An antigen-binding fragment against PSMA, an antibody and its application

Abstract

This invention discloses an anti-PSMA antigen-binding fragment, an antibody, and their applications. The heavy chain variable region of the antigen-binding fragment comprises the amino acid sequences shown in SEQ ID NO. 1-2 (CDR1), SEQ ID NO. 3-4 (CDR2), and SEQ ID NO. 5-6 (CDR3); the light chain variable region of the antigen-binding fragment comprises the amino acid sequences shown in SEQ ID NO. 7-8 (CDR1), SEQ ID NO. 9-10 (CDR2), and SEQ ID NO. 11-12 (CDR3). The antigen-binding fragment and antibody of this invention exhibit high specificity and affinity, and can effectively recognize and bind to PSMA-positive cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an antigen-binding fragment, antibody, and its application against PSMA. Background Technology

[0002] Prostate cancer is a common type of malignant tumor among men, and early detection and timely treatment are crucial for improving patient survival rates. PSMA, or prostate-specific membrane antigen, is a membrane protein highly expressed on the surface of prostate cancer cells, making it a key target for prostate cancer diagnosis and treatment. Traditional treatments mainly include surgical resection, radiotherapy, and chemotherapy; however, these methods often come with significant side effects and limitations. In recent years, CAR-T therapy, as an emerging immunotherapy approach, uses genetic engineering to modify the patient's T cells, enabling them to specifically recognize and kill tumor cells, and has shown significant efficacy in the treatment of certain hematologic malignancies. However, applying 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 against tumor cells.

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

[0004] To address the shortcomings of existing technologies and practical needs, this invention provides an anti-PSMA antigen-binding fragment, an antibody, and their applications. The antigen-binding fragment and antibody can specifically bind to the PSMA protein, enabling the preparation of chimeric antigen receptors and CAR-T cells, which exhibit significant cytotoxicity against target cells expressing the PSMA protein.

[0005] To achieve this objective, the present invention employs the following technical solution:

[0006] In a first aspect, the present invention provides an antigen-binding fragment against PSMA, 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, the CDR2 includes the amino acid sequence shown in SEQ ID NO.9 or SEQ ID NO.10, and the CDR3 includes the amino acid sequence shown in SEQ ID NO.11 or SEQ ID NO.12.

[0008] The antigen-binding fragment and antibody described in this invention possess high specificity and affinity, enabling them to effectively recognize and bind to PSMA-positive cells. Through genetic engineering, the gene sequence encoding the antigen-binding fragment is inserted into T cells, constructing T cells capable of expressing specific anti-PSMA antibodies. These modified T cells can specifically recognize and kill PSMA-positive tumor cells, thus demonstrating significant potential in treating prostate cancer and other PSMA-expressing tumors. This invention also provides related preparation methods and applications, offering a new strategy for cancer treatment.

[0009] Preferably, 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;

[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, the CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.2, the CDR2 is the amino acid sequence shown in SEQ ID NO.4, and the 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, said anti-PSMA antibody comprising the antigen-binding fragment as described in the first aspect.

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

[0040] Meanwhile, the present invention also provides a method for preparing anti-PSMA antibody as described in the second aspect, specifically including the following steps:

[0041] (1) Hybridoma preparation was carried out by immunizing BALB / c mice with PSMA antigen. Five healthy female BALB / c mice aged 7-8 weeks were selected for immunization. After a certain immunization time, the serum titer of the immunized mice was detected. Spleen cells of mice whose serum titers met the requirements of the fusion experiment were selected and fused with myeloma cells SP2 / 0 in an appropriate ratio under the action of a fusion agent to prepare hybridoma monoclonal cells.

[0042] (2) Hybridoma cells were cultured in selective medium R1640-HAT for 7-10 days, and then cultured in HT medium for 3-4 days. On days 10-14, the hybridoma supernatant samples were detected by ELISA to obtain positive clones.

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

[0044] Thirdly, the present invention provides a nucleic acid molecule that encodes the antigen-binding fragment against PSMA as described in the first aspect or the anti-PSMA antibody as described in the second aspect.

[0045] Fourthly, the present invention provides an expression vector comprising the nucleic acid molecule described in the third aspect.

[0046] Fifthly, the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the anti-PSMA antibody described in the second aspect.

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

[0048] Preferably, the signal peptide includes CD8α signal peptide and / or IgGκ light chain signal peptide, and more preferably IgGκ light chain signal peptide.

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

[0050] Preferably, the transmembrane domain includes CD8α and / or CD28, with CD8α being the most preferred.

[0051] Preferably, the signal conduction structure domain includes the CD3ζ signal conduction domain.

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

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

[0054] In this 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 signal transduction domain sequence.

[0055] The amino acid sequence (SEQ ID NO.19) of the IgGκ light chain signal peptide is as follows:

[0056] MDMRVPAQLLGLLLLWLRGARC;

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

[0058] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

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

[0060] IYIWAPLAGTCGVLLLSLVITLYC;

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

[0062] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

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

[0064] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR.

