CN114835781B - WTN polypeptide and application thereof in detection and treatment of cancer - Google Patents

Abstract

The invention provides a WTN polypeptide and application thereof in detection and treatment of cancers, and also provides a polypeptide compound comprising the WTN polypeptide, a coding DNA molecule thereof, a carrier, a host cell and a pharmaceutical composition in which the polypeptide compound is positioned, and a kit for detecting PSMA, wherein the application of the kit comprises detection and treatment of prostate cancer.

Description

WTN polypeptide and application thereof in detection and treatment of cancer

Technical Field

The invention relates to the technical field of biology, in particular to a WTN polypeptide and application thereof in detecting and treating cancers.

Background

Malignant tumors are one of the major diseases that endanger human health. Traditional tumor treatment modalities such as surgery, radiation therapy, and chemotherapy have been the main strategies for tumor treatment in recent decades, however, patients develop resistance to drugs and radiation therapy, resulting in the recurrence of high frequency tumor patients.

The tumor cell therapy has received great attention due to its advantages of specific targeting, remarkable effect, few side effects, etc., and gradually becomes an important means in the comprehensive treatment of tumors, which is called green therapy of tumors in the industry and is also a hotspot and development direction of the current basic research and clinical application of tumor therapy. There have been many important advances in the development of immune cell-based tumor cell therapy. Among the many different types of tumor cell therapy, one of the most promising tumor cell therapies being developed is immune cells expressing chimeric antigen receptors (CAR-T cells, CAR-NK cells).

Chimeric Antigen Receptors (CARs) are genetically engineered receptors that are designed to target specific antigens (e.g., tumor antigens). This targeting specificity can lead to cytotoxicity to the tumor, e.g., so that immune cells expressing the CAR can specifically target and kill tumor cells.

The development of Chimeric Antigen Receptor T cell (CAR-T) technology, CAR-T can be divided into three generations. The first generation CAR-T cells consist of an extracellular binding region-single chain antibody (scFV), a transmembrane domain (TM), and a signaling domain-immunoreceptor tyrosine-based activation motif (ITAM), wherein the chimeric antigen receptor portions are linked as follows: scFv-TM-CD3 ζ. This CAR-T cell can elicit anti-tumor cytotoxic effects, but cytokine secretion is relatively low and does not elicit a lasting anti-tumor effect in vivo. Subsequently developed second generation CAR-T cells incorporate the co-stimulatory domain of CD28 or CD137 (also known as 4-1 BB), where the chimeric antigen receptor portions are linked as follows: scFv-TM-CD28-ITAM or scFv-TM-CD137-ITAM. The costimulation effect of B7/CD28 or 4-1BBL/CD137 generated by the signal transduction structural domain causes the continuous proliferation of T cells, and can improve the level of cytokines such as IL-2 and IFN-gamma secreted by the T cells, and simultaneously improve the survival cycle and the anti-tumor effect of CAR-T in vivo. A third generation CAR-T cell developed in recent years in which the chimeric antigen receptor portions are linked as follows: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further improve the survival cycle of CAR-T in vivo and its anti-tumor effects. At present, the structural pattern of CAR used in CAR-NK is essentially followed by the design of CAR-T.

The WTN polypeptide with better specific binding efficiency with the tumor is screened by a phage display technology, and the specificity of the polypeptide is verified. After being combined with a detectable marker, the WTN polypeptide can be used for detecting a cancer marker PSMA, and CAR-T or CAR-NK cells prepared from the WTN polypeptide can accurately target cells expressing PSMA.

Disclosure of Invention

The invention provides WTN polypeptides specifically binding to PSMA (prostate specific membrane antigen), and complexes, conjugates, DNA molecules, vectors, and host cells thereof.

In a first aspect, the invention provides a WTN polypeptide specifically binding to PSMA, wherein the WTN polypeptide has a homology of 90% or more with the polypeptide shown in SEQ ID NO. 1, and specifically, the polypeptide has a homology of 90%, 91%,92%,93%,94%,95%,96%,97%,98%,99%,100% with the polypeptide shown in SEQ ID NO. 1.

