CN110964112B

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

Info

Publication number: CN110964112B
Application number: CN201811158683.8A
Authority: CN
Inventors: 张巍; 单娟娟; 赵永春; 徐艳敏; 黄霞; 赵文旭; 陈军; 张茜真
Original assignee: Chongqing Jingzhun Biological Industrial Technology Institute Co ltd; Chongqing Precision Biotech Co ltd
Current assignee: Chongqing Jingzhun Biological Industrial Technology Institute Co ltd; Chongqing Precision Biotech Co ltd
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2023-05-02
Anticipated expiration: 2038-09-30
Also published as: CN110964112A

Abstract

The invention belongs to the field of genetic engineering, and in particular relates to a humanized antibody for enhancing the activity of an anti-PSCA chimeric antigen receptor and application thereof. The humanized antibodies of the invention recognize PSCA and are capable of enhancing anti-tumor activity of anti-PSCA chimeric antigen receptors. The chimeric antigen receptor combined by the humanized antibody can be stably expressed in T lymphocytes from patients, does not cause self-activation phenomenon without being stimulated by PSCA positive target cells, has better capability of eliminating tumor cells, and is used for adoptive cell therapy for solid tumors.

Description

Humanized antibody for enhancing activity of anti-PSCA chimeric antigen receptor 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 an anti-PSCA chimeric antigen receptor and application thereof, and a PSCA-recognizing chimeric antigen receptor comprising the antibody.

Background

Chimeric antigen receptor (chimeric antigen receptor, CAR) is an artificial receptor that mimics TCR function, consisting of antigen recognition domain, hinge region, transmembrane region and intracellular signaling domain linked in sequence. The intracellular signaling domain is typically a CD3 zeta chain or FcRgamma, or is linked to one or more costimulatory molecules, such as 4-1BB (CD 137), CD28, ICOS (CD 278). When an antigen (receptor) on the surface of a tumor cell is combined with an antibody (ligand) of a chimeric antigen receptor, a signal is transmitted into cells through a hinge region and a transmembrane region, and the intracellular signal domain converts the signal into an activation signal to activate effector cells, and the effector cells proliferate and produce cytokines so as to kill the tumor cells.

In recent years, T lymphocyte chimeric antigen receptor (CAR-T) has demonstrated significant therapeutic effects in tumor treatment, especially in the treatment of CD19 positive malignant tumors or leukemia; research into solid tumors using CAR-T is also gaining attention, but most CAR-T is not satisfactory in treating solid tumors. Compared with the capability of killing tumor in the treatment of CAR-T, the safety and the capability of effectively recognizing tumor cells of the CAR-T are more important factors, and the inventor team discovers 2 murine antibodies which are more suitable for being targeted to PSCA CAR antigen recognition areas through early-stage researches, and the murine antibodies have higher killing effect on PSCA positive tumor cells in the high-efficiency target ratio and weaker killing effect in the low-efficiency target ratio; the inventor team also found that most of the CAR-T cells combined with the murine antibody have the possibility of self-activation in normal culture in vitro, but the problem of self-activation of the CAR-T cells is one of the key problems threatening the safety of the CAR-T, no good solution is provided for the CAR-T self-activation at present, whether the selection or the humanization degree of a hinge region or an scFv has an influence on the CAR-T cell self-activation is not reported, and whether the self-activation is a big factor influencing the killing activity of the CAR-T cell is not confirmed.

In view of the foregoing, a solution to reduce CAR-T self-activation was sought to achieve more potent CARs and CAR-T by engineering scFv, which is necessary for CAR-T solid tumor treatment.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a humanized single-chain antibody recognizing PSCA, which can enhance the target anti-PSCA CAR-T activity, reduce the CAR-T cell self-activation, and activate and clear target cells only upon PSCA positive target cell stimulation.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

the humanized single-chain antibody for recognizing PSCA is prepared by using 6 sections of FR region amino acid sequences of the light chain and heavy chain of a monoclonal antibody of mouse anti-human PSCA, performing sequence analysis and comparison by using an IMGT/BLAST database, and selecting a human antibody sequence with highest homology as a reconstruction template; PCR site-directed mutagenesis was performed on the humanized altered sequence.

