CN110964112B - Humanized antibody for enhancing anti-PSCA chimeric antigen receptor activity and application thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and in particular relates to a humanized antibody for enhancing the activity of anti-PSCA chimeric antigen receptor and its application. The humanized antibody of the present invention recognizes PSCA and can enhance the anti-tumor activity of the anti-PSCA chimeric antigen receptor. Furthermore, the chimeric antigen receptor of the humanized antibody combination can be stably expressed in patient-derived T lymphocytes, and will not be self-activated without being stimulated by PSCA-positive target cells, and has a better ability to eliminate tumor cells. Adoptive cell therapy for solid tumors.

Description

Humanized antibody for enhancing anti-PSCA chimeric antigen receptor activity and application thereof

technical field

The invention belongs to the field of genetic engineering, and relates to a humanized antibody for enhancing the activity of anti-PSCA chimeric antigen receptor and its application, and also relates to a chimeric antigen receptor for recognizing PSCA comprising the antibody.

Background technique

Chimeric antigen receptor (CAR) is an artificial receptor that mimics the function of TCR, and is composed of antigen recognition domain, hinge region, transmembrane region and intracellular signal domain in sequence. The intracellular signaling domain is usually CD3ζ chain or FcRγ, or linked to one or more co-stimulatory molecules, such as 4-1BB (CD137), CD28, ICOS (CD278). When the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal is transmitted into the cell through the hinge region and the transmembrane region, and the intracellular signal domain converts the signal into an activation signal, activating Effector cells, effector cells proliferate and produce cytokines to kill tumor cells.

In recent years, T lymphocyte chimeric antigen receptor (CAR-T) has shown significant therapeutic effects in tumor treatment, especially in the treatment of CD19-positive malignant tumors or leukemia; the use of CAR-T for solid tumor research is also continuing. However, the effect of most CAR-T in the treatment of solid tumors is not satisfactory. Compared with the ability to kill tumors in the treatment of CAR-T, safety and whether CAR-T can effectively recognize tumor cells are more important factors. The inventor team found two more suitable as targeted PSCA CARs through previous research. The murine antibody in the antigen recognition region has a high killing effect on PSCA-positive tumor cells at a high-efficiency target ratio, but the killing is weak at a low-efficiency target ratio; the inventor team also found that CAR-T of most mouse antibody combinations There is a possibility of self-activation of cells in normal culture in vitro, and the self-activation of CAR-T cells is also one of the key issues that threaten the safety of CAR-T. At present, there is no good solution for CAR-T self-activation. It has not been reported whether the choice of region or scFv or the degree of humanization will affect the self-activation of CAR-T cells, and whether self-activation is a major factor affecting the killing activity of CAR-T cells has not yet been confirmed.

To sum up, it is necessary for the treatment of CAR-T solid tumors to obtain more effective CAR and CAR-T by modifying scFv, and to seek a solution to reduce CAR-T self-activation.

Contents of the invention

In view of this, one of the objects of the present invention is to provide a humanized single-chain antibody that recognizes PSCA. The humanized single-chain antibody described in the present invention can enhance the target anti-PSCA CAR-T activity and reduce the risk of CAR-T cells. Self-activation, only under the stimulation of PSCA-positive target cells will it be activated to clear the target cells.

To achieve the above object, the technical solution of the present invention is:

The humanized single-chain antibody that recognizes PSCA, the amino acid sequence of the 6 FR regions of the light and heavy chains of the mouse anti-human PSCA monoclonal antibody is analyzed and compared with the IMGT/BLAST database, and the human antibody sequence with the highest homology is selected As a transformation template; perform PCR site-directed mutagenesis on the humanized transformation sequence.

Humanized antibody means that the variable region part of the antibody (ie VH and VL regions) or the antibody is entirely encoded by human antibody genes. Humanized antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body, and the form of humanized antibodies has gradually developed from chimeric antibodies and modified antibodies to humanized antibodies. However, the conventional humanized transformation method of CDR transplantation usually leads to the loss of the original antigen-binding activity of the transformed antibody. Therefore, it is necessary to repeatedly modify the key residues that affect antigen-antibody binding, and then detect the specificity and affinity of a large number of antigen-antibody binding. , so as to screen for active antibody sequences.

