Fully humanized single-chain antibody aiming at PSCA and application of fully humanized single-chain antibody

Abstract

The invention belongs to the technical field of cellular immune engineering, and particularly relates to a fully human-derived polypeptide for recognizing PSCA and application of the fully human-derived polypeptide. The polypeptide specifically recognizes PSCA and is good in affinity performance, and the polypeptide specifically targeting PSCA can mark PSCA positive cells, can be combined with PSCA antigens and PSCA positive cells and can serve as a PSCA molecule detection reagent and an in-vivo tracing marking reagent. The polypeptide specifically targeting PSCA provided by the invention can also be used in an extracellular antigen recognition region of a chimeric antigen receptor, is used in immune cell treatment drugs targeting PSCA, and is used for killing malignant tumors expressing PSCA. The polypeptide for specifically recognizing the PSCA is a natural full-humanized antibody source, compared with a mouse antibody, a chimeric antibody and a humanized antibody, the immunogenicity of the polypeptide is greatly reduced, and the safety can be guaranteed to the maximum extent in clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor cell immunotherapy, and specifically relates to a fully human ScFv targeting PSCA and an application thereof. Background Art

[0002] Chimeric antigen receptor (CAR) is an artificial receptor that simulates TCR function. 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 the CD3ζ chain or FcRγ, or is connected to one or more co-stimulatory molecules, such as 4-1BB, CD28, and ICOS (CD278).

[0003] PSCA (prostate stem cell antigen) is a glycosylphosphatidylinositol (GPI)-linked cell membrane antigen that is only expressed in the prostate and bladder in normal tissues. It is overexpressed in most prostate tumors, bladder cancer, pancreatic cancer, non-small cell lung cancer, and gastric cancer. It is a good therapeutic target for solid tumors. Faced with the current problem of poor efficacy and poor prognosis of solid tumors, immunotherapy targeting PSCA may be a breakthrough in the future treatment of prostate cancer and a variety of solid tumors expressing PSCA.

[0004] The extracellular antigen recognition domain can also be called the extracellular antigen binding domain, which can specifically recognize tumor surface antigens. The CAR molecule transmits immune cell activation signals through the hinge region and transmembrane region by using the characteristics of specific recognition of tumor antigens by the antigen binding domain. The most common extracellular antigen recognition domain is ScFv (single-chain antibody) that recognizes tumor antigens. The selection of ScFv is crucial for the function of CAR. Therefore, for immunotherapy targeting PSCA, the screening of anti-PSCA antibodies or anti-PSCA ScFv is very necessary. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a fully humanized single-chain antibody targeting PSCA and its use in detection reagents and anti-tumor drugs. The ScFv provided by the present invention can specifically recognize PSCA, recognize and detect PSCA expressed by cells and its ratio, and can be used as a detection reagent or in vivo tracer marker molecule for PSCA; the ScFv provided by the present invention can be used as the antigen recognition domain of the chimeric antigen receptor to recognize PSCA on the surface of the tumor, construct chimeric antigen receptor immune cells, and effectively inhibit the growth of tumors in vivo, and the anti-tumor effect of expressing PSCA targets is significant. In addition, it can be used as an effective ingredient of anti-tumor drugs for malignant tumors with high PSCA expression, and effectively control 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, 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.

[0007] The antigen-binding polypeptide targeting PSCA comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, as shown in the amino acid sequences of SEQ ID NOs. 2-4; and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, as shown in the amino acid sequences of SEQ ID NOs. 5-7.

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

[0009] The antigen-binding polypeptide targeting PSCA and the heavy chain variable region of the single-chain antibody are 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, 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 certain specific embodiments, the method for preparing antigen-binding polypeptides targeting PSCA is to construct a fully humanized single-chain antibody library by phage display technology, enrich phages carrying certain fragments with specific affinity by affinity panning by appropriate methods, and obtain their gene sequences by sequencing. In order to screen humanized antibodies targeting PSCA antigens, a fully humanized single-chain antibody library was constructed, normal human PBMC was used as raw materials, total RNA was extracted and reverse transcribed into cDNA, antibody variable regions were amplified, heavy chain variable region VH and light chain variable region VL were connected by linker peptides, constructed into phagemid vectors, electrotransferred to display strains, and antibody libraries were constructed. The library was panned by PSCA extracellular segment antigen, and the selected clones were identified, and finally ScFv with specific binding to PSCA antigen was obtained. When constructing chimeric antigen receptors or other uses, ScFv can be artificially synthesized by PCR to obtain a large number of ScFv targeting PSCA, that is, the extracellular domain including the amino acid sequence shown in SEQ ID NO: 1 or its functional variant is obtained.

[0012] Furthermore, the antigen-binding polypeptide targeting PSCA further comprises a Linker sequence. In some embodiments, the Linker amino acid sequence is GSTSGSGKPGSGEGSTKG or (GGGGS)n, where n=1-5.

[0013] Furthermore, the antigen-binding polypeptide targeting PSCA further comprises a Linker sequence, preferably the sequence shown in SEQ ID NO:10.

[0014] An antibody that recognizes PSCA, said antibody comprising the CDRs of SEQ ID NOs: 2-7.

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

[0016] Another object of the present invention is to provide a chimeric antigen receptor targeting PSCA.