Humanized antibodies refer to antibodies in which the variable region portions (i.e., VH and VL regions) or the antibodies are encoded entirely by human antibody genes. Humanized antibodies can greatly reduce the immune side effects of heterologous antibodies on the human body, and the forms of humanized antibodies are also gradually developed from original chimeric antibodies, remodelled antibodies and the like to humanized antibodies. However, the conventional humanized modification method of CDR grafting generally causes the modified antibody to lose the original antigen binding activity, so that repeated modification of key residues affecting the antigen-antibody binding is required, and then a large number of antigen-antibody binding specificities and affinity detection are carried out, so that the antibody sequence with activity is obtained through screening.

Accordingly, it is an object of the present invention to provide a humanized single-chain antibody recognizing PSCA.

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

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

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

The second purpose of the invention is to provide the application of the humanized single-chain antibody in preparing medicines for treating solid tumors.

Further, the solid tumor cells express PSCA.

Further, chimeric antigen receptors combined by the humanized single chain antibodies are provided.

The inventor groups find that the selection of scFv and hinge region (hinge region) are key factors influencing the self-activation of the CAR-T through a large number of CAR-T expression researches, and how to design the scFv so as to better retain the affinity activity of the scFv to antigen and how to select the hinge sequence to be combined with the scFv to optimally recognize PSCA chimeric antigen receptor is a difficult problem. The inventors group screened scFv and humanized scFv designed by the presently reported monoclonal antibodies against PSCA by exclusion without more effective technical cues by constructing CARs (chimeric antigen receptors) in combination with different range sequences; but the chimeric antigen receptor recognizing PSCA, which is protected by the present invention, has unexpected technical effects. The combination mode of the CAR can be stably expressed in T lymphocytes from patients after test, does not cause self-activation phenomenon after PSCA positive target cell stimulation, has better capability of eliminating tumor cells, and is used for adoptive cell therapy for solid tumors.

Further, the chimeric antigen receptor is 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 amino acid sequence of the PSCA-recognizing single-chain antibody is shown as SEQ ID NO. 13.

Further, the chimeric antigen receptor is characterized in that the amino acid sequence of the hinge region is shown as SEQ ID NO. 14 or SEQ ID NO. 15; the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 16; the intracellular signaling domain is a CD3 zeta chain and/or fcrγ and/or CD28 and/or CD137 and/or CD278.

Preferably, the intracellular signaling domains are CD28, CD137 and cd3ζ in that order.

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

It is a further object of the present invention to provide a vector comprising the above sequence, wherein the vector is selected from the group consisting of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector and a plasmid.

It is also an object of the present invention to provide a vector-infected cell as described, which can be selected from the group consisting of: stem cells, T cells, NK cells, monocytes or macrophages.

Further, the invention also aims at providing the application of the cells in preparing medicines for treating solid tumors, which is characterized in that the cells of the solid tumors 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 invention also aims at providing the application of the amino acid sequence shown in SEQ ID NO. 13 in preparing a CAR-T framework.

In general, the humanized single-chain antibody provided by the invention can enhance the effectiveness of killing tumors by the CAR-T cells, and the CAR-T cells expressing the CAR combined by the scFv can maintain the positive rate of the target PSCA in the cell culture process of patients, can avoid potential safety hazards caused by self-activation after the CAR-T cells are infused back into the human body, can enhance the proliferation and the tumor killing capacity of the CAR-T, has no toxic or side effect on antigen-negative cells, and can be used for targeted treatment of tumors.

The invention has the beneficial effects that:

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

2) The PSCA chimeric antigen receptor containing the humanized single-chain antibody provided by the invention can be expressed in T lymphocytes more stably, has better capability of eliminating tumor cells, and can be used for adoptive cell therapy for solid tumors in preparation of medicines for treating the solid tumors.

3) The CAR-T cells obtained by recognizing the PSCA chimeric antigen receptor and containing the humanized single-chain antibody provided by the invention can not be automatically activated and proliferated without target cell stimulation and activation, and can maintain the positive rate of the PSCA-targeted chimeric antigen receptor in the cell culture process of patients, and the safety is good and the effectiveness is high.

Drawings

FIG. 1 sequence targeting human PSCA humanized scFv

Figure 2 is a schematic diagram of a different CAR structure.

FIG. 3 PSCA expression in different tumor cells.

FIG. 4 PSCA-CAR transfection T lymphocyte ability assay.

Figure 5 CAR-T cells combined with different scFv kill tumor cells expressing different degrees of PSCA.