Preferably, the WTN polypeptide is substituted, deleted or added at one or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 of the polypeptide shown in SEQ ID NO. 1.

Preferably, the WTN polypeptide is a polypeptide represented by SEQ ID NO. 1.

Preferably, the WTN polypeptide is a product obtained by chemically modifying amino, carboxyl, sulfhydryl, phenolic hydroxyl, imidazolyl, guanidino, indolyl, methylthio and other sites at the tail end of the main chain or side chain of the polypeptide shown in SEQ ID NO. 1.

Preferably, the chemical modification includes acetylation, amidation, glycosylation, polyethylene glycol (PEG) modification, fatty acid modification, and other polypeptide modification techniques known in the art.

Preferably, the acetylation and amidation include acetylation of the N-terminal end of the polypeptide backbone and amidation of the C-terminal end of the polypeptide backbone.

Preferably, the glycosylation modification includes N-glycosylation, O-glycosylation, S-glycosylation, C-glycosylation and glycosylphosphatidylinositol modification.

Preferably, the N-glycosylation is attachment of the amide nitrogen of the side chain by asparagine.

Preferably, the O-glycosylation is linked to an oxygen on a serine or threonine residue.

Preferably, the sugar structures include various monosaccharides, oligosaccharides, and polysaccharides.

Preferably, the PEG modification types include linear PEG, branched PEG, homo-bifunctional PEG derivatives, hetero-functional disubstituted PEG derivatives and multi-arm functional PEG derivatives.

Preferably, the fatty acid modifications can be divided into unsaturated fatty acid and saturated fatty acid modifications.

Preferably, the saturated fatty acids include myristic acid, palmitic acid.

Preferably, the unsaturated fatty acid modification comprises oleic acid, linoleic acid.

In a second aspect, the invention provides a polypeptide complex comprising a domain linked by peptide bonds at the amino-and/or carboxy-terminus of the WTN polypeptide;

preferably, the domain is composed of amino acids;

preferably, the domain comprises a hinge region, a transmembrane domain and/or a signaling domain;

preferably, the hinge region comprises a combination of one or more of a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, an ICOS hinge region.

Preferably, the hinge region is a CD8 alpha hinge region, the amino acid sequence of which is shown in SEQ ID NO. 3, and the corresponding coding nucleic acid sequence is shown in SEQ ID NO. 4.

Preferably, the transmembrane domain comprises a transmembrane domain of a protein comprising: 2B4 gene expression, the α, β or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 and CD154.

Preferably, the transmembrane domain is a protein expressed from the 2B4 gene (i.e., a 2B4 transmembrane structure), the amino acid sequence of which is shown in SEQ ID NO. 5, and the corresponding coding nucleic acid sequence of which is shown in SEQ ID NO. 6.

Preferably, the signalling domain comprises a co-stimulatory domain and/or a primary signalling domain.

Preferably, the costimulatory domain includes 2B4, CD3 zeta, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), and 4-1BB (CD 137) functional signaling domains.

Preferably, the co-stimulatory domain is a 2B4 co-stimulatory signal, the amino acid sequence of which is shown in SEQ ID NO. 7, and the corresponding coding nucleic acid sequence of which is shown in SEQ ID NO. 8.

Preferably, the primary signaling domain comprises signaling regions of one or any combination of proteins of NKG2D primary signaling, CD 3-zeta, fcsri gamma, fcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD 66D.

Preferably, the primary signaling domain is the NKG2D primary signal, the amino acid sequence of which is shown in SEQ ID NO. 9 and the corresponding coding nucleic acid sequence of which is shown in SEQ ID NO. 10.

Preferably, the polypeptide complex is a tumor antigen binding peptide.

Preferably, the WTN polypeptide region comprises a linker between the plurality of repeats of the WTN polypeptide and the plurality of repeats of the WTN polypeptide.

Preferably, the plurality may be 1, 2, 3, 4, 5.

Preferably, the plurality is 3.

Preferably, the 3 WTN polypeptides are linked by a linker.

Preferably, the amino acid sequence of the linker is GGGS.

Preferably, the composition of the tumor antigen binding peptide is WTN polypeptide region-hinge region-transmembrane domain-costimulatory domain-primary signaling domain or WTN polypeptide region-hinge region-transmembrane domain-primary signaling domain.