[0017] The chimeric antigen receptor comprises an extracellular antigen recognition domain that can recognize a PSCA antigen, wherein the extracellular antigen recognition domain that recognizes a PSCA antigen comprises a CDR of a sequence of SEQ ID NOs: 2-7; in some embodiments, the extracellular antigen recognition domain that recognizes a PSCA antigen comprises a sequence that may also comprise a light or heavy chain sequence of SEQ ID NOs: 8 and 9; in some embodiments, the extracellular antigen recognition domain that recognizes a PSCA antigen comprises an amino acid sequence as shown in SEQ ID NO: 1; in some embodiments, the extracellular antigen recognition domain that recognizes a PSCA antigen comprises a Linker sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

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

[0019] Furthermore, the hinge region preferably comprises one of the following optional amino acid sequences or its functional variants: SEQ ID NO.13, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.26, so that the obtained chimeric antigen receptor can have a better cell killing effect.

[0020] Furthermore, the transmembrane region may be derived from: α, β or chain of T cell receptor, 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.12, SEQ ID NO.27.

[0022] The chimeric antigen receptor also comprises an intracellular signaling domain, which comprises a signaling region and a co-stimulatory domain; the intracellular signaling domain comprises a signaling region, which is derived from a primary cytoplasmic signaling sequence containing ITAM of protein molecules including but not limited to 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.17.

[0024] The chimeric antigen receptor further comprises 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), signaling lymphocyte activation molecule (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 of their functional variants: SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.20.

[0026] In certain 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.13 or a functional variant thereof, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO.12 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 and the amino acid sequence of SEQ ID NO.17 or a functional variant thereof;

[0028] Combination 2): the hinge region comprises the amino acid sequence as shown in SEQ ID NO.26 or a functional variant thereof, the transmembrane region comprises the amino acid sequence as shown in SEQ ID NO.27 or a functional variant thereof, and the intracellular signaling domain comprises the amino acid sequence as shown in SEQ ID NO.15 or a functional variant thereof and the amino acid sequence as shown in SEQ ID NO.14 or a functional variant thereof and the amino acid sequence as shown in SEQ ID NO.17 or a functional variant thereof;

[0029] Combination 3): the hinge region comprises the amino acid sequence shown in SEQ ID NO.18 or a functional variant thereof, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO.12 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 and the amino acid sequence of SEQ ID NO.17 or a functional variant thereof;

[0030] Combination 4): the hinge region comprises the amino acid sequence shown in SEQ ID NO.26 or a functional variant thereof, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO.12 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 and the amino acid sequence of SEQ ID NO.17 or a functional variant thereof;

[0031] Combination 5): The hinge region comprises the amino acid sequence shown in SEQ ID NO.19 or a functional variant thereof, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO.27 or a functional variant thereof, and the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO.15 or a functional variant thereof and the amino acid sequence of SEQ ID NO.17 or a functional variant thereof.

[0032] The chimeric antigen receptor further comprises a CD8 leader peptide, which comprises an amino acid sequence as shown in SEQ ID NO. 28 or a functional variant thereof. The PSCA-targeted antibody is localized on the cell membrane under the guidance of the CD8 leader peptide. As known to those skilled in the art, all other signal peptides that can be used in the field of CAR or antibodies can be used in the present invention. The extracellular segment generally comprises a CD8α or GM-CSFRα leader peptide.

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

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

[0035] The nucleic acid sequence of the chimeric antigen receptor comprises the nucleic acid sequence of the 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.44.

[0037] Furthermore, the chimeric antigen receptor also includes a promoter, and the nucleic acid sequence of the promoter is shown in SEQ ID NO.47 or SEQ ID NO.48.

[0038] Furthermore, the nucleic acid sequence of the chimeric antigen receptor may also be as shown in SEQ ID NO.45 or SEQ ID NO.46.

[0039] The present invention also aims to provide a multi-nucleic acid vector.

[0040] Furthermore, the expression vector is selected from any one 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.

[0041] In certain 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 certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR encompassed by the present invention, and a right (3') retroviral LTR.

[0043] The polynucleic acid vector comprises a TIGIT mutant nucleotide sequence as shown in any one of SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.42.

[0044] In some embodiments, the TIGIT mutant contained in the above-mentioned multi-nucleic acid vector can also be connected to the transmembrane of human CD28, the intracellular signal of human CD28 and the CD3ζ signal region to form a TIGIT mutant fusion protein.

[0045] The present invention also aims to provide a multi-target CAR structure, comprising a nucleotide sequence of a second chimeric antigen receptor targeting a non-PSCA target; the second chimeric antigen receptor target of the non-PSCA target is CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCM A, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), CD155 or TAG-72.

[0046] In the multi-target CAR structure, the nucleotide sequence of the second chimeric antigen receptor includes the extracellular antigen recognition region shown in sequences such as SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.42.

[0047] In certain embodiments, the chimeric antigen receptor targeting PSCA or the multi-target CAR described above comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).

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

[0049] In certain embodiments, the gene expression vector may comprise a secretory anti-PD-1 ScFv; in certain embodiments, the gene expression vector comprises a PD-1 conjugated transduction peptide (such as a PD-1-CD28-CD137-CD3 signal structure); in certain embodiments, the gene expression vector may comprise a plurality of CAR combinations, such as a combination of two CARs targeting different antigens or different recognition sites of the same antigen.