Figure 6 detection of cytokine release after CAR-T cell killing of different scFv combinations.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental methods in the preferred embodiments, in which specific conditions are not noted, are generally carried out according to conventional conditions, for example, those described in the guidelines for molecular cloning experiments (third edition, J. Sam Brookfield et al), or according to the manufacturer's recommendations. The examples are presented for better illustration of the present invention, but are not intended to limit the scope of the present invention to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

This part of the experiment was performed on single chain antibodies of two different monoclonal antibodies (different epitope peptides) in the "prior art", and although in theory "single chain antibodies" could be used to prepare chimeric antigen receptors, not every single chain antibody could actually be used to prepare chimeric antigen receptors, and the differences in the hinge regions that make up the chimeric antigen receptor would also have a significant effect on the function of the chimeric antigen receptor. This requires the inventors to exercise creative efforts to find products with unexpected effects in the combination of numerous CARs.

EXAMPLE 1 design of humanized Single-chain antibodies against human PSCA antigen

The 6-segment FR area amino acid sequences of the light chain and the heavy chain of the monoclonal antibody of the mouse anti-human PSCA antigen are subjected to sequence analysis and comparison by an IMGT/BLAST database, and the sequence of the human antibody with highest homology is selected as a reconstruction template. And carrying out PCR site-directed mutagenesis on the humanized modified sequence, obtaining 4 new modified scFVs after site-directed mutagenesis, and selecting P-3h1 for CAR structural modification, wherein the amino acid sequence of the 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 aiming at PSCA, two existing murine antibodies are utilized to construct scFv thereof, four groups of CAR structures of PSCA1-G4h, PSCA1-8h, PSCA2-G4h and PSCA2-8h are designed, and humanized modified scFv is utilized to construct a combination of P-3h1-G4h and P-3h1-8h CAR. The CAR structure is shown in fig. 2.

Synthesis of different combinations of gene sequences targeting the chimeric antigen receptor of PSCA

Synthesizing different single-chain antibody scFv sequentially containing leader peptide (also called signal peptide) (called LP for short) and anti-human PSCA antigen, igG4 hinge region (called G4h for short) or CD8 hinge region (called 8h for short), CD28 transmembrane region (called TM for short), and CD28, CD137 and CD3 intracellular signal domain, wherein the nucleic acid sequence for encoding the leader peptide is shown as SEQ ID NO 9; the nucleic acid sequence of the single-chain antibody of the anti-human PSCA antigen is shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 20; the nucleic acid sequence of the hinge region is shown as SEQ ID NO. 3 or SEQ ID NO. 4; the nucleic acid sequence of the CD28 transmembrane region is shown as SEQ ID NO. 5; the nucleic acid sequences of intracellular signal molecules are shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 respectively. The CAR structure is shown in fig. 1.

Construction of lentiviral vectors expressing chimeric antigen receptors

The primer nucleic acid sequences are shown as SEQ ID NO. 10 and SEQ ID NO. 11, and are synthesized by Nanjing Jinsri Biotech company.

Then, PCR amplification is carried out by taking the sequences as primers and the synthesized chimeric antigen receptor sequences as templates, a reaction system is used for loading according to the instruction of KOD FX NEO DNA polymerase (purchased from TOYOBO company), the amplified products are identified, then DNA fragments are recovered by a recovery kit (Promega company), the specific method is shown in the instruction, the chimeric antigen receptor is obtained by recovery, and the DNA recovered fragments are sent to Nanjing Jinsri biotechnology company for sequencing.

The gene sequence of the chimeric antigen receptor obtained by cloning is subjected to double enzyme digestion, and enzyme digestion reaction is carried out according to instructions. The digested product is separated by agarose gel electrophoresis, DNA fragment recovery is carried out by using an agarose gel DNA fragment recovery kit, and then the target fragment and the carrier fragment are connected by T4 ligase (purchased from Promega company) to obtain the lentiviral vector for expressing the chimeric antigen receptor. The lentiviral vector was transformed into E.coli TOP10, and after 12h of selection and culture, the plasmid was extracted by a plasmid extraction kit (Invitrogen company) as described in the specification.

Packaging of lentiviruses

The lentivirus is packaged by adopting a calcium phosphate method, and specific steps are shown in molecular cloning experimental guidelines (third edition, J. Sam Brookfield et al).