Therefore, one of the objectives of the present invention is to provide a humanized single-chain antibody that recognizes PSCA.

The light chain amino acid sequence of the humanized single-chain antibody (scFv) recognizing PSCA is shown in SEQ ID NO:22, and the heavy chain amino acid sequence of the humanized single-chain antibody is shown in SEQ ID NO:23.

Further, the amino acid sequence of the humanized antibody is shown in SEQ ID NO: 13.

Further, the nucleotide sequence of the humanized antibody is shown in SEQ ID NO:2.

The second object of the present invention is to provide the application of the humanized single-chain antibody in the preparation of medicaments for treating solid tumors.

Further, the solid tumor cells express PSCA.

Further, a chimeric antigen receptor combined with the humanized single-chain antibody is provided.

After a large number of CAR-T expression studies, the inventor team found that the selection of scFv and the hinge region (hinge region) are the key factors affecting the self-activation of CAR-T. How to design scFv so that it can better retain its affinity for the antigen Activity, how to choose the hinge sequence to combine with scFv for the best recognition of PSCA chimeric antigen receptor is a difficult problem. In the absence of more effective technical tips, the inventor team combined the scFv and humanized scFv of the currently reported murine anti-PSCA monoclonal antibody design by the exclusion method, and constructed CAR by combining with different hinge sequences (embedded However, only the chimeric antigen receptor that recognizes PSCA protected by the present invention has unexpected technical effects. The combination of CARs protected by the present invention can be stably expressed in patient-derived T lymphocytes after testing, and will not self-activate without being stimulated by PSCA-positive target cells and has better ability to eliminate tumor cells. Adoptive cell therapy for solid tumors.

Further, the chimeric antigen receptor is characterized in that the chimeric antigen receptor comprises a single-chain antibody that recognizes PSCA, a hinge region, a transmembrane region, and an intracellular signaling domain; the single-chain antibody that recognizes PSCA The amino acid sequence is shown in SEQ ID NO:13.

Further, the chimeric antigen receptor is characterized in that the amino acid sequence of the hinge region is shown in SEQ ID NO: 14 or SEQ ID NO: 15; the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 16 shown; the intracellular signaling domain is CD3ζ chain and/or FcRγ and/or CD28 and/or CD137 and/or CD278.

As a preference, the intracellular signaling domains are CD28, CD137 and CD3ζ in sequence.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:21 or SEQ ID NO:24.

The third object of the present invention is to provide a vector containing the above sequence, which is selected from lentiviral expression vectors, retroviral expression vectors, adenoviral expression vectors, adeno-associated virus expression vectors, DNA vectors, RNA vectors or plasmid.

The object of the present invention is also to provide a cell infected by the vector, which can be selected from: stem cells, T cells, NK cells, monocytes or macrophages.

Further, the object of the present invention is also to provide the application of said cells in the preparation of drugs for the treatment of solid tumors, characterized in that said solid tumor cells can express PSCA

In some embodiments, the solid tumor is prostate cancer, bladder cancer, cervical cancer.

In addition, returning to the origin of the invention, the purpose of the present invention is also to provide the application of the amino acid sequence shown in SEQ ID NO: 13 in the preparation of the CAR-T backbone.

In general, the humanized single-chain antibody provided by the present invention can enhance the effectiveness of CAR-T cells in killing tumors, and the CAR-T cells expressing the CAR of the scFv combination can not only maintain the CAR targeting PSCA in The positive rate in the patient's cell culture process can avoid the safety hazard caused by the self-activation of CAR-T cells after reinfusion into the human body, and at the same time can strengthen the proliferation and tumor killing ability of CAR-T, and is non-toxic to antigen-negative cells Side effects, can be used for targeted therapy of tumors.