Preferably, the tumor antigen binding peptide consists of the WTN polypeptide region-hinge region-transmembrane domain-co-stimulatory domain-primary signaling domain,

preferably, the tumor antigen binding peptide consists of WTN polypeptide region-CD 8 alpha hinge region-2B 4 transmembrane domain-2B 4 costimulatory domain-NKG 2D primary signaling domain, and the amino acid sequence thereof is shown in SEQ ID NO. 11, and the corresponding coding nucleic acid sequence is shown in SEQ ID NO. 12.

In a third aspect, the invention provides a DNA molecule encoding the WTN polypeptide described above.

Preferably, the DNA molecule sequence of the WTN polypeptide is shown in SEQ ID NO. 2.

In a fourth aspect, the invention provides a DNA molecule encoding the aforementioned polypeptide complex.

Preferably, the polypeptide complex is the aforementioned tumor antigen binding peptide.

In a fifth aspect, the present invention provides a vector comprising the aforementioned DNA molecule encoding a polypeptide and/or the aforementioned DNA molecule of a polypeptide complex.

Preferably, the vector is an expression vector.

Preferably, the expression vector further comprises a promoter and a transcription termination sequence operably linked thereto.

Preferably, the expression vector is a plasmid expression vector or a viral expression vector.

The plasmid expression vectors include, but are not limited to, pcDNA3.1+/-, pcDNA4/HisMax B, pSecTag 2A, pVAX1, pBudCE4.1, pTracer CMV2, pcDNA3.1 (-)/Myc-His A, pcDNA6-Myc/His B, pCEP4, pIRES, pIRESneo, pIRES hyg3, pCMV-Myc, pCMV-HA, pIRES-puro3, pIRES-neo3, pCAGGS, pSilencerr 1.0, pSilencerr 2.1-U6 hygro, pSilencerr 3.1-H1neo, and pSilencerr 4.1-CMV neo.

Preferably, the viral expression vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral expression vector or other types of viral vectors.

Preferably, the viral expression vector includes, but is not limited to, pLKO.1, pLVX-IRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCV-MCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC-CMV 1, pBHGE3, pAAV-MCS, pAAV-RC, pHelper, pALKZ-LacZ, pLK0.1-Puro, p0.1-CMV-1, pLKO-LKO-1, pLKO 0.1-CMV-Neo, pLKO 0.1-Neo, pLKO 1-Neo-CMV-tGFP, pLKO 1-Puro-CMV-tagCFP, pLKO 1-Puro-CMV-tagYFP, pLKO l-Puro-CMV-tagGFP, pLKO 1-Puro-CMV-tagFP635, pLKO. PLKO-Puro-IPTG-1xLacO, pLKO-Puro-IPTG-3xLacO, pLPl, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminshin, pcDNAl,2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP and Lenti6.2/N-Lumio/V5-GW/lacZ.

In a sixth aspect, the invention provides a host cell comprising one or more of the aforementioned polypeptide, the aforementioned polypeptide complex, the aforementioned DNA molecule encoding a WTN polypeptide, the aforementioned DNA molecule of the aforementioned polypeptide complex, and the aforementioned vector.

Preferably, the host cell includes prokaryotic cells and eukaryotic cells.

Preferably, the prokaryotic cell is a bacterial cell, such as Agrobacterium, E.coli.

Preferably, the bacterial cells comprise gram negative and gram positive microorganisms.

Preferably, the eukaryotic cell is a fungal cell, i.e., a yeast cell.

Preferably, the eukaryotic cell is a mammalian cell, an insect cell, a plant cell, or an algal cell.

Preferably, the mammalian cells include cells of human or non-human origin.

Preferably, the human cell is an immune cell.

Preferably, the immune cells comprise one or more of T cells, B cells, K cells and NK cells.

Preferably, the immune cell is an NK cell or a T cell.

Preferably, the immune cells are autologous or allogeneic.

Preferably, the immune cells are derived from mononuclear cells of autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood and the like.