[0065] Preferably, the amino acid sequence of the chimeric antigen receptor includes the sequences 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 (Humanized 27D11 Amino Acid Sequence):

[0071] MALPPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQSVRNDVGWYQQKPGKAPKLLIYFASNRYTGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQ GTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCKASGYTFANFGMNWVRQAPGKGLEWMGWISTYTGEPTYTDSFKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARND YPYWYFDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0072] In a sixth aspect, the present invention provides a host cell comprising 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 described in the second aspect.

[0074] Preferably, the pharmaceutical composition further includes an antitumor drug.

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

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

[0077] Eighthly, the present invention provides the 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 treatment drugs.

[0078] Preferably, the cancer includes cancers that are positive for PSMA expression.

[0079] Preferably, the cancer includes any one of stomach 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 this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all 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 fragment and anti-PSMA antibody provided by the present invention can specifically bind PSMA proteins from multiple species (including humans, mice and cynomolgus monkeys), providing more options for the selection of subsequent animal models. The anti-PSMA antibody of the present invention can specifically bind PSMA without binding to other non-target proteins, and has obvious specificity.

[0083] (2) This invention provides two chimeric antigen receptors (CARs) against PSMA and one humanized chimeric antigen receptor against PSMA. After these chimeric antigen receptors are transfected into T cells via lentiviral vectors, CAR-T cells expressing anti-PSMA are obtained. The CAR-T cells have significant cytotoxicity against cells that stably express PSMA protein. Therefore, they have significant therapeutic value for cancers that are positive for PSMA expression, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. Attached Figure Description

[0084] Figure 1 The graph shows the results of the antibody-cell line binding ability test.

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

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

[0087] Figure 4 The results of combining humanized 27D11 (Hu27D11) CAR with PSMA are shown in the figure. Detailed Implementation

[0088] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0089] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0090] Example 1

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

[0092] Subsequently, flow cytometry screening was performed to obtain subclones, and sequencing yielded two correct sequence clones of anti-PSMA antibodies, 12B1 and 27D11. Sequencing identification confirmed the anti-PSMA antibody sequences 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 detection.

[0103] 1. Antigen coating: The PSMA antigen is coated onto 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: Different concentrations of anti-PSMA antibodies are incubated with the coated antigen at 37°C to simulate the environment inside the human body.

[0105] 3. Binding assay: Biotin-labeled PSMA antigen is added, followed by streptavidin-labeled HRP. The binding assay must be performed at room temperature to ensure enzyme activity.

[0106] 4. Colorimetric reaction: Add TMB substrate and perform a colorimetric reaction based on the color change. The colorimetric reaction must be carried out under dark conditions to prevent substrate decomposition.

[0107] 5. Data Analysis: OD was measured using an ELISA reader. 450 The values ​​were determined, and 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 antibody affinity constant (KD value). The results are shown in Table 1.

[0109] Table 1

[0110]

[0111] Affinity test results for the two antibodies (12B1 and 27D11): The binding rate constants were comparable to those of the control antibody. Both antibodies (12B1 and 27D11) showed 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 antibody of this invention to human PSMA can be measured by flow cytometry. 5 × 10⁵ 5 LNCaP cells (human prostate cancer cells) were incubated in PBS + 5% BSA at 4°C for 30 min. They were then incubated together with experimental antibodies at concentrations from 1 μg / mL (10-fold serial dilutions) down to the lowest concentration of 0.01 μg / mL + 5% BSA in PBS at 4°C for 60 min. After centrifugation and washing, the cells were incubated with secondary antibody (FITC-labeled, 1:200, Sigma, F9512) in PBS + 5% BSA on ice for 30 min (protected from light). The cells were washed three times and analyzed by flow cytometry. Results Figure 1 As shown: EC2 strains of two chimeric antibodies 50 They are 27D11 (0.2482 μg / mL) and 12B1 (2.476 μg / mL), respectively.

[0115] Example 4

[0116] In this embodiment, a chimeric antigen receptor against PSMA and its expression vector were constructed.

[0117] (1) Sequence Design

[0118] This chimeric antigen receptor includes an IgGκ light chain signal peptide sequence (Leader), an antibody sequence that specifically binds to the PSMA antigen (scFv), a CD8a hinge region and transmembrane region sequence, a 4-1BB co-stimulatory domain sequence, and a CD3ζ signal transduction domain sequence.

[0119] The specific structure is as follows: Figure 2 As shown in the figure; the amino acid sequences of each part are shown in Table 2 below.

[0120] Table 2

[0121]

[0122] (2) Constructing a chimeric antigen receptor expression vector against PSMA

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

[0124] Then, the pCDH-EF1 vector was ligated to the CAR gene fragment, and the specific ligation system is shown in Table 3 below:

[0125] Table 3

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

[0127] Ligation was performed at 22℃ for 1 hour. The ligation product was directly transformed into Stbl3 E. coli competent cells. 200 μL of the transformation product was spread onto ampicillin-resistant LB plates and incubated upside down in an incubator at 37℃ overnight.