The beneficial effects of the present invention are:

1) The humanized single-chain antibody provided by the present invention can specifically recognize PSCA and enhance the anti-PSCA chimeric antigen receptor activity.

2) The PSCA-recognizing chimeric antigen receptor comprising the humanized single-chain antibody of the present invention provided by the present invention can be more stably expressed in T lymphocytes and has a better ability to eliminate tumor cells, and can be used to prepare Adoptive cell therapy for solid tumors in drugs for the treatment of solid tumors.

3) The CAR-T cells obtained by recognizing the PSCA chimeric antigen receptor comprising the humanized single-chain antibody described in the present invention cannot activate and proliferate by themselves without stimulation and activation of target cells, and can maintain the chimeric antigen receptor targeting PSCA. The positive rate of combined antigen receptors in the patient's cell culture process is good in safety and high in effectiveness.

Description of drawings

Figure 1 Sequence of humanized scFv targeting human PSCA

Figure 2 Schematic diagram of different CAR structures.

Figure 3 Expression of PSCA in different tumor cells.

Figure 4 Detection of PSCA-CAR ability to transfect T lymphocytes.

Figure 5. CAR-T cells with different scFv combinations can kill tumor cells expressing PSCA to different degrees.

Figure 6 Detection of cytokine release after cell killing by CAR-T cells with different scFv combinations.

Detailed ways

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental method that does not indicate the specific conditions in the preferred embodiment is usually according to the conventional conditions, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.), or according to the conditions suggested by the manufacturer . The examples given are for better description of the content of the present invention, but the content of the present invention is not limited to the examples given. Therefore, non-essential improvements and adjustments to the implementation by those skilled in the art based on the content of the invention above still fall within the protection scope of the present invention.

This part of the experiment was obtained through the single-chain antibodies of two different monoclonal antibodies (different epitope peptides) in the "prior art", although theoretically "single-chain antibodies" can be used to prepare chimeric antigen receptors, However, not every single-chain antibody can be used to prepare chimeric antigen receptors, and the difference in the hinge region of chimeric antigen receptors also has a major impact on the function of chimeric antigen receptors. This requires the inventor to pay creative work to find a product with unexpected effects in many combinations of CARs.

Embodiment 1 Design the humanized single chain antibody of anti-human PSCA antigen

The amino acid sequences of the 6 FR regions of the light and heavy chains of the mouse anti-human PSCA monoclonal antibody were analyzed and compared through the IMGT/BLAST database, and the human antibody sequence with the highest homology was selected as the transformation template. PCR site-directed mutagenesis was performed on the humanized sequence, and four new modified scFVs were obtained after site-directed mutation. P-3h1 was selected for CAR structural modification. The amino acid sequence of P-3h1 is shown in Figure 1.

Example 2 Construction of chimeric antigen receptor virus containing scFv recognizing PSCA

In order to construct a more effective chimeric antigen receptor against PSCA, we used two existing murine antibodies to construct its scFv and designed four groups of CAR structures: PSCA1-G4h, PSCA1-8h, PSCA2-G4h, and PSCA2-8h. The p-3h1-G4h and P-3h1-8h CAR combination was constructed by deriving the transformed scFv. The CAR structure is shown in Figure 2.

Synthesis of Gene Sequences of Different Combinations of PSCA-targeting Chimeric Antigen Receptors

Synthesize different single-chain antibody scFvs containing leader peptide (also known as signal peptide) (abbreviated as LP), anti-human PSCA antigen, IgG4 hinge region (referred to as G4h) or CD8 hinge region (abbreviated as 8h), CD28 transmembrane region (referred to as TM), and CD28, CD137 and CD3 intracellular signal domains, wherein the nucleic acid sequence encoding the leader peptide is shown in SEQ ID NO: 9; the nucleic acid sequence of the single-chain antibody against human PSCA antigen is shown in SEQ ID NO: 1, SEQ ID NO: Shown in ID NO:2, SEQ ID NO:20; The nucleic acid sequence of the hinge region is shown in SEQ ID NO:3 or SEQ ID NO:4; The nucleic acid sequence of CD28 transmembrane region is shown in SEQ ID NO:5; Intracellular The nucleic acid sequences of the signal molecules are respectively shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. The CAR structure is shown in Figure 1.