Preferably, the host cell includes commercially available cell lines, such as 293 cells, 293T cells, 293FT cells, 293LTV cells, 293EBNA cells, SW480 cells, u87MG cells, HOS cells, C8166 cells, MT-4 cells, molt-4 cells, heLa cells, HT1080 cells, TE671 cells, COS1 cells, COS7 cells, CV-1 cells, BMT10 cells.

In a seventh aspect, the invention provides a pharmaceutical composition comprising one or more of the aforementioned polypeptide, the aforementioned polypeptide complex, the aforementioned DNA molecule encoding a WTN polypeptide, the aforementioned DNA molecule of the aforementioned polypeptide complex, the aforementioned vector, and the aforementioned host cell.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients and/or additives.

Preferably, the excipients include, but are not limited to, buffer systems, thickeners, stabilizers, neutralizing agents, humectants.

Preferably, the additives include, but are not limited to, fillers, binders, moisturizers, glidants, stabilizers, preservatives, emulsifiers.

Preferably, the route of administration of the pharmaceutical composition is enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oromucosal, ocular, pulmonary and respiratory, dermal, vaginal, rectal, and the like.

Preferably, the pharmaceutical composition may be administered in a liquid, solid or semi-solid dosage form.

Preferably, the liquid dosage form can be solution (including true solution and colloidal solution), emulsion (including o/w type, w/o type and double emulsion), suspension, injection (including water injection, powder injection and infusion), eye drop, nose drop, lotion, liniment, etc.

Preferably, the solid dosage form can be tablet (including common tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (including hard capsule, soft capsule, enteric-coated capsule), granule, powder, pellet, dripping pill, suppository, pellicle, patch, aerosol (powder), etc.

Preferably, the semi-solid dosage form may be an ointment, gel, paste, or the like.

Preferably, the pharmaceutical composition can be prepared into common preparations, and can also be prepared into sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.

In an eighth aspect, the invention provides a conjugate of a WTN polypeptide that specifically binds to PSMA.

Preferably, the conjugate is a detectable label attached to the WTN polypeptide.

Preferably, the linkage is covalent bonding or physisorption.

Preferably, the linkage is a non-peptide linkage.

Preferably, the detectable label does not include a natural amino acid component.

Preferably, the detectable label comprises a radioactive label, a chemiluminescent label, a fluorescent label, a quantum dot, a thermometric label, or an immunopolyase chain reaction label.

Preferably, the radioactive labels include, for example, 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho and 153Sm.

Preferably, the chemiluminescent label comprises an acridinium ester, a thioester, a sulfonamide, luminol, isoluminol, a phenanthridinium ester.

Preferably, the fluorescent marker comprises 5-fluorescein, 6-carboxyfluorescein, 3' 6-carboxyfluorescein, 5 (6) -carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, rhodamine, phycobiliprotein and R-phycoerythrin.

Preferably, the fluorescent marker is a fluorescent molecule.

Preferably, the fluorescent molecule is FITC.

Preferably, the detectable label may emit a detectable signal; preferably, the detectable signal comprises an optical signal or an electrical signal. Preferably, the optical signal comprises a fluorescent signal, a light absorption signal, an infrared absorption signal, a raman scattering signal or a chemiluminescent signal.

In a ninth aspect, the present invention provides a method for producing the host cell, the method comprising the step of introducing one or more of the DNA molecule encoding WTN polypeptide, the DNA molecule of the polypeptide complex, and the vector into the cell.

Preferably, the method for introducing the cells includes heat shock method, calcium phosphate precipitation, transfection method, particle bombardment, microinjection, electroporation, and the like.

In a tenth aspect, the invention provides a method of producing a WTN polypeptide as hereinbefore defined, the method comprising culturing a host cell as hereinbefore defined in a culture environment suitable for high-level expression of the protein;

preferably, the method further comprises the step of extracting and purifying the polypeptide.

In an eleventh aspect, the invention provides a method for detecting PSMA, the method comprising the step of contacting a conjugate of a WTN polypeptide as hereinbefore described with a sample to be detected.

Preferably, the method further comprises the step of processing the sample.

Preferably, the detection is for non-diagnostic purposes.

Preferably, the sample to be detected is a sample suspected of containing PSMA.