4 purification of lentiviruses

Collecting virus, centrifuging for 10min at 3000r/min, filtering, and centrifuging for 10min at 3000r/min to obtain a new 50ml centrifuge tube; according to the virus supernatant, PEG6000 and 0.3M NaCl with the final concentration of 8.5% are used for settling the virus; then, the virus was resuspended in 200. Mu.l of DMEM medium and the supernatant was centrifuged at 5000r/min for 30min at 4℃and the virus was kept for further use at-80℃by sub-packaging with 1.5ml of EP tubes, 40. Mu.l of each tube. The prepared viruses are respectively named as follows:

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

Lentivirus titer assay

293T cells were added with 1. Mu.l of Polybrene (Polybrene) solution, then 1. Mu.l and 9. Mu.l of diluted virus solution were added to 293T cells, respectively, and 24 hours later, the virus infection titer was estimated by detecting Protein-L expression by a flow method after 72 hours of infection with DMEM medium containing 10% FBS (wt); protein-L is a Protein capable of binding to an antibody light chain, and the expression of a CAR containing a single-chain antibody on the surface of an infected cell can be reflected by detecting the expression of a Protein-L molecule on the surface of the infected cell; the calculation results show that the virus titer is greater than1× 10 ⁸ TU/ml。

Example 3 detection of the positive proportion of PSCA expressed by different tumor cells

Tumor cells Hela, PC3, T24 and RT4 cultured for 10 days are respectively collected into a 10ml centrifuge tube, 300g/min and centrifuged for 5min, and the supernatant is discarded to collect the cells; resuspend cells with PBS solution containing 1% fetal calf serum by volume fraction and adjust the density of cells to 1X 106 cells/ml; and (3) after the collected cells are subpackaged, labeling an anti-PSCA flow detection antibody, incubating for 30min at 4 ℃, washing for 2 times by using a PBS (phosphate buffered saline) solution, and detecting by using an up-flow cytometer.

As shown in FIG. 3, PC3, RT4 and Hela were PSCA-positive cells, the expression positive rate was increased in sequence, and T24 was PSCA-negative cell.

Example 4 PSCA-CAR transfection T lymphocyte Capacity assay

1. Isolation of human peripheral blood mononuclear cells

Collecting peripheral blood by a blood collecting tube added with an anticoagulant, sub-packaging the peripheral blood in a 50ml centrifuge tube, adding 7.5ml of hydroxyethyl starch for dilution, naturally settling for about 30min at room temperature (18-25 ℃), collecting upper plasma, and centrifuging the collected upper plasma for 15min at 1400 rpm/min; then separating lymphocyte layer by gradient centrifugation, collecting second white lymphocyte layer, washing with physiological saline for 2 times, re-suspending cells, and culturing in RPMI 1640 complete medium containing 10% FBS to obtain human peripheral blood mononuclear cells.

Infection of T lymphocytes with lentiviral vectors

Isolated human peripheral blood mononuclear cells are activated by anti-CD 3 and anti-CD 28 antibodies and then subjected to viral infection, and uninfected T lymphocytes are used as a blank; after 24h, the medium was changed to RPMI 1640 complete medium containing 500IU/ml recombinant human IL-2 and the culture was continued for 7-14 days. PSCA1-G4h, PSCA1-8h, PSCA2-G4h, PSCA2-8h, P-3h1-G4h and P-3h1-8h virus infected T cells were designated PSCA-CAR-1, PSCA-CAR-2, PSCA-CAR-3, PSCA-CAR-4, (P-3 h 1) -CAR-1 and (P-3 h 1) -CAR-2, respectively.

1) And detecting the positive rate of Protein-L by using flow cytometry on the T cells infected with the cultured viruses in the culture process, wherein the detection result shows that different PSCA-CAR combinations express the positive rate on T lymphocytes on the 10 th day of culture.

The results are shown in FIG. 4, where PSCA-CAR-2, (P-3 h 1) -CAR-1 and (P-3 h 1) -CAR-2 CAR were positive at a higher rate, except that PSCA-CAR-4 transfected T lymphocytes were low in positive rate, and other PSCA-CARs were highly expressed in T cells.