Construction of Lentiviral Vectors Expressing Chimeric Antigen Receptors

The nucleic acid sequences of the primers were designed as shown in SEQ ID NO: 10 and SEQ ID NO: 11, and were synthesized by Nanjing GenScript Biotechnology Company.

Then use the above-mentioned sequences as primers and the above-mentioned synthetic chimeric antigen receptor sequences as templates for PCR amplification. The reaction system is loaded according to the instructions of KOD FX NEO DNA polymerase (purchased from TOYOBO company), and the amplified products are identified. Afterwards, DNA fragments were recovered with a recovery kit (Promega Company). The specific method was described in the instruction manual. Chimeric antigen receptors were recovered, and the recovered DNA fragments were sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing.

The gene sequence encoding the chimeric antigen receptor obtained by cloning was double digested, and the digestion reaction was carried out according to the instructions. After the digested product was separated by agarose gel electrophoresis, the DNA fragment was recovered with an agarose gel DNA fragment recovery kit, and then the target fragment and the vector fragment were connected by T4 ligase (purchased from Promega Company) to obtain the expression mosaic. Lentiviral vectors that bind antigen receptors. The lentiviral vector was transformed into Escherichia coli TOP10, a single clone was picked and cultured for 12 hours, and then the plasmid was extracted with a plasmid extraction kit (Invitrogen Company). For details, see the instructions.

Lentiviral Packaging

In this example, the calcium phosphate method was used to package the lentivirus. For specific steps, see Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.).

4 Purification of lentivirus

Collect the virus and centrifuge it at 3000r/min for 10min and then filter it. Centrifuge the filtrate at 3000r/min for 10min and put it into a new 50ml centrifuge tube; according to the amount of virus supernatant, use PEG6000 with a final concentration of 8.5% and 0.3M NaCl to settle the virus; Centrifuge for 30 min at 1/min, discard the supernatant, resuspend the virus in 200 μl DMEM medium, aliquot into 1.5 ml EP tubes, 40 μl per tube, and store at -80°C for later use. The obtained viruses were named as:

PSCA1-G4h, PSCA1-8h, PSCA2-G4h, PSCA2-8h, P-3h1-G4h and P-3h1-8h.

Lentivirus titer determination

293T cells were added with 1 μl polybrene (Polybrene) solution, and then 1 μl and 9 μl of the diluted virus solution were added to 293T cells respectively, and after 24 hours, the medium was replaced with DMEM medium containing 10% FBS (wt), and after 72 hours of infection, the The virus infection titer was calculated by detecting the expression of Protein-L by flow cytometry; Protein-L is a protein that can bind to the light chain of an antibody, and the expression of Protein-L molecules on the surface of infected cells can reflect the CAR containing single-chain antibodies on the surface of infected cells expression; calculation results showed that the virus titer was greater than 1×10 ⁸ TU/ml.

Example 3, different tumor cells express PSCA positive ratio detection

The tumor cells Hela, PC3, T24, and RT4 cultured to 10 days were collected into 10ml centrifuge tubes, centrifuged at 300g/min for 5min, and the supernatant was discarded to collect the cells; resuspended in PBS solution containing 1% fetal bovine serum cells, and adjust the density of the cells to 1×106/ml; the collected cells were aliquoted and labeled with anti-PSCA flow detection antibody, incubated at 4°C for 30 min, washed twice with PBS solution, and tested on a flow cytometer.

The results were shown in Figure 3, PC3, RT4, and Hela were PSCA-positive cells, and the expression positive rate increased sequentially, and T24 was PSCA-negative cells.