Preferably, the sample may be a cell, but also a specimen or culture (including a microbial culture), even including specimens of synthetic origin.

Preferably, the sample is derived from a virus, bacterium, microorganism, soil, water source, human, animal, plant, or the like.

Preferably, the samples include tissue sections, such as biopsy and autopsy samples, and frozen sections for histological purposes. Such samples include bodily fluids such as blood and blood components or products (e.g., serum, plasma, platelets, red blood cells, etc.), sputum, tissue, cultured cells (e.g., primary cultures, explants and transformed cells) stool, urine, synovial fluid, joint tissue, synovial cells, fibroblast-like synovial cells, macrophage-like synovial cells, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, and the like.

Preferably, the method uses an immunoassay method including, but not limited to, an enzyme linked immunoassay, a radioimmunoassay, a fluoroimmunoassay, a chemiluminescent immunoassay, an electrochemiluminescent immunoassay, and the like.

According to a twelfth aspect of the invention, there is provided a kit for detecting PSMA, the kit comprising a conjugate of the WTN polypeptide as defined above;

preferably, the kit further comprises one or more of a sample processing solution, a buffer solution, an ionic strength regulator, a surfactant and a preservative.

Preferably, the kit further comprises the instruments or devices required for the detection of PSMA.

In a thirteenth aspect, the invention provides the use of the aforementioned polypeptide, the aforementioned polypeptide complex, the aforementioned DNA molecule encoding a WTN polypeptide, the aforementioned DNA molecule of a polypeptide complex, or the aforementioned vector or the aforementioned host cell in the preparation of the aforementioned pharmaceutical composition.

In a fourteenth aspect, the present invention provides use of the aforementioned polypeptide, the aforementioned polypeptide complex, the aforementioned DNA molecule encoding a WTN polypeptide, the aforementioned DNA molecule of the polypeptide complex, or the aforementioned vector for producing the aforementioned host cell.

The fifteenth aspect of the invention provides the use of the DNA molecule encoding a WTN polypeptide, the DNA molecule of the polypeptide complex, as described above, in the preparation of the vector as described above.

In a sixteenth aspect, the present invention provides the use of the polypeptide, the polypeptide complex, the DNA molecule encoding a WTN polypeptide, the DNA molecule of the polypeptide complex, the vector, the host cell or the pharmaceutical composition for the manufacture of a medicament for the treatment of cancer.

Preferably, the cancer comprises cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, colorectal cancer, breast cancer, skin squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, thyroid cancer, bladder transitional epithelial cancer, leukemia, brain tumor, stomach cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, cancer of the female reproductive tract, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma;

preferably, the cancer is prostate cancer.

The seventeenth aspect of the present invention provides the use of the aforementioned polypeptide, the aforementioned polypeptide complex, the aforementioned DNA molecule encoding a WTN polypeptide, the aforementioned DNA molecule of the polypeptide complex, the aforementioned vector, the aforementioned host cell, or the aforementioned pharmaceutical composition for the manufacture of a kit for diagnosing cancer.

Preferably, the cancer is prostate cancer.

Preferably, the diagnosis is detected PSMA.

Preferably, the kit is the aforementioned kit.

An eighteenth aspect of the present invention provides a method for treating cancer, which comprises administering the aforementioned polypeptide, the aforementioned polypeptide complex, the aforementioned DNA molecule encoding a WTN polypeptide, the aforementioned DNA molecule of the aforementioned polypeptide complex, the aforementioned vector, the aforementioned host cell, or the aforementioned pharmaceutical composition to a subject.

Drawings

FIG. 1 is a graph showing the results of immunofluorescence of Lncap cell lines with higher expression levels of polypeptides WTN and PSMA.

FIG. 2 is a graph showing the immunofluorescence results of PC3 cell lines with low expression of polypeptides WTN and PSMA.

FIG. 3 is a graph showing the results of flow assays of C4-2 GFP cells and TABP-EIC-WTN cells cultured together for 2, 6, 12, and 24 hours.

FIG. 4 is a graph showing the results of measuring IFN-. Gamma.secretion levels by ELISA.

