Fully human single-chain antibody against PSCA and application thereof

Abstract

The present application belongs to the technical field of cellular immunization engineering, and particularly relates to a polypeptide specifically recognizing PSCA and application thereof. The polypeptide provided by the present application specifically recognizes PSCA and has good affinity performance. The polypeptide specifically targeting PSCA provided by the present application can mark PSCA positive cells, combine with PSCA antigen and PSCA positive cells, and can be used as a detection reagent for PSCA molecules and an in-vivo tracing labeling reagent. The polypeptide specifically targeting PSCA provided by the present application can also be used in an extracellular antigen recognition region of a chimeric antigen receptor, and can be used in an immune cell treatment drug targeting PSCA to kill malignant tumors expressing PSCA. The polypeptide specifically recognizing PSCA of the present application is derived from a natural full human antibody, and compared with a mouse-derived antibody, a chimeric antibody and a humanized antibody, the immunogenicity is greatly reduced, and the safety can be maximally ensured in clinical application.

Description

Technical Field

[0001] This invention belongs to the field of tumor cell immunotherapy technology, specifically relating to a fully human ScFv that targets PSCA and its applications. Background Technology

[0002] Chimeric antigen receptors (CARs) are artificial receptors that mimic the function of TCRs. CAR molecules generally contain an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signaling domain. The intracellular signaling domain is usually a CD3ζ chain or FcRγ, or linked to one or more co-stimulatory molecules, such as 4-1BB, CD28, ICOS (CD278).

[0003] PSCA (prostate stem cell antigen) is a glycosylphosphatidylinositol (GPI)-linked cell membrane antigen expressed only in the prostate and bladder in normal tissues. Overexpression is highly observed in most prostate tumors, bladder cancer, pancreatic cancer, non-small cell lung cancer, and gastric cancer, making it a promising therapeutic target for solid tumors. Given the current challenges of poor treatment outcomes and prognoses for solid tumors, immunotherapy targeting PSCA may represent a breakthrough for the future treatment of prostate cancer and various PSCA-expressing solid tumors.

[0004] The extracellular antigen recognition domain, also known as the extracellular antigen-binding domain, specifically recognizes tumor surface antigens. CAR molecules utilize this antigen-binding domain to specifically recognize tumor antigens, transmitting immune cell activation signals through the hinge region and transmembrane region. The most common extracellular antigen recognition domain employs ScFv (single-chain antibody) that recognizes tumor antigens. The selection of ScFv is crucial for the effective functioning of CARs; therefore, for immunotherapies targeting PSCA, screening for anti-PSCA antibodies or anti-PSCA ScFvs is essential. Summary of the Invention

[0005] In view of this, the present invention aims to provide a fully humanized single-chain antibody targeting PSCA and its use in detection reagents and antitumor drugs. The ScFv provided by the present invention can specifically recognize PSCA, identify and detect the expression of PSCA in cells and its proportion, and can be used as a PSCA detection reagent or an in vivo tracer marker molecule. The ScFv provided by the present invention can act as the antigen recognition domain of a chimeric antigen receptor to recognize PSCA on the tumor surface, constructing chimeric antigen receptor immune cells, which can effectively inhibit tumor growth in vivo and show significant anti-PSCA target tumor effects. Furthermore, it can serve as an effective component of antitumor drugs targeting malignant tumors with high PSCA expression, effectively controlling the proliferation of malignant tumors with high PSCA expression.

[0006] The antigen-binding polypeptide targeting PSCA comprises a heavy chain variable region and a light chain variable region. The light chain variable region comprises L-CDR1, L-CDR2, and L-CDR3. The amino acid sequence of L-CDR1 is QGILTY, the amino acid sequence of L-CDR2 is AAS, and the amino acid sequence of L-CDR3 is LQDYNFPRT. The amino acid sequence of L-CDR1 is GYTFTDYY, the amino acid sequence of L-CDR2 is INPYNGGT, and the amino acid sequence of L-CDR3 is ARSVYDYPFDY.

[0007] The antigen-binding polypeptide targeting PSCA comprises a heavy chain variable region and a light chain variable region. The light chain variable region comprises L-CDR1, L-CDR2, and L-CDR3, as shown in the amino acid sequences SEQ ID NO. 2-4. The heavy chain variable region comprises H-CDR1, H-CDR2, and H-CDR3, as shown in the amino acid sequences SEQ ID NO. 5-7.

[0008] The antigen-binding polypeptide targeting PSCA, the light chain variable region of the single-chain antibody is shown in SEQ ID NO:8.

[0009] The antigen-binding polypeptide targeting PSCA has a heavy chain variable region as shown in SEQ ID NO:9.

[0010] The single-chain antibody that recognizes PSCA comprises a heavy chain variable region and a light chain variable region, wherein the sequence of the light chain variable region is shown in SEQ ID NO:8 or a variant thereof, and the sequence of the heavy chain variable region is shown in SEQ ID NO:9 or a variant thereof.

[0011] In some specific embodiments, the method for preparing antigen-binding peptides targeting PSCA involves constructing a fully human single-chain antibody library using phage display technology. Through affinity panning using appropriate methods, phages carrying a specific affinity fragment are enriched, and their gene sequences are obtained through sequencing. To screen for human antibodies targeting PSCA antigens, a fully human single-chain antibody library was constructed. Using normal human PBMCs as raw materials, total RNA was extracted and reverse-transcribed into cDNA. The antibody variable region was amplified, and the heavy chain variable region VH was linked to the light chain variable region VL using a linker. This was then incorporated into a phage vector, electroporated into a display strain, and an antibody library was constructed. The library was panned using PSCA extracellular antigens, and the selected clones were identified, ultimately yielding ScFvs that specifically bind to PSCA antigens. For constructing chimeric antigen receptors or other applications, ScFvs can be artificially synthesized by PCR to obtain a large number of PSCA-targeting ScFvs, i.e., extracellular domains including the amino acid sequence shown in SEQ ID NO:1 or its functional variants.

[0012] Furthermore, the antigen-binding polypeptide targeting PSCA also contains a linker sequence. In some embodiments, the linker amino acid sequence is GSTGSGSGKPGSGEGSTKG or (GGGGS)n, where n=1-5.

[0013] Furthermore, the antigen-binding polypeptide targeting PSCA also contains a linker sequence, preferably the sequence shown in SEQ ID NO:10.

[0014] An antibody that recognizes PSCA, said antibody comprising the CDR of SEQ ID NO:2-7.

[0015] An antibody that recognizes PSCA may also contain light and heavy chain sequences of SEQ ID NO:8 and SEQ ID NO:9.

[0016] Another objective of this invention is to provide a chimeric antigen receptor that targets PSCA.

[0017] The chimeric antigen receptor includes an extracellular antigen recognition domain capable of recognizing the PSCA antigen, the extracellular antigen recognition domain recognizing the PSCA antigen comprising the CDR of sequences SEQ ID NO:2-7; in some embodiments, the extracellular antigen recognition domain recognizing the PSCA antigen may also comprise the light and heavy chain sequences of SEQ ID NO:8 and SEQ ID NO:9; in some embodiments, the extracellular antigen recognition domain recognizing the PSCA antigen comprises the amino acid sequence as shown in SEQ ID NO:1; in some embodiments, the extracellular antigen recognition domain recognizing the PSCA antigen comprises the linker sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0018] The chimeric antigen receptor further includes a hinge region, a transmembrane region, and an intracellular recognition domain. The hinge may be derived from human IgG4, human CD8, human CD4, human CD28, human CD7, etc.

[0019] Furthermore, the hinge region preferably includes one of the following optional amino acid sequences or a functional variant thereof: SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.25, which can enable the obtained chimeric antigen receptor to have a better cell-killing effect.

[0020] Furthermore, the transmembrane region may originate from: α, β or chains of T cell receptors, CD8, CD28, CD3ε, CD4, CD16, CD137, CD80, and CD86, etc.

[0021] The transmembrane region preferably comprises one of the following optional amino acid sequences or a functional variant thereof: SEQ ID NO.11, SEQ ID NO.21.

[0022] The chimeric antigen receptor further includes an intracellular signaling domain, which comprises a signal transduction region and a co-stimulatory domain; the intracellular signaling domain comprises a signal transduction region derived from a primary cytoplasmic signal transduction sequence containing ITAM, including but not limited to protein molecules such as CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS (CD278), FcεRI, CD66d, DAP10, and DAP12.

[0023] The signal transduction region is derived from CD3ζ, and its amino acid sequence is shown in SEQ ID NO.16.

[0024] The chimeric antigen receptor further includes an intracellular signaling domain, which comprises a signal transduction region and a co-stimulatory domain; the co-stimulatory domain may be derived from: CD28, 41BB, OX40, CD27, DAP10, 2B4 (SLAMF4, CD244), CD3γ, CD3δ, FcεRI CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signal transduction lymphocyte activation molecules (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, LFA-1 (CD11a / CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), etc.