Example 4 Detection of PSCA-CAR transfection ability of T lymphocytes

1 Isolation of human peripheral blood mononuclear cells

Collect peripheral blood with anticoagulant-added blood collection tubes and divide into 50ml centrifuge tubes, add 7.5ml hydroxyethyl starch to dilute, then settle naturally at room temperature (18-25°C) for about 30min, collect the upper layer of plasma, and put the collected upper layer of plasma in Centrifuge at 1400rpm/min for 15min; then use gradient centrifugation to separate the lymphocyte layer, take the second layer of white lymphocyte layer, wash twice with normal saline, resuspend the cells, add RPMI 1640 complete medium containing 10% FBS and culture , to obtain human peripheral blood mononuclear cells.

lentiviral vector infection of T lymphocytes

The isolated human peripheral blood mononuclear cells were activated by anti-CD3 and anti-CD28 antibodies and then infected with the virus, and the uninfected T lymphocytes were used as blank control; after 24 hours, the medium was replaced with RPMI 1640 containing 500IU/ml recombinant human IL-2 Complete medium, continue to culture for 7-14 days. PSCA1-G4h, PSCA1-8h, PSCA2-G4h, PSCA2-8h, P-3h1-G4h and P-3h1-8h virus-infected T cells were named PSCA-CAR-1, PSCA-CAR-2, PSCA-CAR, respectively -3, PSCA-CAR-4, (P-3h1)-CAR-1 and (P-3h1)-CAR-2.

1) During the culture process, the positive rate of Protein-L was detected by flow cytometry on the cultured T cells infected with the virus, and the test results indicated the positive rate of expression of different PSCA-CAR combinations in T lymphocytes on the 10th day of culture.

The results are shown in Figure 4. Except for the low positive rate of PSCA-CAR-4 transfected T lymphocytes, other PSCA-CARs were highly expressed in T cells, among which PSCA-CAR-2, (P-3h1)-CAR -1 and (P-3h1)-CAR-2 CAR positive rates were higher.

Example 5. Effect of Virus Infection of CAR-T Cells on Tumor Cell Killing Ability

The killing ability of different scFv chimeric antigen receptors to target cells was completed by ACEA xCELLigence RTCA MP instrument, and the experimental steps were carried out according to the instrument manual; on the first day, the target cells (tumor cells expressing CEA) were treated with 2-5*10 ⁴ per The wells are plated in the 96-well plate equipped with the instrument, and the tumor cells attached to the bottom of the wells are recorded every 15 minutes with the resistance index as the data. After 24 hours, the corresponding CAR-T cells are plated in each well according to the pre-designed effect-target ratio. After the CAR-T cells were plated, the resistance index was recorded every 15 minutes, and the proliferation or death of the adherent target cells was judged by the resistance index. The formula for analyzing the results by resistance index is: CAR-T cell killing rate = baseline resistance index - real-time resistance index.

The experimental results are shown in Figure 5: the 24-hour CAR-T killing results at low-efficiency target ratios of 1:1 and 1:2 show that (P-3h1)-CAR-1 and (P-3h1)-CAR-2 have better killing Effect, especially at 1:2 effect-to-target ratio, only (P-3h1)-CAR-1 and (P-3h1)-CAR-2 had killing effect on PSCA expressing cells, and (P-3h1)- The amino acid sequences of CAR-1 and (P-3h1)-CAR-2 are respectively shown in SEQ ID NO:21 or SEQ ID NO:24. The 24-hour killing percentage statistics of CAR-T cells with an effect-to-target ratio of 1:2 and 1:1 are shown in Table 1 and Table 2:

Table 1 Effect-to-target ratio 1:2 Different PSCA-CAR-T kill tumor cell percentage

Table 2 Effect-to-target ratio 1:1 Different PSCA-CAR-T kill tumor cell percentage

Example 6 Detection of cytokine release after cell killing by CAR-T cells with different scFv combinations

Cytokine IFN-γ was detected using the method of Elisa and kits from BD Company. Detection kit product number: 555142 Production batch number 6266958, see kit instructions for specific steps.