FIG. 5 is a graph showing the results of measuring the expression level of PD-L1 on C4-2 cells at different concentrations of IFN-. Gamma.;

FIG. 6 is a graph showing the results of measuring the expression level of PD-L1 on C4-2 cells co-cultured with TABP-EIC-WTN cells for 24 hours.

FIG. 7 is a graph showing the results of measuring the expression level of PD-L1 in TABP-EIC-WTN cells co-cultured with C4-2 cells.

FIG. 8 is a graph showing the results of measuring the expression level of PD-1 in TABP-EIC-WTN cells co-cultured with C4-2 cells.

FIG. 9 is a graph showing the results of measuring the expression level of PD-L1 in TABP-EIC-WTN cells stimulated by IFN-. Gamma.at different concentrations.

FIG. 10 is a graph showing the results of flow cytometry plots and summary data, in which the expression amount of PD-L1 on TABP-EIC-WTN cells cultured using a common medium and the supernatant obtained after co-culturing TABP-EIC-WTN + C4-2 cells for 12 hours showed no significant difference in the percentage of PD-L1 expression in TABP-EIC-WTN cells cultured in both media.

FIG. 11 is a schematic view of a cell co-incubation device in which C4-2 cells are seeded at the bottom and TABP-EIC-WTN cells are seeded at the upper region, the inner and outer spaces being separated by a filter membrane which allows cytokines to pass through but prevents TABP-EIC-WTN cells from coming into direct contact with C4-2 cells.

Fig. 12 is a graph of flow cytometry plots and the results of summary data.

FIG. 13 is a graph showing the results of analysis of the transcription differences between co-cultured/cultured TABP-EIC-WTN/C4-2 cells alone, in which: volcano plot of deregulated genes between co-cultured TABP-EIC-WTN and separately cultured TABP-EIC-WTN cells, panel B: volcano patterns of deregulated genes between co-cultured C4-2 and C4-2 cells cultured alone.

Figure 14 is a graph of the results of KEGG pathway enrichment analysis, panel a: up-regulated genes in co-cultured TABP-EIC-WTN, panel B: down-regulated genes in co-cultured TABP-EIC-WTN, panel C: upregulation of genes in co-cultured C4-2 cells.

FIG. 15 is a diagram showing the results of flow assay, panel A: expression level of NKG2D on TABP-EIC-WTN cells, FIG. B: MICA/B expression level on C4-2 cells.

FIG. 16 is a graph showing the results of Western blot analysis of proteins involved in the signal pathway of PD-L1 expression, in FIG. A: graph of assay results without addition of NKG2D blocking agent, graph B: graph showing the results of detection when NKG 2D-blocking agent was added.

FIG. 17 is a graph showing the statistical results of the expression levels of proteins involved in the signal pathway of PD-L1 expression, A: PD-L1, panel B: p-PI3K, panel C: p-AKT, panel D: p-mTOR, E Panel: p-JAK1, panel F: p-JAK2, G panels: p-STAT1.

FIG. 18 is a graph showing the results of measurement of bioluminescence intensity (BLI) of C4-2 GFP cells co-cultured with TABP-EIC-WTN, in which: test result chart, B chart: and (5) a statistical result graph.

FIG. 19 is a graph showing the results of increasing the tumor cell inhibition rate of TABP-EIC-WTN in terms of atezolizumab or nivolumab, in which A is: e: t =1:1, B diagram: e: t =5:1.

FIG. 20 is a graph showing the results of measurement of IFN-. Gamma.secretion from TABP-EIC-WTN cells when Atezolizumab or nivolumab was added.

Fig. 21 is a graph of flow cytometry plots and summary data results, panel a: flow cytometry plot, panel B: FIG. 1 is a graph showing the expression of CD107a induced by C4-2 cells on TABP-EIC-WTN cells (when atezolizumab or nivolumab is added), the expression of TABP-EIC-WTN cells and C4-2 cells being expressed at a ratio of 1:1 ratio at 37 ℃ for 20 hours, followed by collection and treatment of TABP-EIC-WTN cells with atezolizumab (20. Mu.g/mL) or nivolumab (20. Mu.g/mL), and then detection.