[0025] The co-stimulatory domain comprises one or more of the following optional amino acid sequences or one or more functional variants thereof: SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.19.

[0026] In some specific embodiments, the hinge region, transmembrane region, and intracellular signaling domain of the chimeric antigen receptor include one of the following optional combinations:

[0027] Combination 1): The hinge region comprises the amino acid sequence shown in SEQ ID NO.12 or a functional variant thereof, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO.11 or a functional variant thereof, and the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO.13 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO.16 or a functional variant thereof;

[0028] Combination 2): The hinge region comprises the amino acid sequence shown in SEQ ID NO.25 or a functional variant thereof, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO.21 or a functional variant thereof, and the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO.14 or a functional variant thereof, the amino acid sequence shown in SEQ ID NO.13 or a functional variant thereof, and the amino acid sequence shown in SEQ ID NO.16 or a functional variant thereof;

[0029] Combination 3): The hinge region contains the amino acid sequence shown in SEQ ID NO.17 or a functional variant thereof, the transmembrane region contains the amino acid sequence shown in SEQ ID NO.11 or a functional variant thereof, and the intracellular signaling domain contains the amino acid sequence shown in SEQ ID NO.13 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO.16 or a functional variant thereof;

[0030] Combination 4): The hinge region contains the amino acid sequence shown in SEQ ID NO.25 or a functional variant thereof, the transmembrane region contains the amino acid sequence shown in SEQ ID NO.11 or a functional variant thereof, and the intracellular signaling domain contains the amino acid sequence shown in SEQ ID NO.13 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO.16 or a functional variant thereof;

[0031] Combination 5): The hinge region contains the amino acid sequence shown in SEQ ID NO.18 or a functional variant thereof, the transmembrane region contains the amino acid sequence shown in SEQ ID NO.21 or a functional variant thereof, and the intracellular signaling domain contains the amino acid sequence shown in SEQ ID NO.14 or a functional variant thereof and the amino acid sequence shown in SEQ ID NO.16 or a functional variant thereof.

[0032] The chimeric antigen receptor described above further comprises a CD8 leader peptide sequence or a functional variant thereof. The PSCA-targeting antibody is localized to the cell membrane under the guidance of the CD8 leader peptide. As is known to those skilled in the art, all other signal peptides that can be used in the CAR or antibody fields can be used in this invention, and the extracellular segment generally comprises a CD8α or GM-CSFRα leader peptide.

[0033] The present invention aims to provide a nucleic acid sequence encoding the aforementioned chimeric antigen receptor.

[0034] In some embodiments, the nucleic acid sequence is used as the target gene and combined with a vector to transfect immune cells to obtain engineered immune cells.

[0035] The nucleic acid sequence of the chimeric antigen receptor includes the nucleic acid sequence of an antigen-binding polypeptide targeting PSCA, as shown in SEQ ID NO.39.

[0036] Furthermore, the nucleic acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.41.

[0037] Furthermore, the chimeric antigen receptor also includes a promoter, the nucleic acid sequence of which is shown in SEQ ID NO.44 or SEQ ID NO.45.

[0038] Furthermore, the nucleic acid sequence of the chimeric antigen receptor may also be as shown in SEQ ID NO.42 or SEQ ID NO.43.

[0039] Another objective of this invention is to provide a polynucleotide vector.

[0040] Furthermore, the expression vector is selected from any one of lentiviral expression vectors, retroviral expression vectors, adenovirus expression vectors, adeno-associated virus expression vectors, DNA vectors, RNA vectors, and plasmids.

[0041] In some embodiments, the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0042] In some embodiments, the vector comprises a left (5') retroviral LTR, a Psi(Ψ) packaging signal, a central polypurine segment / DNA flap (cPPT / FLAP), a retroviral export element, a promoter operatively linked to a polynucleotide encoding a CAR covered by the present invention, and a right (3') retroviral LTR.

[0043] The aforementioned polynucleotide vector contains a TIGIT mutant nucleotide sequence as shown in any of the sequences SEQ ID NO.37, SEQ ID NO.38, and SEQ ID NO.39.

[0044] In some embodiments, the TIGIT mutant contained in the polynucleotide vector described above can also be linked to the transmembrane region of human CD28, the intracellular signaling region of human CD28, and the CD3ζ signaling region to form a TIGIT mutant fusion protein.

[0045] A further objective of this invention is to provide a multi-target CAR structure comprising a nucleotide sequence targeting a second chimeric antigen receptor that is not a PSCA target; wherein the second chimeric antigen receptor target is CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, or CDH17. Any of the following targets: Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), CD155, or TAG-72.

[0046] The multi-target CAR structure described herein includes an extracellular antigen recognition region in the nucleotide sequence of the second chimeric antigen receptor, as shown in SEQ ID NO. 37, SEQ ID NO. 38, and SEQ ID NO. 39.

[0047] In some embodiments, the chimeric antigen receptor or multi-target CAR that targets PSCA described above includes a hepatitis B virus post-transcriptional regulatory element (HPRE) or a prairie dog post-transcriptional regulatory element (WPRE) and an optimized prairie dog post-transcriptional regulatory element (oPRE).

[0048] In some embodiments, the nucleic acid sequence encoding the aforementioned chimeric antigen receptor comprises an optimized Kozark sequence.

[0049] In some embodiments, the gene expression vector may contain a secreted anti-PD-1 ScFv; in some embodiments, the gene expression vector may contain a PD-1 conjugated transducing peptide (such as a PD-1-CD28-CD137-CD3 signaling structure); in some embodiments, the gene expression vector may contain multiple CAR combinations, such as two CAR combinations targeting different antigens or different recognition sites of the same antigen.

[0050] The present invention also aims to provide an engineered cell that expresses a polypeptide or single-chain antibody comprising any one of SEQ ID NO. 1-9, or a chimeric antigen receptor comprising any one of the foregoing, or the cell comprising a nucleic acid or vector as described in any one of the foregoing.

[0051] The engineered cells also express fusion proteins.

[0052] Furthermore, the fusion protein is TIGIT or a functional variant thereof.

[0053] Furthermore, the amino acid sequence of the expressed TIGIT or its functional variant protein is as shown in any one of SEQ ID NO.22, SEQ ID NO.23, or SEQ ID NO.24.

[0054] Furthermore, the expressed TIGIT fusion protein has the structure of TIGIT-28TM-28, TIGIT-8TM-134, or TIGIT-8TM-BB, where 28TM and 8TM are transmembrane structures derived from human CD28 and human CD8, respectively, and 28 and 134 are intracellular signaling structures derived from human CD28 and human CD134, respectively.

[0055] The engineered cells also include chimeric antigen receptors targeting non-PSCA targets, which are tumor surface antigens, including but not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), CD155, or TAG-72, or any combination of multiple targets.

[0056] Furthermore, the non-PSCA target is CD155.

[0057] Furthermore, the chimeric antigen receptor targeting CD155 recognizes the extracellular antigen recognition region of CD155 as TIGIT and its mutated functional proteins.

[0058] Furthermore, the chimeric antigen receptor targeting CD155 recognizes the extracellular antigen recognition region sequence of CD155 as shown in any one of SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24.

[0059] The engineered cells mentioned in any of the preceding claims are immune cells such as T cells, T cell precursors, NK cells, DC cells, and macrophages.

[0060] Preferably, the immune cells are T lymphocytes.

[0061] The present invention aims to further provide a cell product comprising the engineered cells described in any of the preceding embodiments.

[0062] Furthermore, the cell product also includes other active agents that can enhance CAR expression activity.

[0063] In some specific embodiments, the active agent is an immunosuppressant such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506; antibodies or other immunoablative agents such as CAMPATH; anti-CD3 antibodies or other antibody therapy; cyclophosphamide; fludarabine; cyclosporin; FK506; rapamycin; mycophenolic acid; steroids; FR901228; cytokines; and radiation.

[0064] In some specific embodiments, the active agent that enhances the activity of CAR-expressing cells can be an active agent that blocks inhibitory molecules. Inhibitory molecules such as PD1 can reduce the ability of CAR-expressing cells to initiate immune effector responses in some embodiments. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFRβ), and TGFRβ. The extracellular domain of the inhibitory molecule can be fused to the transmembrane domain and the intracellular signal transduction domain, such as PD1 CAR.

[0065] Furthermore, a variety of other therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab, pembrolizumab, pidilizumab, and atezolizumab.

[0066] Specifically, other therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib, ofa tumuma b, rituximab, bevacizumab, trastuzumab, imatinib, cetuximab, and panitumumab. Catuximab, ibritumomab, oflamumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitini b, pazopanib, sunitinib Sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, pona tini b, rad otini b, bosutini b Lestaurtinib, ruxolitinib, pacritinib, cobimetinib, simetinibSelumetinib, Trametinib, Binimetinib, Alectinib, Ceritinib, Crizotinib, Aflibercept, Adipotide, and Dinni-interleukin.