Measurement: measure the absorbance (OD value) of each well sequentially at a wavelength of 450nm with a blank air conditioner, and the measurement should be carried out within 15 minutes after adding the stop solution.

The results are shown in Figure 6, showing that the effect-to-target ratio is 1:1. After 24 hours of killing target cells, CAR-T cells use CD8 as the hinge sequence such as: composed of chimeric antigen receptors such as PSCA1-8h or PSCA2-8h to kill PSCA positive Tumor cells release higher IFN-γ (IFN-γ can also be written as IFN-g), and the expression level of IFN-γ reflects the degree of activation and killing ability of CAR-T cells. The statistical results are shown in Table 3

Table 3 IFN-g secretion of different PSCA-CAR-T cells after killing tumor target cells for 24 hours (in pg/ml)

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Sequence Listing

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   Chongqing Institute of Precision Bioindustry Technology Co., Ltd.

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<210> 13

<211> 247

<212> PRT

<213> Artificial

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Thr Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Leu Lys Leu Asn Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Lys

115 120 125

Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser

145 150 155 160

Trp Val Arg Gln Ala Pro Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile

165 170 175

His Asn Gly Gly Gly His Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met

195 200 205

Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Arg

210 215 220

Met Tyr Tyr Gly Asn Ser His Trp Tyr Phe Asp Val Trp Gly Ala Gly

225 230 235 240

Thr Ser Val Thr Val Ser Ser

245

<210> 14

<211> 47

<212> PRT

<213> Artificial

<400> 14

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 15

<211> 229

<212> PRT

<213> Artificial

<400> 15

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 16

<211> 27

<212> PRT

<213> Artificial

<400> 16

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 17

<211> 41

<212> PRT

<213> Artificial

<400> 17

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 18

<211> 44

<212> PRT

<213> Artificial

<400> 18

Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

1 5 10 15

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

20 25 30

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

35 40

<210> 19

<211> 111

<212> PRT

<213> Artificial

<400> 19

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

1 5 10 15

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

20 25 30

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

35 40 45

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

50 55 60

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

65 70 75 80

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

85 90 95

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 20

<211> 748

<212> DNA

<213> Artificial

<400> 20

gacatccagg atatccagat gacccagagc ccaagctccc tgtctgccag cgtgggcgac 60

agggtgacca tcacatgccg cacatcccag gatatctcta actacctgaa ttggtatcag 120

cagaagcccg gcaaggcccc taagctgctg atctactata ccctgaagct gaacagcgga 180

gtgccctccc ggttctccgg atctggcagc ggcaccgact tcacctttac aatctctagc 240

ctgcagcccg aggatatcgc cacatactat tgccagcagt ccaagaccct gccttggaca 300

tttggcggcg gcaccaaggt ggagatcaag ggctccacat ctggaagcgg caagcctgga 360

tctggagagg gaagcaccaa gggacaggtg aagctgcagg agagcggagg aggactggtg 420

cagccaggag gctccctgaa gctgtcttgc gtggccagcg gcttcacctt ttcctcttac 480

acaatgtcct gggtgcggag aacaccagag aagagactgg agtgggtggc ctatatccac 540

aacggcggcg gccacaccta ctatcccgac acaatcaagg gccggttcac catcagcaga 600

gataacgcca agaatacact gtttctggag atgagctccc tgaagtccga ggacaccgcc 660

atgtactatt gtacaaggcg catgtactat ggcaattctc actggtactt cgacgtgtgg 720

ggagcaggca cctccgtgac agtgtctc 748

<210> 21

<211> 699

<212> PRT

<213> Artificial

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Thr Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Leu Lys Leu Asn Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Lys