[0050] The present invention also aims to provide an engineered cell, wherein the cell expresses a polypeptide or a single-chain antibody comprising any one of SEQ ID NOs. 1-9, or a chimeric antigen receptor as described in any one of the foregoing, or the cell comprises a nucleic acid or a 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.23, SEQ ID NO.24, and SEQ ID NO.25.

[0054] Furthermore, the expressed TIGIT fusion protein structure is TIGIT-28TM-28 or 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 signal structures derived from human CD28 and human CD134, respectively.

[0055] Furthermore, the amino acid sequence of the expressed TIGIT fusion protein is as follows: the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51.

[0056] The engineered cells further include chimeric antigen receptors targeting non-PSCA targets, wherein the non-PSCA targets 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, PDL 1. Any one or more targets including EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma associated antigen 1), CD155 or TAG-72.

[0057] Furthermore, the non-PSCA target is CD155.

[0058] Furthermore, the chimeric antigen receptor targeting CD155 recognizes that the extracellular antigen recognition region of CD155 is TIGIT and its mutated functional protein.

[0059] 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.23, SEQ ID NO.24, and SEQ ID NO.25.

[0060] The engineered cells described in any of the above items are immune cells such as T cells, T cell precursors or NK cells, DC cells, macrophages, etc.

[0061] Preferably, the immune cells are T lymphocytes.

[0062] The present invention further aims to provide a cell product, which comprises any of the engineered cells described above.

[0063] Furthermore, the cell product also includes other active agents that can enhance the expression activity of CAR.

[0064] In certain 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, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation.

[0065] In certain specific embodiments, the activating agent for enhancing the activity of CAR-expressing cells can be an activating 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 a transmembrane domain and an intracellular signaling domain, such as PD1 CAR.

[0066] Further, a variety of additional 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.

[0067] In particular, additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib, ofatumumab, rituximab, bevacizumab, trastuzumab, bemtansine, imatinib, cetuximab, panitumumab, and trastuzumab. b) Catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitini b, pazopanib b, sunitinib b), 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, bosutinibb), lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, and denileukin.

[0068] Furthermore, the cell product can also be used in combination with some therapeutic means, and the therapeutic means can be surgery, chemotherapy, and radiation.

[0069] The present invention further aims to provide a pharmaceutical composition, which comprises any of the above-mentioned polypeptides or single-chain antibodies, the nucleic acid sequence of a chimeric antigen receptor or any of the above-mentioned expression vectors or any of the above-mentioned engineered cells.

[0070] The present invention also aims to provide a use of any of the above-mentioned 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 drugs targeting tumors expressing PSCA targets.

[0071] Specifically, the anti-tumor drugs include but are not limited to anti-renal cancer drugs, anti-prostate cancer drugs, anti-thymic tumor drugs, anti-ovarian cancer drugs, anti-glioblastoma drugs, anti-pancreatic cancer drugs, anti-bladder cancer drugs, anti-nasopharyngeal cancer drugs, anti-gastric cancer drugs, anti-colorectal cancer drugs and other malignant solid tumor drugs.

[0072] In the present invention, the term "functional variant" generally refers to an amino acid sequence having substantially the same function (e.g., having the properties of the chimeric antigen receptor) and having 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 therewith. In certain embodiments, the variant of the amino acid sequence has substantially the same function therewith. The beneficial effects of the present invention are

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

[0074] The ScFv provided by the present invention can be used as the antigen recognition domain of the chimeric antigen receptor to recognize PSCA on the surface of the tumor, construct a chimeric antigen receptor immune cell, effectively inhibit the growth of tumors in vivo, and has a significant effect on tumors expressing PSCA targets. In addition, it can be used as an effective ingredient of an anti-tumor drug for malignant tumors with high PSCA expression, and effectively control the proliferation of malignant tumors with high PSCA expression. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0076] Figure 2 shows the in vitro functional validation of different CAR-T constructed by targeting PSCA ScFv in PSCA-expressing malignancies such as pancreatic cancer, prostate cancer and bladder cancer; Figure 2A The in vitro killing effect of CAR1 in three cell models is shown in Figure 2. Figure 2B This is the in vitro killing of CAR2 in PC3-PSCA-Luc-GFP; Figure 2C The in vitro killing results of three CARs on HPAC-Luc-GFP.

[0077] FIG3 is an in vivo functional verification of CAR-T with different structures constructed by targeting PSCA ScFv in malignant tumors expressing PSCA, such as pancreatic cancer, prostate cancer and bladder cancer; Figure 3A The in vivo efficacy of CAR1 in the HPAC-Luc-GFP (pancreatic cancer) peritoneal tumor 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 the PC3-PSCA-Luc-GFP (prostate cancer) peritoneal tumor model.

[0078] Figure 4 is the functional verification of dual-target CAR-T; Figure 4A The in vitro killing effects of the two CARs in the two cell models are shown; Figure 4B :In vitro killing effects of four CARs in three cell models.

[0079] FIG5 is a functional validation of CAR combined with fusion protein targeting PSCA; Figure 5A The in vitro killing effects of CAR16 and CAR17 on two target cells; Figure 5B The in vitro killing results of four CARs in three cell models.

