using Tiangen's agarose gel kit;

[0124] Then, the pCDH-EF1 vector and the CAR gene fragment were connected. The specific connection system is shown in Table 3 below:

[0125] Table 3

[0126] Reagents Usage pCDH-EF1 vector 2μL (50ng) CAR gene 10μL (150ng) T4DNALigaseBuffer 2μL T4 DNA Ligase (NEB) 1μL <![CDATA[ddH2O]]> 5μL Total 20μL

[0127] The cells were connected at 22°C for 1 hour, and the ligation product was directly transformed into Stbl3 E. coli competent cells. 200 μL of the transformation product was spread on an ampicillin-resistant LB plate, and the LB plate was inverted and cultured in an incubator at 37°C overnight.

[0128] The next morning, three single clones were randomly selected for colony PCR identification, and the positive clones were sent for sequencing to obtain the sequence of the anti-PSMA chimeric antigen receptor lentiviral expression plasmid chimeric antigen receptor, among which the humanized CAR vector was as follows Figure 3 shown.

[0129] Example 5

[0130] Flow cytometry was used to detect the binding of PSMA protein to 293T cells transiently transfected with CAR plasmid.

[0131] The binding of 293T cells to PSMA protein after transient transfection of CAR plasmid was further studied. First, 293T cells were prepared and cultured to an appropriate density. Then, the CAR plasmid DNA was mixed with the transfection reagent and operated according to the manufacturer's instructions. The mixture was added to the culture medium containing 293T cells, and the culture dish was gently shaken to ensure even distribution. Next, the cells were placed in an incubator and continued to be cultured, giving appropriate conditions to promote transfection efficiency. 24 hours after transfection, the cells were collected and washed with PBS. Subsequently, the cells were labeled with a solution containing PSMA protein and incubated. Afterwards, the labeled cells were detected using a flow cytometer to analyze the binding of CAR plasmid-transfected 293T cells to PSMA protein. Finally, the flow cytometry data were collected and analyzed to evaluate the transfection efficiency and the expression level of CAR protein. The results are shown in Figure 2. Figure 4 As shown, humanized 27D11 (Hu27D11) can recognize both human and monkey PSMA antigens, with binding rates of 28.44% and 69.98% for human and monkey PSMA, respectively.

[0132] In summary, the anti-PSMA antibodies provided by the present invention can specifically bind to PSMA proteins from a variety of sources, including human and monkey sources, and have substantially no binding ability to other proteins, and are highly specific. Therefore, the anti-PSMA antibodies provided by the present invention have specific therapeutic effects on diseases targeting PSMA proteins.

[0133] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-PSMA antigen-binding fragment, characterized in that: The CDR1 of the heavy chain variable region of the antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, the CDR2 comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.6; The CDR1 of the light chain variable region of the antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.7 or SEQ ID NO.8, CDR2 includes the amino acid sequence shown in SEQ ID NO.9 or SEQ ID NO.10, and CDR3 includes the amino acid sequence shown in SEQ ID NO.11 or SEQ ID NO.

12.

2. The antigen-binding fragment according to claim 1, characterized in that The CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.1, the CDR2 is the amino acid sequence shown in SEQ ID NO.3, and the CDR3 is the amino acid sequence shown in SEQ ID NO.5; The CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.7, the CDR2 is the amino acid sequence shown in SEQ ID NO.9, and the CDR3 is the amino acid sequence shown in SEQ ID NO.11; Preferably, CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.2, CDR2 is the amino acid sequence shown in SEQ ID NO.4, and CDR3 is the amino acid sequence shown in SEQ ID NO.6; The CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.8, the CDR2 is the amino acid sequence shown in SEQ ID NO.10, and the CDR3 is the amino acid sequence shown in SEQ ID NO.

12.

3. An anti-PSMA antibody, characterized in that The anti-PSMA antibody comprises the antigen-binding fragment of claim 1 or 2; Preferably, the amino acid sequence of the heavy chain variable region of the anti-PSMA antibody is as shown in SEQ ID NO.13-SEQ ID NO.15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.16-SEQ ID NO.18; Preferably, the anti-PSMA antibody further comprises a constant region.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-PSMA antigen-binding fragment of claim 1 or 2 or the anti-PSMA antibody of claim 3.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 4.

6. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the anti-PSMA antibody of claim 3; Preferably, the chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane domain and a signaling domain; Preferably, the signal peptide comprises a CD8α signal peptide and / or an IgGκ light chain signal peptide, preferably an IgGκ light chain signal peptide; Preferably, the hinge region comprises any one of CD8α, CD28, human IgG1, IgG2, IgG4 or IgA, preferably CD8α; Preferably, the transmembrane domain comprises CD8α and / or CD28, preferably CD8α; Preferably, the signaling domain comprises a CD3ζ signaling domain; Preferably, the signal transduction domain further comprises a co-stimulatory domain, and the co-stimulatory domain comprises any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.

7. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 4, the expression vector of claim 5 or the chimeric antigen receptor of claim 6.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-PSMA antibody of claim 3; Preferably, the pharmaceutical composition further comprises an anti-tumor drug; Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.

9. Use of the anti-PSMA antigen-binding fragment of claim 1 or 2, the anti-PSMA antibody of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the chimeric antigen receptor of claim 6, the host cell of claim 7 or the pharmaceutical composition of claim 8 in the preparation of cancer detection reagents and / or cancer therapeutic drugs.

10. The use according to claim 9, characterized in that: The cancer includes PSMA-positive cancer; Preferably, the cancer includes any one of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer or gallbladder cancer.

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