115 120 125

Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser

145 150 155 160

Trp Val Arg Gln Ala Pro Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile

165 170 175

His Asn Gly Gly Gly His Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met

195 200 205

Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Arg

210 215 220

Met Tyr Tyr Gly Asn Ser His Trp Tyr Phe Asp Val Trp Gly Ala Gly

225 230 235 240

Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro

245 250 255

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

260 265 270

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

275 280 285

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

290 295 300

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

305 310 315 320

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

325 330 335

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

340 345 350

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

355 360 365

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

370 375 380

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

385 390 395 400

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

405 410 415

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

420 425 430

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

435 440 445

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

450 455 460

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Phe Trp Val Leu

465 470 475 480

Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val

485 490 495

Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His

500 505 510

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

515 520 525

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

530 535 540

Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

545 550 555 560

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

565 570 575

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

580 585 590

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

595 600 605

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

610 615 620

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

625 630 635 640

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

645 650 655

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

660 665 670

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

675 680 685

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

690 695

<210> 22

<211> 107

<212> PRT

<213> Artificial

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Thr Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Leu Lys Leu Asn Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 122

<212> PRT

<213> Artificial

<400> 23

Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile His Asn Gly Gly Gly His Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Met Tyr Tyr Gly Asn Ser His Trp Tyr Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 24

<211> 517

<212> PRT

<213> Artificial

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Thr Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Leu Lys Leu Asn Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Lys

115 120 125

Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser

145 150 155 160

Trp Val Arg Gln Ala Pro Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile

165 170 175

His Asn Gly Gly Gly His Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met

195 200 205

Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Arg

210 215 220

Met Tyr Tyr Gly Asn Ser His Trp Tyr Phe Asp Val Trp Gly Ala Gly

225 230 235 240

Thr Ser Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg

245 250 255

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

260 265 270

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

275 280 285

Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val

290 295 300

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

305 310 315 320

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

325 330 335

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

340 345 350

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Arg Gly Arg Lys

355 360 365

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

370 375 380

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Glu

385 390 395 400

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

405 410 415

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

420 425 430

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

435 440 445

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

450 455 460

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

465 470 475 480

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

485 490 495

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

500 505 510

Ala Leu Pro Pro Arg

515

Claims (10)

1. The humanized single-chain antibody that recognizes PSCA is characterized in that, the light chain amino acid sequence of described humanized single-chain antibody is as shown in SEQ ID NO:22, and the heavy chain amino acid of described humanized single-chain antibody The sequence is shown in SEQ ID NO:23. 2. The humanized single-chain antibody of claim 1, wherein the amino acid sequence of the humanized single-chain antibody is as shown in SEQ ID NO:13. 3. The application of the humanized single-chain antibody described in any one of claims 1-2 in the preparation of a drug for the treatment of solid tumors, the solid tumor cells express PSCA, and the solid tumors are prostate cancer, bladder cancer , Cervical cancer. 4. A chimeric antigen receptor of the humanized single-chain antibody combination according to any one of claims 1-2. 5. The chimeric antigen receptor of claim 4, characterized in that, the chimeric antigen receptor comprises a single-chain antibody recognizing PSCA, a hinge region, a transmembrane region and an intracellular signaling domain; the recognition PSCA The amino acid of the single chain antibody is shown in SEQ ID NO:13. 6. The chimeric antigen receptor of claim 5, wherein the amino acid sequence of the hinge region is as shown in SEQ ID NO:14 or SEQ ID NO:15; the amino acid sequence of the transmembrane region is as SEQ ID Shown in NO:16; the intracellular signaling domain is CD3ζ chain and/or FcRγ and/or CD28 and/or CD137 and/or CD278. 7. The chimeric antigen receptor according to claim 6, wherein the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:21 or SEQ ID NO:24. 8. A vector containing the nucleotide sequence encoding the humanized single-chain antibody according to any one of claims 1-2, or the chimeric antigen receptor according to any one of claims 4-7, said vector It is selected from lentiviral expression vectors, adenoviral expression vectors, and adeno-associated virus expression vectors. 9. The vector-infected cell of claim 8, which may be selected from the group consisting of stem cells, T cells, NK cells, monocytes or macrophages. 10. the application of the cell described in claim 9 in the medicine that is used for the preparation treatment solid tumor, is characterized in that, the cell of described solid tumor can express PSCA, and described solid tumor is prostate cancer, bladder cancer, cervical cancer .

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