[0128] The following morning, three single clones were randomly selected for colony PCR identification. Positive clones were sent for sequencing to obtain the chimeric antigen receptor sequence of the anti-PSMA chimeric antigen receptor lentiviral expression plasmid, including the humanized CAR vector, such as... Figure 3 As shown.

[0129] Example 5

[0130] Flow cytometry was used to detect the binding of PSMA protein in 293T cells after transient transfection with CAR plasmid.

[0131] To further investigate the binding of PSMA protein in 293T cells after transient transfection with the CAR plasmid, we first prepared 293T cells and cultured them to an appropriate density. Then, the CAR plasmid DNA was mixed with the transfection reagent according to the manufacturer's instructions. The mixture was added to the culture medium containing 293T cells, and the culture dish was gently agitated to ensure uniform distribution. The cells were then placed in an incubator and cultured under appropriate conditions to promote transfection efficiency. Twenty-four hours after transfection, the cells were collected and washed with PBS. Subsequently, the cells were labeled with a solution containing PSMA protein and incubated. Flow cytometry was then used to analyze the binding of PSMA protein to the CAR plasmid-transfected 293T cells. Finally, the flow cytometry data were collected and analyzed to assess transfection efficiency and CAR protein expression levels. Results are as follows: Figure 4 As shown, humanized 27D11 (Hu27D11) can recognize both human and monkey PSMA antigens, binding to human and monkey PSMA at rates of 28.44% and 69.98%, respectively.

[0132] In summary, the anti-PSMA antibody provided by this invention can specifically bind to PSMA proteins from multiple sources, including human and monkey proteins, and has virtually no binding ability to other proteins, exhibiting high specificity. Therefore, the anti-PSMA antibody provided by this invention has a specific therapeutic effect on diseases targeting PSMA proteins.

[0133] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the 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 antigen-binding fragment against PSMA, characterized in that, The heavy chain variable region of the antigen-binding fragment has the following amino acid sequence: CDR1 is shown in SEQ ID NO.1, CDR2 is shown in SEQ ID NO.3, and CDR3 is 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; or, The heavy chain variable region of the antigen-binding fragment has the following amino acid sequence: CDR1 is shown in SEQ ID NO.2, CDR2 is shown in SEQ ID NO.4, and CDR3 is 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.

2. An anti-PSMA antibody, characterized in that, The anti-PSMA antibody includes the antigen-binding fragment as described in claim 1.

3. The anti-PSMA antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-PSMA antibody is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

16. or, The amino acid sequence of the heavy chain variable region of the anti-PSMA antibody is shown in SEQ ID NO.14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

17.

4. The anti-PSMA antibody according to claim 2, characterized in that, The anti-PSMA antibody also includes a constant region.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antigen-binding fragment of the anti-PSMA according to claim 1 or the anti-PSMA antibody according to any one of claims 2-4.

6. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 5.

7. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor includes the anti-PSMA antibody according to any one of claims 2-4.

8. The chimeric antigen receptor according to claim 7, characterized in that, The chimeric antigen receptor also includes a signal peptide, a hinge region, a transmembrane domain, and a signal transduction domain.

9. The chimeric antigen receptor according to claim 8, characterized in that, The signal peptides include CD8α signal peptide and / or IgGκ light chain signal peptide.

10. The chimeric antigen receptor according to claim 9, characterized in that, The signal peptide is an IgGκ light chain signal peptide.

11. The chimeric antigen receptor according to claim 8, characterized in that, The hinge region includes any one of CD8α, CD28, human IgG1, IgG2, IgG4, or IgA.

12. The chimeric antigen receptor according to claim 11, characterized in that, The hinge region is CD8α.

13. The chimeric antigen receptor according to claim 8, characterized in that, The transmembrane domains include CD8α and / or CD28.

14. The chimeric antigen receptor according to claim 13, characterized in that, The transmembrane domain is CD8α.

15. The chimeric antigen receptor according to claim 8, characterized in that, The signal conduction structure domain includes the CD3ζ signal conduction domain.

16. The chimeric antigen receptor according to claim 15, characterized in that, The signal transduction domain further includes a co-stimulatory domain, which includes any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10, or OX40.

17. A host cell, characterized in that, The host cell includes the nucleic acid molecule of claim 5, the expression vector of claim 6, or the chimeric antigen receptor of any one of claims 7-16.

18. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anti-PSMA antibody according to any one of claims 2-4.

19. The pharmaceutical composition according to claim 18, characterized in that, The pharmaceutical composition also includes an antitumor drug.

20. The pharmaceutical composition according to claim 19, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

21. The use of the anti-PSMA antigen-binding fragment of claim 1, the anti-PSMA antibody of any one of claims 2-4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the chimeric antigen receptor of any one of claims 7-16, the host cell of claim 17, or the pharmaceutical composition of any one of claims 18-20 in the preparation of prostate cancer detection reagents and / or prostate cancer treatment drugs.

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