[0067] Furthermore, the cell products can also be used in combination with some treatment methods, such as surgery, chemotherapy, and radiation.

[0068] The present invention aims to further provide a pharmaceutical composition comprising any of the aforementioned polypeptides or single-chain antibodies, a chimeric antigen receptor nucleic acid sequence, an expression vector, or an engineered cell as described above.

[0069] The present invention aims to provide the use of any of the preceding polypeptides or single-chain antibodies, chimeric antigen receptors or nucleic acid sequences of chimeric antigen receptors or expression vectors or engineered cells or cell products or pharmaceutical compositions in a tumor drug targeting PSCA.

[0070] Specifically, the antitumor drugs include, but are not limited to, drugs for malignant solid tumors such as drugs for renal cancer, prostate cancer, thymic cancer, ovarian cancer, glioma, pancreatic cancer, bladder cancer, nasopharyngeal carcinoma, gastric cancer, and colorectal cancer.

[0071] In this invention, the term "functional variant" generally refers to an amino acid sequence that has substantially the same function as the chimeric antigen receptor (e.g., may possess the properties of the chimeric antigen receptor) and has at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity. In some embodiments, the variant of the amino acid sequence has substantially the same function.

[0072] The beneficial effects of this invention are as follows:

[0073] The ScFv provided by this invention can specifically recognize PSCA, identify and detect the PSCA expressed in cells and its proportion, and can be used as a PSCA detection reagent or in vivo tracer marker molecule.

[0074] The ScFv provided by this invention can recognize PSCA on the tumor surface as the antigen recognition domain of a chimeric antigen receptor, constructing chimeric antigen receptor immune cells that can effectively inhibit tumor growth in vivo and have a significant effect against tumors expressing PSCA targets. Furthermore, it can serve as an effective component of anti-tumor drugs targeting malignant tumors with high PSCA expression, effectively controlling the proliferation of such malignant tumors. Attached Figure Description

[0075] Figure 1 The expression of PSCA in HT1376-Luc-GFP.

[0076] Figure 2 shows the in vitro functional validation of different CAR-T cells constructed targeting PSCA ScFv in malignant tumors such as pancreatic cancer, prostate cancer, and bladder cancer; among which... Figure 2A This describes the in vitro killing effect of CAR1 in three cell models. Figure 2B The in vitro killing effect of CAR2 in PC3-PSCA-Luc-GFP; Figure 2C The in vitro killing effect of three CARs on HPAC-Luc-GFP was investigated.

[0077] Figure 3 shows the in vivo functional validation of different CAR-T structures constructed targeting PSCA ScFv in malignant tumors such as pancreatic cancer, prostate cancer, and bladder cancer; among which... Figure 3A The in vivo efficacy of CAR1 in the HPAC-Luc-GFP (pancreatic cancer) peritoneal tumorigenesis model; Figure 3B The in vivo efficacy of CAR1 in the HT1376-Luc-GFP (bladder cancer) peritoneal tumor model; Figure 3C The in vivo efficacy of CAR2 in a PC3-PSCA-Luc-GFP (prostate cancer) peritoneal tumorigenesis model.

[0078] Figure 4 shows the functional validation of dual-target CAR-T; where Figure 4A The in vitro killing effect of two CARs in two cell models; Figure 4B In vitro killing effects of four CARs in three cell models.

[0079] Figure 5 shows the functional validation of the CAR-coupled fusion protein targeting PSCA; Figure 5A The in vitro killing effect of CAR16 and CAR17 on two types of target cells; Figure 5B The in vitro killing effect of four CARs in three cell models is shown.

[0080] Figure 6 shows the recognition and detection of PSCA target by the screened single-chain antibodies; Figure 6AThe recognition status of PSCA antigen for clone numbers ScFv of AK-24-A08, AK-25-B11, AK-25-D02, and AK25-H08; Figure 6B The recognition status of PSCA antigen for clone number ScFv of AK-25-D03. Detailed Implementation

[0081] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0082] In this embodiment of the invention, the method for preparing a fully human antibody targeting PSCA is as follows:

[0083] (1) Construction of a fully human single-chain antibody library

[0084] PBMCs were separated using Ficoll separation solution. The Ficoll separation solution was slowly added to collected normal human blood, ensuring a clear separation interface between the Ficoll separation solution and the normal human blood. A 50mL centrifuge tube containing the blood and separation solution was centrifuged at approximately 15°C for 20 minutes. After centrifugation, the liquid surface separated into four layers: an upper layer of plasma mixture, a lower layer of red blood cells and granulocytes, and a middle layer of Ficoll liquid. At the boundary between the upper and middle layers, there was a narrow band of white, cloudy layer dominated by PBMCs, which was the PBMC cell layer. The separated PBMCs were obtained by aspirating them using a new sterile Pasteur pipette.

[0085] Total RNA was extracted using conventional methods and reverse transcribed into cDNA. Based on the similarity of the heavy and light chain germline gene sequences, degenerate primers were designed at both ends of the variable region (Li Xiaolin, Construction and Preliminary Screening of a Large-Capacity Non-Immune Human Fab Phage Antibody Library, Master's Thesis, Peking Union Medical College, June 2007). PCR was used to obtain the heavy chain and light chain variable region gene fragments of the antibody. The ScFv nucleic acid fragment was amplified using conventional overlap PCR (PCR method referenced from *Molecular Cloning: A Laboratory Manual* (3rd Edition), Joe Sambrook and David Russell, Science Press, USA). The ScFv nucleic acid fragment was ligated into the phage vector pComb3xss, and the product was transformed into TGI strains using an electroporator to obtain a fully human single-chain antibody library.

[0086] (2) Preparation of phage-displayed fully human single-chain antibody library

[0087] The bacterial culture was revived in fresh LB liquid medium. VSCM13 helper phages were added at a VCSM13:bacteria ratio of 50:1, and the mixture was thoroughly mixed and incubated on a shaker. The culture was centrifuged, the supernatant was discarded, and the precipitate was resuspended in ampicillin- and kanamycin-resistant SOB medium and incubated overnight. The bacterial culture was centrifuged at 8000 rpm for 20 min at 4°C, the supernatant was collected, and 1 / 5 volume of 20% PEG 8000 2.5 mmol / L NaCl solution was added. The mixture was incubated on ice for 1 hour, then centrifuged at 12000 rpm for 30 min at 4°C. The precipitated phages were resuspended in PBS and filtered through a 0.22 μm filter.

[0088] (3) PSCA antigen screening

[0089] PSCA protein with Fc tag is co-incubated with proteinG magnetic beads to prepare PSCA-proteinG coupled magnetic beads. The coupled magnetic beads are then extracted into the phage panning of the prepared fully human single-chain antibody library. After 3-4 rounds of panning, washing and elution, specific monoclonal antibodies against the antigen can be enriched.

[0090] (4) Screening of positive clones

[0091] After the initial selection, the final batch of monoclonal plaques were chosen for ELISA screening. The test results are as follows: Figure 1 As shown, phage clones that bind to PSCA antigen were obtained, and ScFv targeting PSCA were obtained through panning, which have the specific binding ability to human PSCA antigen.

[0092] In this embodiment of the invention, the specific implementation process of antigen screening is as follows:

[0093] (1) Blocking: 5% skim milk powder was dissolved in PBS, filtered and used as blocking solution. The phage and CD70-protein G-conjugated magnetic beads were resuspended in an appropriate amount of blocking solution and mixed by rolling.

[0094] (2) Co-incubation: Place the PSCA-proteinG coupled magnetic beads in a magnetic rack, discard the supernatant, resuspend the magnetic beads with phage, and incubate by rolling.

[0095] (3) Cleaning: Place the magnetic bead phage mixture in a magnetic rack, discard the supernatant and add cleaning solution to clean the magnetic beads. The number of cleaning cycles is determined according to the number of different panning wheels.

[0096] (4) Elution: Place the cleaned magnetic beads on a magnetic rack, aspirate the supernatant, add Gly-HCl 4, mix well, incubate at room temperature for 7 min, add about Tris-HCl to adjust the pH to near neutral, and finally place the mixture on a magnetic rack and transfer the phage supernatant to a new 1.5 mL EP tube to complete one round of panning;

[0097] (5) Enrichment: Inoculate the phage into the TG1 bacterial solution for infection, centrifuge after quiescence, resuspend the precipitate, spread it on a 2YTAG plate, and incubate it upside down overnight;

[0098] (6) Washing the plates: Wash off the bacterial plaques from the overnight culture plates to use as seed culture for the next round of library packaging.