[0080] FIG6 is a diagram showing the recognition and detection of the screened single-chain antibodies against the PSCA target; Fig. 6AThe recognition of PSCA antigen by ScFv clones AK-24-A08, AK-25-B11, AK-25-D02, and AK25-H08; Figure 6B The figure shows the recognition of PSCA antigen by ScFv clone AK-25-D03. DETAILED DESCRIPTION

[0081] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0082] In an embodiment of the present 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] Ficoll separation solution is used for PBMC separation. Ficoll separation solution is slowly added to the collected normal human blood so that the Ficoll separation solution and the normal human blood maintain a clear separation interface. The 50mL centrifuge tube containing the blood and separation solution is centrifuged at about 15°C for 20min. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid. There is a narrow band of white cloud layer dominated by PBMC at the junction of the upper and middle layers, i.e., the PBMC cell layer. PBMC is sucked out with a new sterile Pasteur pipette to obtain separated PBMC.

[0085] Total RNA was extracted by conventional methods and reverse transcribed into cDNA. Based on the similarity of heavy chain and light chain germline gene sequences, degenerate primers were designed at both ends of the variable region ((Li Xiaolin, Construction and Preliminary Screening of 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 variable region gene fragment and light chain variable region gene fragment of the antibody, and the ScFv nucleic acid fragment was amplified by conventional overlapping PCR (PCR method reference "Molecular Cloning: A Laboratory Manual" (third edition), the United States, Joe Sambrook, David Russell. Science Press), the ScFv nucleic acid fragment was connected to the phagemid vector pComb3xss, and the product was transformed into the TGI strain by an electroporator to obtain a fully human single-chain antibody library.

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

[0087] The library bacterial solution was added to fresh LB liquid medium for recovery, and VSCM13 helper phage was added at a multiplicity of infection of 50:1 for VCSM13: bacteria, and the mixture was thoroughly mixed and continued to be cultured in a shaker. The culture was centrifuged and the supernatant was discarded. The precipitate was resuspended in SOB medium with ampicillin and kanamycin resistance and cultured overnight. The bacterial solution was centrifuged at 4°C and 8000rpm for 20min, the supernatant was collected and 1 / 5 volume of 20% PEG 8000 2.5mmol / L NaCl solution was added, incubated on ice for 1 hour, and then centrifuged at 4°C and 12000rpm for 30min. The precipitated phage was resuspended in PBS and filtered with a 0.22μm filter membrane.

[0088] (3) PSCA antigen selection

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

[0090] (4) Screening of positive clones

[0091] After the panning, the monoclonal plaques finally released from the library were selected for ELISA screening. The test results were as follows: Figure 1 As shown, a phage clone binding to PSCA antigen was obtained, and a ScFv targeting PSCA was obtained by panning, which had the specific binding ability to human PSCA antigen.

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

[0093] (1) Blocking: Dissolve 5% skim milk powder in PBS, filter and use as blocking solution. Resuspend phage and CD70-protein G-coupled magnetic beads in appropriate amount of blocking solution and mix by rolling.

[0094] (2) Co-incubation: Place PSCA-protein G-coupled magnetic beads on a magnetic rack, discard the supernatant, resuspend the magnetic beads with phage, and roll over for co-incubation;

[0095] (3) Washing: Place the magnetic bead-phage mixture on a magnetic rack, discard the supernatant, and add a washing solution to wash the magnetic beads. The number of washing times is determined according to different rounds of panning;

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

[0097] (5) Enrichment: The phages were inoculated into the TG1 bacterial solution for infection, centrifuged after resting, the precipitate was resuspended, spread on a 2YTAG plate, and inverted for overnight culture;

[0098] (6) Plate washing: The bacterial plaques on the plates cultured overnight are washed off with the culture medium and used as the seed bacterial solution for the next round of library packaging.

[0099] In the embodiment of the present 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 cover and incubate 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: Wash the antigen-coated plate three times with PBS on a plate washer, add skim milk powder blocking solution to each well, and block at 37°C;

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

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

[0105] (6) Color development: Wash the plate six times with a plate washer, add color developing solution to each well, and place at room temperature away from light for 25 min to develop color;

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

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

[0108] In the embodiments of the present invention, the sequences of the vector structural elements are 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 primary signal transduction domain CD3ζ derived from CD3.

[0109] In the examples of the present invention, PSCA (9) is the ScFv AK-25-D03 targeting PSCA, and TIGIT (mut5), TIGIT (mut2), and TIGIT (mut6) are mutated TIGIT peptides. Table 1 Sequences of vector structural elements

[0110] Example 1 ScFv sequence that specifically recognizes PSCA

[0111] The above-described fully human antibody preparation method was used to screen and obtain the ScFv sequences shown in Table 2 below:

[0112] Table 2 Single chain antibodies targeting PSCA: The bold and underlined sequences in the above table are the CDR sequences of the screened ScFv.

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

[0114] The pComb3xss plasmids constructed with the above ScFv gene sequences were transformed into Rosetta gami B competent cells, spread on LB plates containing ampicillin resistance, and cultured at 37°C overnight; the next day, single clones were picked and placed in 4ml LB medium containing ampicillin resistance for 8h, and then the bacterial solution was transferred to 200mL LB medium for further culture. When the bacterial solution OD reached 0.5-1.0, IPTG with a final concentration of 1mM was added, and expression was induced at 37°C for 12h. The bacteria were collected by centrifugation and PBS was added to resuspend the precipitate, ultrasonically broken, centrifuged for 10min, the precipitate was discarded, and the supernatant was collected for protein purification, which was purified using a GE Ni Sepharose excel purification column.