Example 5 effect of Virus-infected CAR-T cells on tumor cell killing Capacity

The killing capacity of chimeric antigen receptors of different scfvs on target cells is completed by a ACEA xCELLigence RTCA MP instrument, and experimental steps are carried out according to the instruction manual of the instrument; target cells (CEA expressing tumor cells) were treated at 2-5 x 10 on day one ⁴ Each well is plated in a 96-well plate equipped with an instrument, tumor cells attached to the bottom of the well are recorded every 15 minutes by taking the resistance index as data, corresponding CAR-T cells are plated in each well according to a pre-designed effective target ratio after 24 hours, the resistance index is recorded every 15 minutes after the CAR-T cells are plated in, and proliferation or death of the attached target cells is judged through the resistance index. The formula of the analysis result by using the resistance index is as follows: CAR-T cell killing = baseline resistance index-real-time resistance index.

The experimental results are shown in fig. 5: at a low efficiency target ratio of 1: the results of killing by CAR-T in 1 and 1:2 for 24 hours show that (P-3 h 1) -CAR-1 and (P-3 h 1) -CAR-2 have better killing effect, especially only (P-3 h 1) -CAR-1 and (P-3 h 1) -CAR-2 have killing effect on PSCA expression cells when killing for 24 hours at 1:2 effective target ratio, and the amino acid sequences of (P-3 h 1) -CAR-1 and (P-3 h 1) -CAR-2 are shown as SEQ ID NO:21 or SEQ ID NO:24 respectively. The statistics of the percent 24 hour killing of CAR-T cells at effective target ratios of 1:2 and 1:1 are shown in tables 1 and 2:

TABLE 1 effective target ratio 1:2 different PSCA-CAR-T killer tumor cell percentages

Table 2 effective target ratio 1:1 different PSCA-CAR-T killing tumor cell percentages

Example 6 detection of cytokine release after CAR-T cell killer cells with different scFv combinations

Cytokine IFN-gamma detection was performed using the Elisa method using the BD company kit. Detection kit cargo number: 555142, production lot 6266958, and specific steps are shown in the instruction book of the kit.

And (3) measuring: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with blank air-conditioner zero, and the measurement was performed within 15 minutes after the addition of the stop solution.

The results are shown in FIG. 6, showing that after killing target cells for 24 hours with a 1:1CAR-T cell effective target ratio, CD8 as a range sequence as follows: the chimeric antigen receptor such as PSCA1-8h or PSCA2-8h releases higher IFN-gamma when killing PSCA positive tumor cells (IFN-gamma can also be written as IFN-g), and the IFN-gamma expression level reflects the activation degree and killing capacity of CAR-T cells. The statistical results are shown in Table 3

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

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

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Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser

85 90 95

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

100 105 110

Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser

115 120 125

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

130 135 140

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

145 150 155 160

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

165 170 175

Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe

180 185 190

Gln Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

195 200 205

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

210 215 220

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

225 230 235 240

<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

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

1. The humanized single-chain antibody for recognizing PSCA is characterized in that the light chain amino acid sequence of the humanized single-chain antibody is shown as SEQ ID NO. 22, and the heavy chain amino acid sequence of the humanized single-chain antibody is shown as 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 shown in SEQ ID No. 13.

3. Use of the humanized single-chain antibody of any one of claims 1-2 for the preparation of a medicament for the treatment of solid tumors, said solid tumor cells expressing PSCA, said solid tumors being prostate cancer, bladder cancer, cervical cancer.

4. The chimeric antigen receptor of the humanized single chain antibody combination of any one of claims 1-2.

5. The chimeric antigen receptor of claim 4, wherein the chimeric antigen receptor comprises a single chain antibody that recognizes PSCA, a hinge region, a transmembrane region, and an intracellular signaling domain; the amino acid of the PSCA-recognizing single-chain antibody is shown as SEQ ID NO. 13.

6. The chimeric antigen receptor according to claim 5, wherein the amino acid sequence of the hinge region is shown as SEQ ID NO. 14 or SEQ ID NO. 15; the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 16; the intracellular signaling domain is a CD3 zeta 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 shown as SEQ ID NO. 21 or SEQ ID NO. 24.

8. A vector comprising a nucleotide sequence encoding the humanized single chain antibody of any one of claims 1-2, or the chimeric antigen receptor of any one of claims 4-7, said vector being selected from the group consisting of a lentiviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector.

9. The vector-infected cell of claim 8, which is selected from the group consisting of: stem cells, T cells, NK cells, monocytes or macrophages.

10. Use of the cells of claim 9 for the preparation of a medicament for the treatment of solid tumors, wherein the cells of solid tumors are capable of expressing PSCA, and wherein the solid tumors are prostate cancer, bladder cancer, cervical cancer.