[0099] In this embodiment of the invention, the specific implementation process of ELISA detection is as follows:

[0100] (1) Coating: Dilute PSCA antigen to 1 μg / mL with carbonate coating buffer, add 100 μL / well to a 96-well plate, and seal overnight at 4°C;

[0101] (2) Obtaining phage samples: Centrifuge the overnight cultured phage monoclonal recombinant bacterial solution for 10 min and take the supernatant as the test sample;

[0102] (3) Blocking: After washing the antigen-coated plate three times with PBS on a plate washer, add skim milk blocking solution to each well and block at 37°C;

[0103] (4) Antibody incubation: After washing the plate 3 times with a plate washer, add the recombinant monoclonal bacterial solution of the phage to be tested to each well and incubate at 37°C;

[0104] (5) Add secondary antibody: Wash the plate 3 times with a plate washer, add 100 μL of 1:5000 Anti-M13-HRP secondary antibody to each well, and incubate at 37℃;

[0105] (6) Color development: Wash the plate 6 times with a plate washer, add color development solution to each well, and let it stand at room temperature in the dark for 25 minutes to develop color;

[0106] (7) Termination: Add H2SO4 to each well to terminate the reaction;

[0107] (8) Detection: Place the detection plate in an ELISA reader to detect OD. 450 A phage clone with an absorbance 2.5 times higher than the negative control is considered a positive clone.

[0108] In this embodiment of the invention, the sequence of the carrier structural elements is shown in Table 1 below; 8h / 7h / 8hdc / G4H are different hinge structures; 8TM / 28TM are different transmembrane structures; 28 / BB / CD134 are different intracellular signaling structures; Z and z both refer to the CD3ζ primary signal transduction domain derived from CD3.

[0109] In this embodiment of the invention, PSCA (9) is ScFv AK-25-D03 targeting PSCA, and TIGIT (mut5), TIGIT (mut2), and TIGIT (mut6) are mutated TIGIT peptides. Table 1. Sequence of carrier structural elements 1 PSCA(9) DIVMTQSPSSLSASVGDRVTVTCRASQGILTYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNFPRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGEVQLQQSGPEVKKPGASVKVSCKASGYTFTDYYMNWVRQMHGKGLEWMGVINPYNGGTDYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSVYDYPFDYWGQGTLVTVSS 2 L - CDR1 QGILTY 3 L - CDR2 AAS 4 L - CDR3 LQDYNFPRT 5 H - CDR1 GYTFTDYY 6 H - CDR2 INPYNGGT 7 H - CDR3 ARSVYDYPFDY 8 PSCA(9)VL DIVMTQSPSSLSASVGDRVTVTCRASQGILTYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNFPRTFGQGTKLEIKR 9 PSCA(9)VH EVQLQQSGPEVKKPGASVKVSCKASGYTFTDYYMNWVRQMHGKGLEWMGVINPYNGGTDYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSVYDYPFDYWGQGTLVTVSS 10 Linker GSTSGSGKPGSGEGSTKG 11 CD8TM IYIWAPLAGTCGVLLLSLVITLYC 12 8h(CD8 - derived Hinge) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 13 4 - 1BB intracellular signal KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 14 CD28 intracellular signal RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 15 CD27 intracellular signal YSCPREEEGSTIPIQEDYRKPEPACSP 16 CD3ζ RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 17 7h APPRASALPAPPTGSALPDPQTASALPDPPAASALP 18 G4h ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 19 CD134 intracellular signal ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 20 P2A GSGATNFSLLKQAGDVEENPGP 21 CD28TM FWVLVVVGGVLACYSLLVTVAFIIFWV 22 TIGIT (mut5) MTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAIWNADLGWHISPSFKDRVAPGPGLDLTLQSLTVNDTGEYFCVYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP 23 TIGIT (mut2) MTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAIWNADLGWHIPPSFKDRVAPGPGLGLTLQSLTVNDTGEYSCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP 24 TIGIT (mut6) MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAIWNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP 25 8h (dc) KPTTTPAPRPPTPAPTIASQPLSLRPEARPAAGGAVHTRGLDFAD 26 AK-15-H02 L-CDR1 QSISNY 27 AK-15-H02 L-CDR3 QQLYRYPLT 28 AK-15-H02 H-CDR1 ​ 29 ​ ​ 30 ​ ​ 31 ​ DIQMTQSPSSLSSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLYRYPLTFGGGTKVDIKR 32 AK-15-H02 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDSIWLQHPLFDYWGQGTLVTVSS 33 CAR9 DIVMTQSPSSLSASVGDRVTVTCRASQGILTYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNFPRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGEVQLQQSGPEVKKPGASVKVSCKASGYTFTDYYMNWVRQMHGKGLEWMGVINPYNGGTDYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSVYDYPFDYWGQGTLVTVSSLETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRVDGSGATNFSLLKQAGDVEENPGPTSMALPVTALLLPLALLLHAARPMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAIWNADLGWHIPPSFKDRVAPGPGLGLTLQSLTVNDTGEYSCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPEFFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQSQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 34 PSCA(9)-8h-8TM-BBZ DIVMTQSPSSLSASVGDRVTVTCRASQGILTYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNFPRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGEVQLQQSGPEVKKPGASVKVSCKASGYTFTDYYMNWVRQMHGKGLEWMGVINPYNGGTDYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSVYDYPFDYWGQGTLVTVSSLETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* 35 CAR5 DIVMTQSPSSLSASVGDRVTVTCRASQGILTYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNFPRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGEVQLQQSGPEVKKPGASVKVSCKASGYTFTDYYMNWVRQMHGKGLEWMGVINPYNGGTDYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSVYDYPFDYWGQGTLVTVSSLEKPTTTPAPRPPTPAPTIASQPLSLRPEARPAAGGAVHTRGLDFADEFFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* 36 PSCA(9) gacatcgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccgtcacttgccgggccagtcagggcattctcacttatttagcctggtatcagcaaaaaccagggaaagcccctaacctcctgatctatgctgcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggctctggcacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtctacaagattacaatttccctcggacgttcggccaagggaccaagctggagatcaaacgtggcagcacaagcggaagcggcaaaccaggaagcggagaaggaagcaccaagggagaggtgcagctgcagcagtccggaccagaggtgaagaagccaggagccagcgtgaaggtgtcctgtaaggcctctggctacaccttcacagattactatatgaactgggtgcggcagatgcacggcaagggactggagtggatgggcgtgatcaacccatacaatggcggcaccgattataatcagaagtttaagggcagagtgaccatcacagccgacaagtccacctctacagcctacatggagctgagctccctgaggagcgaggacacagccgtgtactattgtgcccgctccgtgtacgactatccctttgattattggggccagggaaccctggtcaccgtctcctca 37 TIGIT(mut5) atgacaggcacaatagaaacaacggggaacatttctgcagagaaaggtggctctatcatcttacaatgtcacctctcctccaccacggcacaagtgacccaggtcaattgggagcagcaggaccagcttctggccatttggaatgctgacttggggtggcacatctccccatccttcaaggatcgagtggccccaggtcccggcctggacctcaccctccagtcgctgaccgtgaacgatacaggggagtacttctgcgtctatcacacctaccctgatgggacgtacactgggagaatcttcctggaggtcctagaaagctcagtggctgagcacggtgccaggttccagattcca 38 TIGIT(mut2) atgacaggcacaatagaaacaacggggaacatttctgcagagaaaggtggctctatcatcttacaatgtcacctctcctccaccacggcacaagtgacccaggtcaattgggagcagcaggaccagcttctggccatttggaatgctgacttggggtggcacatccccccatccttcaaggatcgagtggccccaggtcccggcctgggcctcaccctccagtcgctgaccgtgaacgatacaggggagtactcctgcatctatcacacctaccctgatgggacgtacactgggagaatcttcctggaggtcctagaaagctcagtggctgagcacggtgccaggttccagattcca 39 TIGIT(mut6) atgatgacaggcacaatagaaacaacggggaacatttctgcagagaaaggtggctctatcatcttacaatgtcacctctcctccaccacggcacaagtgacccaggtcaactgggagcagcaggaccagcttctggccatttggaatgctgacttggggtggcacatctccccatccttcaaggatcgagtggccccaggtcccggcctgggcctcaccctccagtcgctgaccgtgaacgatacaggggagtacttctgcatctatcacacctaccctgatgggacgtacactgggagaatcttcctggaggtcctagaaagctcagtggctgagcacggtgccaggttccagattcca 40 CAR9 atggctctgccagtgacagctctgctgctgcctctggctctgctgctgcacgcagctagacccgacatcgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccgtcacttgccgggccagtcagggcattctcacttatttagcctggtatcagcaaaaaccagggaaagcccctaacctcctgatctatgctgcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggctctggcacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtctacaagattacaatttccctcggacgttcggccaagggaccaagctggagatcaaacgtggcagcacaagcggaagcggcaaaccaggaagcggagaaggaagcaccaagggagaggtgcagctgcagcagtccggaccagaggtgaagaagccaggagccagcgtgaaggtgtcctgtaaggcctctggctacaccttcacagattactatatgaactgggtgcggcagatgcacggcaagggactggagtggatgggcgtgatcaacccatacaatggcggcaccgattataatcagaagtttaagggcagagtgaccatcacagccgacaagtccacctctacagcctacatggagctgagctccctgaggagcgaggacacagccgtgtactattgtgcccgctccgtgtacgactatccctttgattattggggccagggaaccctggtcaccgtctcctcactcgagaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgtcgacggaagcggagctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctactagcatggctctgccagtgacagctctgctgctgcctctggctctgctgctgcacgcagctagacccatgacaggcacaatagaaacaacggggaacatttctgcagagaaaggtggctctatcatcttacaatgtcacctctcctccaccacggcacaagtgacccaggtcaattgggagcagcaggaccagcttctggccatttggaatgctgacttggggtggcacatccccccatccttcaaggatcgagtggccccaggtcccggcctgggcctcaccctccagtcgctgaccgtgaacgatacaggggagtactcctgcatctatcacacctaccctgatgggacgtacactgggagaatcttcctggaggtcctagaaagctcagtggctgagcacggtgccaggttccagattccagaattcttctgggtgctggtcgtggtgggtggcgtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtgaggagcaagcggagcagaggcggccacagcgactacatgaacatgaccccccggaggcctggccccacccggaagcactaccagccctacgcccctcccagggacttcgccgcctaccggagccgggtgaagttcagccggagcgccgacgcccctgcctaccagcagggccagagccagctgtacaacgagctgaacctgggccggagggaggagtacgacgtgctggacaagcggagaggccgggaccctgagatgggcggcaagccccggagaaagaaccctcaggagggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggcggaggggcaagggccacgacggcctgtaccagggcctgagcaccgccaccaaggatacctacgacgccctgcacatgcaggccctgccccctcgctaa 41 PSCA(9)-8h-8TM-BBZ atggctctgccagtgacagctctgctgctgcctctggctctgctgctgcacgcagctagacccgacatcgagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcatttccagccatttaaattggtatcagcagaaatcagggaaagcccctaagctcctgatctatgctgcgtccagtttgcaaggtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtcccccgtggacgttcggccaagggaccaagctggaactcaaacgtggcagcacaagcggaagcggcaaaccaggaagcggagaaggaagcaccaagggagaagtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagataccatgatagtagcaaatgcttttgatatctggggccaagggacaacggtcaccgtctcctcgctcgagaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 42 CAR2 atcccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagatctggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcactaggctagcatggctctgccagtgacagctctgctgctgcctctggctctgctgctgcacgcagctagacccgacatcgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccgtcacttgccgggccagtcagggcattctcacttatttagcctggtatcagcaaaaaccagggaaagcccctaacctcctgatctatgctgcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggctctggcacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtctacaagattacaatttccctcggacgttcggccaagggaccaagctggagatcaaacgtggcagcacaagcggaagcggcaaaccaggaagcggagaaggaagcaccaagggagaggtgcagctgcagcagtccggaccagaggtgaagaagccaggagccagcgtgaaggtgtcctgtaaggcctctggctacaccttcacagattactatatgaactgggtgcggcagatgcacggcaagggactggagtggatgggcgtgatcaacccatacaatggcggcaccgattataatcagaagtttaagggcagagtgaccatcacagccgacaagtccacctctacagcctacatggagctgagctccctgaggagcgaggacacagccgtgtactattgtgcccgctccgtgtacgactatccctttgattattggggccagggaaccctggtcaccgtctcctcactcgagaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 43 CAR1 ggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctactaatacgactcactatagggagaccaccgctagcatggctctgccagtgacagctctgctgctgcctctggctctgctgctgcacgcagctagacccgacatcgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccgtcacttgccgggccagtcagggcattctcacttatttagcctggtatcagcaaaaaccagggaaagcccctaacctcctgatctatgctgcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggctctggcacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtctacaagattacaatttccctcggacgttcggccaagggaccaagctggagatcaaacgtggcagcacaagcggaagcggcaaaccaggaagcggagaaggaagcaccaagggagaggtgcagctgcagcagtccggaccagaggtgaagaagccaggagccagcgtgaaggtgtcctgtaaggcctctggctacaccttcacagattactatatgaactgggtgcggcagatgcacggcaagggactggagtggatgggcgtgatcaacccatacaatggcggcaccgattataatcagaagtttaagggcagagtgaccatcacagccgacaagtccacctctacagcctacatggagctgagctccctgaggagcgaggacacagccgtgtactattgtgcccgctccgtgtacgactatccctttgattattggggccagggaaccctggtcaccgtctcctcactcgagaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa 44 Common promoter ggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctact 45 Hypoxic promoter atcccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagatctggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcactag

[0110] Example 1: ScFv sequence specifically recognizing PSCA

[0111] Using the fully human antibody preparation method described above, the ScFv sequences shown in Table 2 below were obtained through screening:

[0112] Table 2 Single-chain antibodies targeting PSCA: AK-25-B11 <![CDATA[AIRLTQSPSSLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIS RAS NLQNGVPSRFSGSGSETLFTLTVTSLQPEDFATYSC QQYYSYPIT FGPGTKLEIKG]]> <![CDATA[QIQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC ARDLPFTMEDGMDV WGQGTTVTVSS <!-- 14 -->]]> AK-25-D02 <![CDATA[DIQMTQSPSSLSTSVGDRVTITCRAS QSIGTY LNWYQQKPGKAPNLLIF AAS TLQTGVPSRFSGSGSGTDFTLTISNLQPEDFATYHCQQ SYSTPFT FGQGTKVDIKR]]> <![CDATA[EVQLVESGAEVKKPGASVKVSCKAS GYTFTSYD INWVRQATGQGLEWMGW MNPNSGNT GYAQKFQGRVTMTRNTSISTAYMELRSLRSDDTAVYYC ARDHGSYFGSGYYYGMDV WGQGTSLTVSS]]> AK-25-H08 <![CDATA[DIQMTQSPSSLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLSISSLQPDDFATYYC QQYKSYSIT FGQGTRLEIKR]]> <![CDATA[QVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQKFQGRVTITADESTSTAYMELSSLRAEDTAVYYC ARESLGYSSGWY WGQGTLVTVSS]]> AK-25-D03 <![CDATA[DIVMTQSPSSLSASVGDRVTVTCRAS QGILTY LAWYQQKPGKAPNLLIY AAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQDYNFPRT FGQGTKLEIKR]]> <![CDATA[EVQLQQSGPEVKKPGASVKVSCKAS GYTFTDYY MNWVRQMHGKGLEWMGV INPYNGGT DYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYC ARSVYDYPFDY WGQGTLVTVSS]]> The bolded and underlined sequences in the table above are the CDR sequences of the selected ScFv.

[0113] The above ScFv was obtained using prokaryotic expression and purification methods:

[0114] The pComb3xss plasmids containing the aforementioned ScFv gene sequences were transformed into Rosetta gami B competent cells and plated onto LB agar plates containing ampicillin. The cells were incubated overnight at 37°C. The following day, single colonies were picked and cultured in 4 ml of LB medium containing ampicillin for 8 h. The bacterial culture was then transferred to 200 ml of LB medium for further culture. When the OD of the bacterial culture reached 0.5-1.0, IPTG was added to a final concentration of 1 mM, and expression was induced at 37°C for 12 h. The bacterial cells were collected by centrifugation, and the precipitate was resuspended in PBS. The cells were then sonicated, centrifuged for 10 min, the precipitate was discarded, and the supernatant was collected for protein purification using a GE Ni Sepharose Excel purification column.

[0115] Identification of PSCA antigen by ScFv protein:

[0116] 293T and 293T-PSCA cells were aliquoted into 1.5 mL Eppendorf tubes, with 1 × 10^6 cells per tube as target cells. After centrifugation at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μL of the above-mentioned ScFv solution (50 μg / mL, 25 μg / mL, and 12.5 μg / mL, respectively). The group without ScFv and secondary antibody was designated as the Blank group, and the group without ScFv and only with secondary antibody was designated as the Control group. The cells were incubated at 4 °C for 30 min, and then resuspended in 1 mL of PBS. After centrifugation at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 30 μL of Anti-His-647 fluorescent secondary antibody for detection. The cells were incubated at 4 °C in the dark for 30 min. The cells were then resuspended in 1 mL of PBS and washed twice at 400 g for 5 min. The supernatant was discarded, and the cells were resuspended in 100 μL of PBS. The positive rate was detected by flow cytometry. The results are shown in Figure 6. Figure 6A The recognition status of PSCA antigen for clones AK-25-B11, AK-25-D02, and AK25-H08 (ScFv); Figure 6B The recognition of PSCA antigen by ScFv clone AK-25-D03 is shown. The above ScFv can significantly recognize PSCA antigen and can be used to detect PSCA expression, and can be used as a component of a kit for detecting PSCA expression.