[0115] Identification of ScFv protein for PSCA antigen:

[0116] 293T and 293T-PSCA cells were each divided into 1.5mL Ep tubes, with 1×10^6 cells in each tube as target cells. After centrifugation at 400g for 5min, the supernatant was discarded and resuspended with 100μL 50μg / mL, 25μg / mL and 12.5μg / mL of the above ScFv solution, respectively. The group without ScFv and secondary antibody was used as the Blank group, and the group without ScFv and only with secondary antibody was used as the Control group. After incubation at 4℃, 1mL PBS was added to resuspend the cells after 30min, centrifuged at 400g for 5min, the supernatant was discarded, 30μL Anti-His-647 fluorescent secondary antibody for detection was added to resuspend the cells, and incubated at 4℃ for 30min in the dark; resuspended with 1mL PBS and washed twice at 400g for 5min, the supernatant was discarded and resuspended with 100μL PBS, and the flow staining positive rate was detected on the machine. The results are shown in Figure 6. Fig. 6A The recognition of PSCA antigen by ScFv clones AK-25-B11, AK-25-D02, and AK25-H08; Figure 6B The figure shows the recognition of PSCA antigen by ScFv of clone number AK-25-D03. The above ScFv can significantly recognize PSCA antigen, and can be used to detect the expression of PSCA, as a component of a kit for detecting the expression of PSCA.

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

[0118] In order to verify the function of the screened fully human antibodies, we constructed CAR vectors to prepare CAR-T cells to verify the ability of the screened fully human antibodies to recognize PSCA expressed by cells and kill tumor cells expressing PSCA.

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

[0120] The ScFv sequence of the PSCA antibody was obtained by PCR amplification, and then the sequence was connected to the lentiviral vector of the CAR structure by restriction endonuclease digestion. Here, the two structures of 8hdc-28TM-28BBz and 8h-8TM-BBz were mainly screened. The 8hdc-28TM-28BBz structure has been confirmed to have good effectiveness at the PSCA target in patent 201810079289.9. 8h-8TM-BBz is the most traditional CAR structure design. The use of these two structures can fully reflect the function of ScFv. The constructed lentiviral vector was successfully constructed after verification by sequencing comparison.

[0121] The present embodiment packages the lentivirus using the calcium phosphate method, specifically: 293T cells are cultured to an optimal state with DMEM medium containing 10% FBS (w / v), and the packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5 centrifuge tube in a certain ratio, CaCl2 and 2×HBS are added, mixed and allowed to stand at room temperature, and then added to the treated 293T cell culture medium, and the medium is changed to 10mL of DMEM medium containing 10% FBS again after 3-5h, and the cell supernatant is collected after 48h or 72h, and the virus is purified. 15 virus particles in Example 1 are obtained.

[0122] Lymphocytes were separated by gradient centrifugation; after centrifugation, the second layer of white lymphocytes was taken, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then the activated PBMCs were infected at a certain multiplicity of infection (MOI). The positive rate of CAR-T was detected on the 8th day of virus infection. The detection method was flow cytometry, and the antibody was: Protein-L-PE. Protein-L can recognize the light chain of the antibody. 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 the positive rate and expression intensity of CAR. anti-CD3 FITC (Bio legend, Lots: B378781, Cat: 300440), Protein-L-PE (Sino Biological, Lot: HR16SE0901, Cat: 11044-H07E-P).

[0123] The prepared CAR-T cells and PSCA-positive target cells RT4-Luc-GFP were plated at an effector-target ratio of 1:1, and the ACEA xCELLigence RTCA MP instrument was used to detect the killing ability of different CAR-Ts on target cells. The experimental steps were carried out according to the instrument manual. The principle of ACEA xCELLigence RTCA MP is to record the resistance index of tumor cells attached to the bottom of the well every 15 minutes, and judge the proliferation or death of the attached target cells by the resistance index. The formula for analyzing the results using the resistance index is: Cell killing rate (%) = (Cell Index value of the control group - Cell Index value of the experimental group) / (Cell Index value of the control group) × 100%. The abbreviation and structure of CAR and the results of CAR expression and killing detection are shown in Table 3: Table 3 CAR structure, abbreviation, virus titer, CAR expression and CAR-T killing rate

[0124] Table 3 shows that among the four ScFvs, AK-25-B11, AK-25-D02, AK-25-D03, and AK-25-H08, which can normally construct CAR, although the constructed CARs can all be expressed normally, only the CAR-T constructed with AK-25-D03 (also known as PSCA (9)) has a higher killing effect on tumor cells expressing PSCA, and the killing result of 76.22% is significantly higher than that of other ScFvs. Finally, 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); the 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 PSCA antigen: human bladder cancer cell line HT1376 and human pancreatic cancer cell line HPAC that naturally express PSCA were selected, and human prostate cancer cell line PC3-PSCA that exogenously expressed high expression was constructed to test the ability of AK-25-D03 (also known as PSCA (9)) to recognize PSCA antigen.