[0117] Example 2 Screening of functional fully human single-chain antibodies

[0118] To verify the function of the screened fully human antibodies, we constructed a CAR vector to prepare CAR-T cells and verified the recognition of PSCA expressed by the screened fully human antibodies and their ability to kill tumor cells expressing PSCA.

[0119] 1) Screening for ScFvs with excellent killing ability against tumor cells expressing PSCA.

[0120] The ScFv sequence of the PSCA antibody was obtained by PCR amplification, and then ligated into a lentiviral vector with a CAR structure by restriction enzyme digestion. Two structures, 8hdc-28TM-28BBz and 8h-8TM-BBz, were primarily used for screening. The 8hdc-28TM-28BBz structure has been proven to have good efficacy against the PSCA target in patent 201810079289.9, while 8h-8TM-BBz is the most traditional CAR structure design. Using these two structures can fully realize the function of ScFv. The constructed lentiviral vector was successfully constructed after sequencing alignment verification.

[0121] In this embodiment, lentivirus packaging was performed using the calcium phosphate method. Specifically, 293T cells were cultured in DMEM medium containing 10% FBS (w / v) to optimal condition. The packaging plasmid (RRE:REV:2G) and expression plasmid were added to a 1.5 cm centrifuge tube at a specific ratio. CaCl2 and 2×HBS were added, mixed, and allowed to stand at room temperature before being added to the prepared 293T cell culture medium. After 3-5 hours, the medium was changed again to 10 mL of DMEM medium containing 10% FBS. After 48 or 72 hours, the cell supernatant was collected, and the virus was purified. Fifteen viral particles were obtained as in Example 1.

[0122] Lymphocytes were isolated using gradient centrifugation. After centrifugation, the second layer of white lymphocytes was collected, washed with physiological saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMCs. The obtained PBMCs were activated for 24 hours with anti-CD3 and CD28 monoclonal antibodies, and then infected with activated PBMCs at a specific multiple of infection (MOI). The CAR-T positivity rate was detected on day 8 of viral infection using flow cytometry. The antibody used was Protein-L-PE. Protein-L recognizes antibody light chains, and the light chain of the ScFv sequence in the CAR antigen recognition region can be recognized by Protein-L. Therefore, Protein-L can be used to detect CAR positivity rate and CAR expression intensity. Anti-CD3 FITC (Bio legend, Lots: B378781, Cat: 300440) and Protein-L-PE (Sino Biological, Lot: HR16SE0901, Cat: 11044-H07E-P) were also used.

[0123] The CAR-T cells prepared above were seeded with PSCA-positive target cells RT4-Luc-GFP at an effector-to-target ratio of 1:1. The killing ability of different CAR-T cells against target cells was detected using an ACEA xCELLigence RTCA MP instrument, and the experimental procedures were performed according to the instrument's instruction manual. The ACEA xCELLigence RTCA MP instrument works by recording the resistivity index of tumor cells attached to the bottom of the wells every 15 minutes, and using the resistivity index to determine the proliferation or death of adherent target cells. The formula for analyzing the resistivity index results is: Cell killing rate (%) = (Control group Cell Index value - Experimental group Cell Index value) / (Control group Cell Index value) × 100%. Table 3 shows the abbreviation and structure of CAR, as well as the results of CAR expression and kill detection. Table 3. CAR structure, abbreviation, viral titer, CAR expression, and CAR-T cell killing rate PSCA(7)-8h-8TM-BBZ AK-25-B11 3.59E8 68.46 28.99 PSCA(8)-8h-8TM-BBZ AK-25-D02 3.55E8 65.48 25.95 PSCA(9)-8h-8TM-BBZ AK-25-D03 5.6E8 66.68 76.22 PSCA(10)-8h-8TM-BBZ AK-25-H08 2.46E8 34.58 27.25

[0124] Table 3 shows that among the four ScFvs that can normally construct CARs, namely AK-25-B11, AK-25-D02, AK-25-D03, and AK-25-H08, although the constructed CARs can all be expressed normally, only the CAR-T constructed by AK-25-D03 (also known as PSCA (9)) has a higher killing effect on tumor cells expressing PSCA, with a killing result of 76.22%, which is significantly higher than the other ScFvs. Ultimately, the inventors screened and obtained ScFvAK-25-D03 (also known as PSCA (9)) which can recognize PSCA and has significant killing effect on tumor cells expressing PSCA. The light chain CDR sequences are as follows: L-CDR1: QGILTY (SEQ ID NO:2); L-CDR2: AAS (SEQ ID NO:3); L-CDR3: LQDYNFPRT (SEQ ID NO:4); and heavy chain CDR sequences are as follows: H-CDR1: GYTFTDYY (SEQ ID NO:5); H-CDR2: INPYNGGT (SEQ ID NO:6); H-CDR3: ARSVYDYPFDY (SEQ ID NO:7).

[0125] Furthermore, the inventors verified the ability of AK-25-D03 (also known as PSCA (9)) to recognize the PSCA antigen: HT1376, a human bladder cancer cell line that naturally expresses PSCA, and HPAC, a human pancreatic cancer cell line that exogenously expresses high expression of PSCA, were selected to demonstrate the ability of AK-25-D03 (also known as PSCA (9)) to recognize the PSCA antigen.

[0126] After cell culture, cells were harvested, centrifuged at 300 g / min for 5 min, and the supernatant was discarded to collect the cells. Cells were resuspended in PBS containing 1% fetal bovine serum and the cell density was adjusted to 1 × 10⁶ cells / ml. The collected cells were aliquoted with the final labeled antibody, incubated at 4°C for 30 min, washed twice with PBS, and the supernatant was discarded to collect the cells. Cells were resuspended in PBS containing 1% fetal bovine serum, labeled with secondary antibody, incubated at 4°C for 30 min, washed twice with PBS, and analyzed by flow cytometry. Labeled antibody: (Manufacturer: Santa Cruz, Catalog No.: Sc-80654, Lot: J 1821; PSCA interstitial secondary antibody: Alexa Fluor 647 goat anti-mouse IgG (H+L), RFF: A21235, Lot: 2482945). The expression of PSCA antigen was detected, and the results are as follows: Figure 1 As shown in Table 4: Table 4: PSCA expression in HPAC-Luc-GFP and PC3-PSCA-Luc-GFP cells PC3-PSCA-Luc-GFP 100% HPAC-Luc-GFP 60% RT4-Luc-GFP 100%

[0127] like Figure 1 As shown in Table 4, the positive rate of PSCA expression in HT1376-Luc-GFP was 100%, in PC3-PSCA-Luc-GFP it was 100%, in HPAC-Luc-GFP it was 60%, and in RT4-Luc-GFP it was 100%.

[0128] Combining Tables 3 and 4, the AK-25-D03 (also known as PSCA (9)) screened in this invention can specifically identify tumors that express PSCA, such as human bladder cancer, human pancreatic cancer, and human prostate cancer. It can also be used as an effective component of anti-tumor drugs targeting malignant tumors with high PSCA expression, effectively killing malignant tumors with high PSCA expression and controlling tumor proliferation, for example, in the application of CAR-T cell therapy.

[0129] Example 3: Compatibility verification of fully human single-chain antibodies with different CAR structures

[0130] Different CAR structures were designed for the optimal PSCA(9) anti-PSCA fully human ScFv selected above to verify the adaptability of PSCA(9) anti-PSCA fully human ScFv to different CAR structures. 8hdc / G4h / 8h was selected, transmembrane CD28TM was selected, and different co-stimulatory signals including CD28, CD27, and 4-1BB were selected. Z was selected for structure design, resulting in the CAR structure designs shown in Table 5 below: Table 5 Different CAR structural designs CAR1 Common promoter - PSCA(9)-8h-8TM-BBZ CAR2 Hypoxia promoter - PSCA(9)-8h-8TM-BBZ CAR3 PSCA(9)-8h-28TM-28BBCD27Z CAR4 PSCA(9)-G4h-28TM-28BBZ CAR5 PSCA(9)-8hdc-28TM-28BBZ

[0131] Using Control T cells as the control group, the experimental groups were divided into CAR1-CAR5 groups. CAR-T cells were prepared using the method described in Example 2 and seeded with PSCA-positive target cells HT1376-Luc-GFP, PC3-PSCA-Luc-GFP, and HPAC-Luc-GFP PSCA at a 1:1 effector-to-target ratio. The killing ability of different CAR-T cells against target cells was detected using an ACEA xCELLigence RTCA MP instrument. The experimental procedures were performed according to the instrument's instruction manual. The ACEA xCELLigence RTCA MP instrument works by recording the resistivity index of tumor cells attached to the bottom of the wells every 15 minutes, and using the resistivity index to determine the proliferation or death of adherent target cells. The formula for analyzing the resistivity index results is: Cell killing rate (%) = (Control group Cell Index value - Experimental group Cell Index value) / (Control group Cell Index value) × 100%.

[0132] The kill results are shown in Figure 2 below: the horizontal axis represents CAR, and the vertical axis represents the percentage of in vitro kills in different cells. Figure 2A The results showed that CAR1 significantly killed HPAC-Luc-GFP, HT1376-Luc-GFP and PC3-PSCA-Luc-GFP, with a killing percentage of more than 50%, proving that CAR1 can exert significant in vitro pharmacological effects in various solid tumor cell models. Figure 2B The results showed that CAR2 significantly killed PC3-PSCA-Luc-GFP, with a killing percentage of over 50%, demonstrating that CAR2 can exert significant in vitro pharmacological effects on PSCA-positive tumor cells. Figure 2C The results showed that CAR3, CAR4 and CAR5 significantly killed HPAC-Luc-GFP, with a killing percentage of over 70%, proving that the CAR-Ts (CAR3, CAR4 and CAR5) constructed from the fully human ScFv PSCA (9) of this invention can exert significant in vitro pharmacological effects on PSCA-positive tumor cells. The specific killing data are shown in the table below. CAR3 73.01 CAR4 75.18 CAR5 74.45 Control-T 3.77

[0133] Six- to eight-week-old female NCG mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used to establish peritoneal tumor models using HPAC-Luc-GFP, HT1376-Luc-GFP, or PC3-PSCA-Luc-GFP, respectively. Three days after intraperitoneal injection of 3E5 tumor cells, CAR1:5E5 CAR-T cells / mouse were administered intraperitoneally. In vivo imaging of mice was performed every 7 days post-administration. The fluorescence values ​​in the images were statistically analyzed, and the significance was determined using a T-test. The results are shown in Figure 3. Figure 3A and Figure 3B The results showed that CAR1 was significantly effective in both the HPAC-Luc-GFP tumorigenesis model and the HT1376-Luc-GFP tumorigenesis model, proving that CAR1 constructed based on the fully human ScFv PSCA (9) targeting PSCA described in this invention has a good killing ability in human bladder cancer and pancreatic cancer. Figure 3C The results showed that CAR2 had significant efficacy in the PC3-PSCA-Luc-GFP tumorigenesis model, proving that CAR2 constructed based on the fully human ScFv PSCA (9) targeting PSCA described in this invention has a good killing ability in human prostate cancer.

[0134] Based on all the data from Examples 2 and 3, it is demonstrated that PSCA(9), when combined with different promoters (i.e., the ordinary promoter and the hypoxia promoter), different hinges (i.e., 8h, 8hdc, and G4h), different transmembrane 8TM and 28TM, and different intracellular signals CD28, 4-1BB, and CD27), can recognize PSCA-positive tumor cells and exert a killing effect in in vitro functional verification. Furthermore, it exhibits significant tumor cell-killing efficacy in different solid tumor models. Through the verification of the above-mentioned multiple CAR structures and tumor indications, AK-25-D03 (also known as PSCA(9)) can serve as the antigen recognition region of a chimeric antigen receptor and can be used for the treatment of PSCA-expressing tumors such as pancreatic cancer, prostate cancer, bladder cancer, gastric cancer, lung cancer, breast cancer, oral squamous cell carcinoma, and gallbladder cancer. This includes the construction of various CAR-T cell structures for the treatment of pancreatic cancer, prostate cancer, bladder cancer, gastric cancer, lung cancer, breast cancer, oral squamous cell carcinoma, and gallbladder cancer.

[0135] Example 4: Single-chain antibody PSCA(9) was used as the CAR structural antigen recognition region to complete the application verification of dual CAR.

[0136] Based on PSCA(9), a dual CAR was designed. The dual CAR design considered the combination of PSCA and TIGIT. The PSCA(9) single CAR used different hinges 7h, 8h and G4h, different transmembrane 8TM and 28TM, and different intracellular signals including CD28, 4-1BB, and CD28-4-1BB to form a dual CAR with TIGIT. The TIGIT single CAR also used different transmembrane 8TM and 28TM, and different intracellular signals CD28, 4-1BB and CD134. The specific design is shown in Table 6. All structures were tested in vitro to confirm whether they had a significant in vitro killing effect on PSCA positive tumor cells, and to verify the universality of PSCA(9) for different dual-target CAR structures. Table 6: Dual CAR Structure Design CAR6 PSCA(9)-G4h-28TM-28BBCD3Z-P2A-TIGIT(mut6)-28TM-28z CAR7 PSCA(9)-G4h-28TM-28BBCD3Z-P2A-TIGIT(mut6)-8TM-134Z CAR8 PSCA(9)-7h-28TM-28z -P2A-TIGIT(mut6)-8TM-BBZ CAR9 PSCA(9)-8h-8TM-BBZ-P2A-TIGIT(mut2)-28TM-28z CAR10 PSCA(9)-8h-8TM-BBZ-P2A-TIGIT(mut5)-28TM-28z CAR11 TIGIT(mut6)-28TM-28z

[0137] The expression of CD155 and PSCA was detected using the same detection method as the PSCA positive antigen in Example 2. The anti-CD155 antibody was Brilliant Violet anti-human CD155 (Biolegend, Lot: B338257, Cat: 337632), and the anti-PSCA antibody was PSCA (7F5) Santa Cruz, Sc-80654, Lot: J1821. The results are shown in Table 7 below: The human bladder cancer cell line HT-1376-Luc-GFP showed high expression of both CD155 and PSCA; the human pancreatic cancer cell line HPAC-Luc-GFP showed high expression of CD155 and moderate to high expression of PSCA. Both cell lines can be used to validate PSCA dual-target CAR-T therapy for human bladder cancer and human pancreatic cancer indications. Table 7: Expression of CD155 and PSCA in HT1376-Luc-GFP and HPAC-Luc-GFP cells HT-1376-Luc-GFP 100% 100% HPAC-Luc-GFP 100% 60% CAR-T preparation and CAR expression detection were performed using the steps in Example 2, and the results are shown in Table 8 below: Table 8: CAR virus titer and expression data CAR6 1.81E8 27.39 CAR7 3.44E8 40.03 CAR8 2.95E8 49.4 CAR9 5.67E7 16.94 CAR10 6.49E8 23.96 CAR11 5.59E7 68.83

[0138] The in vitro efficacy was validated using the method described in Example 3, verifying the effectiveness of the dual CAR containing PSCA (9) in different indications. The results are shown in Figure 4. Figure 4AThe results showed that CAR6 and CAR11 both had significant killing effects on HPAC-Luc-GFP and HT1376-Luc-GFP. At the same time, the in vitro killing effect of CAR6 was significantly higher than that of CAR11 on HPAC-Luc-GFP and HT1376-Luc-GFP tumor cells, proving that the killing ability of dual CARs was significantly increased compared with that of single CARs. Figure 4B The results showed that CAR7, CAR8, CAR9 and CAR10 all had significant killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP (prostate cancer) and HT1376-Luc-GFP, with a killing percentage of more than 60%, proving that dual CARs with different structures can exert significant in vitro pharmacological effects in various solid tumor cell models.

[0139] In summary, for different indications of PSCA positivity, including but not limited to human pancreatic cancer, prostate cancer, and bladder cancer, any dual CAR structure containing PSCA (9) exhibits significant killing function. In addition to the aforementioned dual CAR designs targeting PSCA and CD155, these dual CAR structures can also be PSCA and targets for solid tumors, including but not limited to MOv-γ, PSMA, IL13Rα2, EGFRvIII, EGFR, EPCAM, GD2, MUC1, HER2, GPC3, CEA, Meso, CD133, NKG2D, CD138, LeY, k-Light, ROR1, CD70, CD47, CLDN18.2, CDH17, Trop2, and B7H3.

[0140] Example 5: The single-chain antibody PSCA (9) served as the extracellular recognition region of the CAR structure, completing the fusion protein form constructed by combining CARs, which can exert better in vivo therapeutic effects.

[0141] In CAR structure design, in addition to traditional single-CAR and dual-CAR structures, there are also combined modes of CAR structures with fusion proteins and functional peptides. These combinations can involve the CAR and the fusion protein / functional peptide being constructed and expressed in the same vector, or the CAR targeting PSCA and the fusion protein / functional peptide being co-transferred to target immune cells in two separate vectors. The fusion protein or functional peptide can be a fusion protein containing extracellular segments or extracellular recognition domains of immune checkpoints such as PD1 / PDL1, TIGIT, SIRPγ, and SIRPα; it can also be a fusion protein containing the full length, functional domains, and / or signaling domains of cytokines such as IL15, IL2, IL21, IL12, and IL7; or it can be a full-length or fusion protein containing chemokines or chemokine receptors such as CXCR3, CXCR2, CCL19, and CCL21.