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

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

[0128] Combined with Table 3 and Table 4, AK-25-D03 (also known as PSCA (9)) screened by the present invention can specifically identify PSCA-expressing tumors such as human bladder cancer, human pancreatic cancer, and human prostate cancer, and can be used as an effective ingredient of anti-tumor drugs for malignant tumors with high PSCA expression, effectively killing malignant tumors with high PSCA expression and controlling tumor proliferation, such as application in CAR-T cell therapy.

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

[0130] Different CAR structures were designed for the optimal PSCA (9) anti-PSCA fully human ScFv screened 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, different co-stimulatory signals including CD28, CD27, 4-1BB were selected, and Z was selected for structural design to form the CAR structural design shown in Table 5 below: Table 5 Different CAR structure designs CAR Name structure 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] With Control T as the control group, the experimental group set up CAR1-CAR5 groups, and prepared CAR-T cells by the method of Example 2, and plated with PSCA-positive target cells HT1376-Luc-GFP, PC3-PSCA-Luc-GFP, and HPAC-Luc-GFP PSCA according to the effect-target ratio of 1:1, and the ACEA xCELLigence RTCA MP instrument was used to detect the killing ability of different CAR-Ts on target cells. The experimental steps were carried out according to the instrument manual. The principle of ACEA xCELLigence RTCA MP is to record the tumor cells attached to the bottom of the well every 15 minutes with the resistance index as the data, and judge the proliferation or death of the attached target cells by the resistance index. The formula for analyzing the results using the resistance index is: cell killing rate (%) = (control group Cell Index value-experimental group Cell Index value) / (control group Cell Index value) × 100%.

[0132] The killing result statistics are shown in Figure 2 below: the horizontal axis represents CAR, and the vertical axis represents the in vitro killing percentage in different cells. Figure 2A The results showed that CAR1 had significant killing effects on HPAC-Luc-GFP, HT1376-Luc-GFP and PC3-PSCA-Luc-GFP, with the killing percentages all exceeding 50%, proving that CAR1 can exert significant in vitro pharmacodynamic effects in various solid tumor cell models; Figure 2B The results showed that CAR2 had significant killing effect on PC3-PSCA-Luc-GFP, and the killing percentage was higher than 50%, proving that CAR2 can exert significant in vitro pharmacodynamic effects on PSCA-positive tumor cells; Figure 2C The results showed that CAR3, CAR4 and CAR5 had significant killing effects on HPAC-Luc-GFP, and the killing percentages were all higher than 70%, proving that the CAR-T (CAR3, CAR4 and CAR5) with different structures constructed by the fully human ScFv PSCA (9) targeting PSCA described in the present invention can exert significant in vitro pharmacodynamic effects on PSCA-positive tumor cells. The specific killing data are shown in the following table. CAR-T cell name Kill percentage (%) CAR3 73.01 CAR4 75.18 CAR5 74.45 Control-T 3.77

[0133] 6-8 week old NCG female mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used to establish an intraperitoneal tumor model using HPAC-Luc-GFP or HT1376-Luc-GFP or PC3-PSCA-Luc-GFP. 3 days after intraperitoneal injection of 3E5 tumor cells, CAR1:5E5 CAR-T / CAR-T cells were intraperitoneally administered. Live imaging of mice was performed every 7 days after administration, and the imaging fluorescence values ​​were statistically analyzed using T Test for significance. 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 tumor model and the HT1376-Luc-GFP tumor model, demonstrating that CAR1 constructed based on the fully human ScFv PSCA (9) targeting PSCA described in the present invention has a good killing ability against human bladder cancer and pancreatic cancer. Figure 3C The results showed that CAR2 had significant efficacy in the PC3-PSCA-Luc-GFP tumor model, demonstrating that CAR2 constructed based on the fully human ScFv PSCA (9) targeting PSCA described in the present invention has a good killing ability against human prostate cancer.

[0134] In summary, all the data of Example 2 and Example 3 prove that PSCA (9) can recognize PSCA-positive tumor cells and kill them in in vitro functional verification when combined with different promoters, i.e., common promoter, hypoxia promoter, different hinges, i.e., 8h, 8hdc and G4h, different transmembrane 8TM and 28TM, and different intracellular signals CD28, 4-1BB, CD27, and can play a significant pharmacodynamic function of killing tumor cells 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 be used as the antigen recognition region of the 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, including the treatment of pancreatic cancer, prostate cancer, bladder cancer, gastric cancer, lung cancer, breast cancer, oral squamous cell carcinoma and gallbladder cancer by constructed CAR-T cells of multiple structures.