[0142] Here, we take the TIGIT fusion protein as an example to verify the feasibility of a combination mode of CAR containing the single-chain antibody PSCA(9) with the fusion protein and functional peptide. The CAR structure design is shown in Table 9 below: Table 9 CAR structure design of the combined fusion protein CAR12 PSCA(9)-G4h-28TM-28BBCD3Z-P2A- TIGIT(mut6)-28TM-28 CAR13 PSCA(9)-G4h-28TM-28BBCD3Z-P2A- TIGIT(mut6)-8TM-134 CAR14 PSCA(9)-7h-28TM-28z-P2A-TIGIT(mut6)-8TM-BB CAR15 PSCA(9)-8h-8TM-BBZ-P2A- TIGIT(mut2)-28TM-28 CAR16 PSCA(9)-8h-8TM-BBZ-P2A- TIGIT(mut5)-28TM-28 CAR17 TIGIT(mut6)-28TM-28

[0143] CAR virus preparation, CAR-T preparation, and in vitro killing verification were completed according to the scheme in Example 2. The expression results of CAR titer are shown in Table 10 below: Table 10 CAR virus titer and expression data CAR12 3.99E8 34.78 CAR13 2.7E8 53.97 CAR14 1.24E8 43.06 CAR15 1.26E8 48.38 CAR16 1.18E8 30.98 CAR17 3.73E8 44.06

[0144] The in vitro killing results are shown in Figure 5. Figure 5A The horizontal axis represents different CARs, and the vertical axis represents the killing effect of CARs with different structures combined with fusion proteins and CAR-T cells expressing fusion proteins alone on three cell types for pancreatic cancer, prostate cancer, and bladder cancer: HPAC-Luc-GFP, PC3-PSCA-Luc-GFP, and HT1376-Luc-GFP. The results showed that CAR16 had significant killing effect on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP, and HT1376-Luc-GFP, while CAR17 had no in vitro killing function. This proves that the fusion protein itself does not play a role in in vitro killing, but the in vitro killing function was significantly enhanced after PSCA single CAR combined with TIGIT to form a fusion protein. Figure 5B The inventors further designed more CAR structures containing PSCA (9) and fusion proteins with different structures for combination verification. The CAR virus preparation was completed according to the scheme of Example 2. The results are shown in Table 12 below. The universality of the combination of CAR structures containing PSCA (9) and fusion proteins was verified. The results showed that CAR12, CAR13, CAR14 and CAR15 all had significant killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP and HT1376-Luc-GFP, proving that CARs in different fusion protein forms can exert significant in vitro pharmacological effects in various solid tumor cell models.

[0145] The above experiments demonstrate that PSCA (9) as an extracellular recognition domain is suitable for any CAR structure, including single CAR and double CAR verified in the above examples, and is suitable for any CAR structure combined with fusion protein or functional peptide.

[0146] It should be noted that although this invention uses T cells and CAR-T cells as examples, those skilled in the art can obviously infer from the content disclosed in this invention that engineered immune cells containing PSCA (9) as described herein can also express other immune cells such as NK cells, macrophages, DC cells, and related precursor cells, and specifically recognize PSCA to exert a killing effect on malignant tumors expressing PSCA. The PSCA (9) described in this application, as a chimeric antigen receptor constructed as one of the extracellular antigen recognition domains, can be expressed on T cells, NK cells, macrophages, DC cells, and related precursor cells; it can be co-expressed with fusion proteins of any structure on T cells, NK cells, macrophages, DC cells, and related precursor cells, and can be used in drugs or drug combinations for expressing PSCA in malignant tumors for tumor treatment.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antigen-binding polypeptide targeting PSCA, characterized in that, The antigen-binding polypeptide comprises a heavy chain variable region and a light chain variable region. The light chain variable region comprises L-CDR1, L-CDR2, and L-CDR3, and the heavy chain variable region comprises H-CDR1, HCDR2, and HCDR3. The amino acid sequence of L-CDR1 is QGILTY, the amino acid sequence of L-CDR2 is AAS, the amino acid sequence of L-CDR3 is LQDYNFPRT, the amino acid sequence of H-CDR1 is GYTFTDYY, the amino acid sequence of H-CDR2 is INPYNGGT, and the amino acid sequence of H-CDR3 is ARSVYDYPFDY.

2. The polypeptide of claim 1, characterized in that, The polypeptide contains amino acids as shown in SEQ ID NO. 8, specifically the light chain variable region.

3. The polypeptide of claim 1, characterized in that, The polypeptide contains amino acids as shown in SEQ ID NO.9, including the heavy chain variable region.

4. The polypeptide of claim 1, characterized in that, The polypeptide is a single-chain antibody that recognizes PSCA. The single-chain antibody contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

9.

5. The polypeptide of claim 4 further comprises a linker sequence having an amino acid sequence as shown in SEQ ID NO.

10.

6. A kit for detecting PSCA expression, characterized in that, It comprises the antigen-binding polypeptide according to any one of claims 1-5.

7. A chimeric antigen receptor, characterized in that, It includes an extracellular antigen recognition domain capable of recognizing PSCA antigens, said extracellular antigen recognition domain comprising an antigen-binding polypeptide targeting PSCA as described in any one of claims 1-5.

8. The chimeric antigen receptor according to claim 7, characterized in that, It also includes a hinge region, a transmembrane region, and an intracellular signaling domain.

9. The chimeric antigen receptor according to claim 8, characterized in that, The hinge region comprises one of the following optional amino acid sequences: SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.

25.

10. The chimeric antigen receptor according to claim 9, characterized in that, The transmembrane region is selected from: SEQ ID NO.11 and SEQ ID NO.

21.

11. The chimeric antigen receptor of claim 10, wherein the intracellular signaling domain comprises an amino acid sequence derived from CD3ζ as shown in SEQ ID NO.

16.

12. The chimeric antigen receptor of claim 11, wherein the intracellular signaling domain further comprises one or more of the following optional amino acid sequences: SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.

19.

13. Nucleic acid, characterized in that, Encoding the chimeric antigen receptor as described in claim 7.

14. The nucleic acid according to claim 13, characterized in that, Its nucleic acid sequence is shown in SEQ ID NO.

41.

15. The nucleic acid according to claim 13, characterized in that, The nucleic acid also includes a promoter, the nucleic acid sequence of which is shown in SEQ ID NO.44 or SEQ ID NO.

45.

16. The nucleic acid according to claim 15, characterized in that, Its nucleic acid sequence is shown in SEQ ID NO.42 or SEQ ID NO.

43.

17. An expression vector comprising the nucleic acid according to any one of claims 13-16.

18. The carrier according to claim 17, characterized in that, It also contains TIGIT mutant nucleotides with sequences such as SEQ ID NO.37, SEQ ID NO.38, and SEQ ID NO.

39.

19. An engineered cell, characterized in that, The cell expresses a polypeptide as described in any one of claims 1-5, or a chimeric antigen receptor as described in any one of claims 7-12, or the cell contains a nucleic acid as described in any one of claims 13-16 or a vector as described in any one of claims 17-18.

20. The engineered cell of claim 19, characterized in that, The amino acid sequence of the expressed TIGIT protein is shown in any one of SEQ ID NO.22, SEQ ID NO.23, or SEQ ID NO.

24.

21. The engineered cell of claim 20, characterized in that, The cells also express a TIGIT fusion protein; specifically, the expressed TIGIT fusion protein has the structure TIGIT-28TM-28, TIGIT-8TM-134, or TIGIT-8TM-BB, wherein 28TM and 8TM are transmembrane structures derived from human CD28 and human CD8, respectively, and 28 and 134 are intracellular signaling structures derived from human CD28 and human CD134, respectively.

22. The engineered cell according to any one of claims 19-21, characterized in that, It also includes chimeric antigen receptors that target non-PSCA targets, which are tumor surface antigens, including but not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), CD155, or TAG-72, or any combination of multiple targets.

23. The engineered cell of claim 22, characterized in that... The cells mentioned are T cells, T cell precursor cells, NK cells, DC cells, and macrophages.

24. The engineered cell of claim 23, characterized in that, The cells in question are T lymphocytes.

25. A cell product, characterized in that, The cell product comprises the engineered cells according to any one of claims 19-24.

26. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a polypeptide according to any one of claims 1-5, a nucleic acid according to any one of claims 13-16, or a carrier according to any one of claims 17-18, an engineered cell according to any one of claims 19-24, or a cell product according to claim 25.

27. The use of the engineered cells of any one of claims 19-24, the cell product of claim 25, the pharmaceutical composition of claim 26, or the chimeric antigen receptor of any one of claims 7-12 in the preparation of drugs targeting pancreatic cancer, prostate cancer, bladder cancer, gastric cancer, lung cancer, breast cancer, oral squamous cell carcinoma, and gallbladder cancer expressing PSCA.

Images