[0135] Example 4 Single-chain antibody PSCA (9) as the CAR structural antigen recognition region completed the application verification of dual CAR

[0136] Dual CARs were designed based on PSCA (9). The dual CAR design took into account the combination of PSCA and TIGIT. The single CAR of PSCA (9) used different hinges 7h, 8h and G4h, different transmembrane 8TM and 28TM, and different intracellular signals including CD28, 4-1BB, and CD28-4-1BB, and was combined with TIGIT to form a dual CAR. The single CAR of TIGIT also used different transmembrane 8TM, 28TM, and different intracellular signals CD28, 4-1BB and CD134. The specific design is shown in Table 6. All structures were confirmed by in vitro killing to have obvious in vitro killing effects on PSCA-positive tumor cells, verifying the versatility of PSCA (9) for different dual-target CAR structures. Table 6: Dual CAR structure design CAR Name structure 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 same detection method as the PSCA positive antigen in Example 2 was used to detect the expression of CD155 and PSCA, wherein the anti-CD155 antibody: Brilliant Violet anti-human CD155 (Biolegend, Lot: B338257, Cat: 337632), the anti-PSCA antibody: PSCA (7F5) Santa Cruz, Sc-80654, Lot: J1821. The detection results are shown in Table 7 below: CD155 and PSCA are highly expressed in the human bladder cancer cell line HT-1376-Luc-GFP; CD155 is highly expressed in the human pancreatic cancer cell line HPAC-Luc-GFP, and PSCA is moderately highly expressed; both cell lines can represent human bladder cancer and human pancreatic cancer indications for the verification of PSCA dual-target CAR-T. Table 7: Expression of CD155 and PSCA in HT1376-Luc-GFP and HPAC-Luc-GFP cells cell CD155 expression positive rate PSCA expression positive rate HT-1376-Luc-GFP 100％ 100％ HPAC-Luc-GFP 100％ 60％ The steps of Example 2 were used to prepare CAR-T cells and detect CAR expression. The results are shown in Table 8 below: Table 8: CAR virus titer and expression data CAR Name CAR titer (TU / mL) CAR expression (%) 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 effectiveness validation was performed according to the method of Example 3 to verify the effectiveness of the dual CAR containing PSCA (9) in different indications. The results are shown in FIG4 . Figure 4AThe results showed that both CAR6 and CAR11 had significant killing effects on HPAC-Luc-GFP and HT1376-Luc-GFP. 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 CAR was significantly higher than that of single CAR. Figure 4B The results showed that CAR7, CAR8, CAR9 and CAR10 had significant killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP (prostate cancer) and HT1376-Luc-GFP, and the killing percentages were all higher than 60%, proving that dual CARs with different structures can exert obvious in vitro pharmacodynamic effects in various solid tumor cell models.

[0139] In summary, for different PSCA-positive indications, including but not limited to human pancreatic cancer, prostate cancer and bladder cancer, any dual CAR structure containing PSCA (9) has significant killing function. In addition to the dual CAR design of PSCA and CD155 targets mentioned above, these dual CAR structures can also be PSCA and solid tumor targets 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, B7H3, etc.

[0140] Example 5: Single-chain antibody PSCA (9) as the extracellular recognition region of the CAR structure completes the fusion protein form of the CAR combination construction and can exert better in vivo therapeutic effects

[0141] In terms of CAR structure design, in addition to the traditional single CAR structure and dual CAR structure, there are also combined modes of CAR structure and fusion protein and functional polypeptide. These combinations can be CAR and fusion protein and functional polypeptide constructed in the same vector for expression, or CAR targeting PSCA and fusion protein and functional polypeptide divided into two vectors for co-transfer to target immune cells. The fusion protein or functional polypeptide can be a fusion protein containing the extracellular segment or extracellular recognition functional domain of immune checkpoints such as PD1 / PDL1, TIGIT, SIRPγ, SIRPα, etc., or a fusion protein containing the full length, functional domain and / or signal domain of cytokines such as IL15, IL2, IL21, IL12, IL7, etc., or a full length or fusion protein containing chemokines and chemokine receptors such as CXCR3, CXCR2, CCL19, CCL21, etc.

[0142] Here, TIGIT fusion protein is used as an example to verify the feasibility of the combined mode of CAR containing single-chain antibody PSCA (9) and fusion protein and functional peptide. The CAR structure design is shown in Table 9 below: Table 9 CAR structure design combined with fusion protein CAR Name structure 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] According to the scheme of Example 2, CAR virus preparation, CAR-T preparation and in vitro killing verification were completed. The expression results of CAR titer are shown in Table 10 below: Table 10 CAR virus titer and expression data CAR Name CAR titer (TU / mL) CAR expression (%) 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 of three cell types HPAC-Luc-GFP, PC3-PSCA-Luc-GFP, and HT1376-Luc-GFP of three indications of pancreatic cancer, prostate cancer, and bladder cancer by CAR combined with fusion proteins of different structures and CAR-T expressing fusion proteins alone. The results showed that CAR16 had obvious killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP, and HT1376-Luc-GFP, while CAR17 had no in vitro killing function, which proved that the fusion protein itself did not play a role in in vitro killing, but the killing function in vitro was significantly enhanced after the PSCA single CAR combined with TIGIT formed a fusion protein. Figure 5B The inventors further designed more CAR structures containing PSCA (9) and fusion proteins of different structures for combination verification, and completed the preparation of CAR virus according to the scheme of Example 2. The results are shown in Table 12 below, which verify the versatility of the combination of CAR structures containing PSCA (9) and fusion proteins. The results show that CAR12, CAR13, CAR14 and CAR15 all have significant killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP and HT1376-Luc-GFP, proving that CARs in the form of fusion proteins of different structures can exert obvious in vitro pharmacodynamic effects in various solid tumor cell models.

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

[0146] It should be noted that although the present invention takes T cells and CAR-T cells as examples, based on the content disclosed in the present invention, a person skilled in the art can obviously infer that other immune cells such as NK cells, macrophages, DC cells and related precursor cells can also express the engineered immune cells containing CAR of PSCA (9) described herein, and specifically recognize PSCA, and play a killing role against malignant tumors expressing PSCA. PSCA (9) described in the present application as a chimeric antigen receptor constructed as an extracellular antigen recognition domain or one of the extracellular antigen recognition domains can be expressed in T cells, NK cells, macrophages, DC cells and related precursor cells; it can be co-expressed with fusion proteins of any structure in T cells, NK cells, macrophages, DC cells and related precursor cells, and used as a drug or drug combination for malignant tumors expressing PSCA for tumor treatment.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in 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 amino acid sequence of the L-CDR1 is QGILTY, the amino acid sequence of the L-CDR2 is AAS, the amino acid sequence of the L-CDR3 is LQDYNFPRT, the amino acid sequence of the H-CDR1 is GYTFTDYY, the amino acid sequence of the H-CDR2 is INPYNGGT, The amino acid sequence of H-CDR3 is ARSVYDYPFDY.

2. The polypeptide of claim 1, comprising a light chain variable region represented by amino acids as shown in SEQ ID NO.

8.

3. The polypeptide of claim 1, comprising a heavy chain variable region having amino acids as shown in SEQ ID NO.

9.

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

9.

5. The single-chain antibody recognizing PSCA according to claim 4, further comprising a linker sequence, the amino acid sequence of which is shown in SEQ ID NO.

10.

6. A kit for detecting PSCA expression, characterized in that: Comprising the antigen-binding polypeptide of claims 1-5.

7. A chimeric antigen receptor, characterized in that It comprises an extracellular antigen recognition domain that can recognize PSCA antigen, and the extracellular antigen recognition domain comprises the antigen-binding polypeptide targeting PSCA according to claims 1-5.

8. The chimeric antigen receptor according to claim 7, characterized in that It also contains 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 or a functional variant thereof: SEQ ID NO.13, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.

26.

10. The chimeric antigen receptor according to claim 9, characterized in that It also comprises a hinge region, a transmembrane region and an intracellular signaling domain, wherein the transmembrane region is selected from all or part of or a mutant sequence of the following protein transmembrane regions: α, β or chain of T cell receptor, CD8, CD28, CD3ε, CD4, CD16, CD137, CD80 or CD86, etc.; preferably, the transmembrane region comprises one of the following optional amino acid sequences or a functional variant thereof: SEQ ID NO.12, SEQ ID NO.

27.

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

17.

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 or one or more of their functional variants: SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.

20.

13. A nucleic acid sequence, characterized in that Encoding the chimeric antigen receptor according to claim 7.

14. The nucleic acid sequence encoding the chimeric antigen receptor according to claim 13, characterized in that: The nucleic acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.

44.

15. The nucleic acid sequence of the chimeric antigen receptor according to claim 13, characterized in that: The chimeric antigen receptor further comprises a promoter, and the nucleic acid sequence of the promoter is shown as SEQ ID NO.47 or SEQ ID NO.

48.

16. The nucleic acid sequence encoding the chimeric antigen receptor according to claim 15, characterized in that: The nucleic acid sequence of the chimeric antigen receptor is shown as SEQ ID NO.45 or SEQ ID NO.

46.

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

18. The vector according to claim 17, characterized in that It also contains a TIGIT mutant nucleotide sequence as shown in any one of SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.

42.

19. An engineered cell, characterized in that The cell expresses the polypeptide or single-chain antibody according to any one of claims 1 to 5, or the chimeric antigen receptor according to any one of claims 7 to 12, or the cell comprises the nucleic acid or vector according to claims 13 to 18.

20. The engineered cell according to claim 19, characterized in that The amino acid sequence of the expressed TIGIT or its functional variant protein is as shown in any one of SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.

25.

21. The engineered cell according to claim 20, characterized in that The cells also express a fusion protein comprising TIGIT or a functional variant thereof; preferably, the expressed TIGIT fusion protein structure is TIGIT-28TM-28 or 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 signal structures derived from human CD28 and human CD134, respectively.

22. The engineered cell of claim 21, characterized in that The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.

51.

23. The engineered cell of claims 19-22, characterized in that: Also included are 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, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma associated antigen 1), CD155 or TAG-72, or any combination of multiple targets.

24. The engineered cell of claim 23, characterized in that The cells are immune cells such as T cells, T cell precursor cells, NK cells, DC cells, macrophages, etc.

25. The engineered cell of claim 24, characterized in that For T lymphocytes.

26. A cell product, characterized in that The cell product comprises the engineered cells of claims 19-25.

27. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide or single-chain antibody according to any one of claims 1-5 or the nucleic acid or vector according to claims 13-18, the engineered cell according to any one of claims 19-25 or the cell product according to claim 26.

28. Use of the polypeptide or single-chain antibody according to any one of claims 1 to 5, the nucleic acid or vector according to claims 13 to 18, the engineered cell according to any one of claims 19 to 25, the cell product according to claim 26, the pharmaceutical composition according to claim 27, or the chimeric antigen receptor according to claims 7 to 12 in the preparation of a drug for tumors expressing PSCA targets.

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