FIG. 22 is a graph showing the results of measurement of tumor sizes in groups of mice on days 7, 14, and 21.

FIG. 23 is a graph showing the results of tumor suppression with and without co-cultured CIK, panel A: tumor suppression without co-cultured CIK, panel B: tumor suppression with co-cultured CIK.

FIG. 24 is a photograph of immunohistochemical staining of tumor cells, panel A: staining result graph, panel B: and (5) a statistical result graph.

Fig. 25 is a graph showing statistical results of statistics of tumor sizes of mice treated with and without co-cultured CIK, respectively, a graph: CIK with co-culture, panel B: no co-cultured CIK.

FIG. 26 is a graph showing a real image of a tumor after resection and a statistical result of tumor weight, in which FIG. A: material object diagram, B diagram: and (6) counting the results.

FIG. 27 shows the measurement of the expression levels of CD3, CD8 and CD56 in CIK co-cultured in Panel A: control, panel B: CD3, panel C: CD8, panel D: CD56, panel E: CD3 CD56, panel F: and (5) a statistical result graph.

FIG. 28 is a graph showing the results of tumor growth in mice from the control group, CIK-treated group and co-cultured CIK-treated group, panel A: tumor growth in mice, panel B: statistical analysis of BLI measurements.

FIG. 29 is a graph showing the statistical results of tumor volumes of mice in the control group, CIK-treated group and co-cultured CIK-treated group on days 5, 10 and 20.

Figure 30 is a graph of serum testosterone and PSA levels after ADT in a prostate cancer patient, panel a: serum testosterone, panel B: and (3) PSA.

Fig. 31 is a graph of HE staining of tumor tissue of prostate cancer patients (100-fold and 200-fold), panel a: 100 times, panel B: 200 times.

Fig. 32 is a flow cytometry plot and summary data histogram, panel a: flow cytometry plot, panel B: statistical result graph, graph C: statistical results are shown in the figure, wherein the expression of PD-L1 on primary prostate cancer cells incubated with TABP-EIC-WTN cells for 24 hours, (right) CCK-8 assay results showing that atezolizumab (20 μ g/mL) rather than nivolumab (20 μ g/mL) significantly enhances the inhibitory effect on TABP-EIC-WTN, E: t =1:1.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can apply the above teachings and modifications to the equivalent embodiments with equivalent variations. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 peptide library screening

1. Purpose of the experiment

The invention adopts a Ph.D. -12 phage display peptide library kit to screen out the polypeptide WTN specifically combined with PSMA.

2. Ph.D. -12 phage display peptide library kit composition

Random dodecapeptide phage display library: 100 μ L, 1.5X 10 ¹³ pfu/mL, stored in TBS solution with 50% Glycerol, complexity 2.7X 10 ⁹ Transforming the cells; -28gIII sequencing primers: 5 '-HOGTATGGGATTTTTGCTAAACAAC-3', 100pmol, 1pmol/. Mu.L; -96gIII sequencing primers: 5'-HOCCCTCATAGTTAGCGTAACG-3',100 pmol/. Mu.L, 1 pmol/. Mu.L; coli ER2738 host strain F' lacIq Δ (lacZ) M15 proA + B + zzf Tn10 (TetR)/fhuA 2 supE thi Δ (lac-proAB) Δ (hsdMS-mcrB) 5 (rk-mk-McrBC-): the strain is provided in the form of a thallus culture containing 50% of glycerol, and non-competent cells are stored at-70 ℃; streptavidin, 1.5mg of freeze-dried powder; biotin: 10mM 100. Mu.L.

3. Experimental method

Day one

The panning experiments were performed in single sterile polystyrene petri dishes, 12 or 24 well plates, 96 well microplates, with at least one plate (or well) coated with each target molecule, depending on the number and type of target molecules on which the panning of the library was performed simultaneously, and the amounts given in the following methods are the amount of 60 x 15mm petri dishes, in brackets the amount of microplates, which was adjusted accordingly for the other medium-sized wells, but in each case the same number of phage was added: 1.5X 10 ¹¹ A virus seed;

(1) A100. Mu.g/mL solution of the target molecule (NaHCO dissolved in 0.1M, pH 8.6) was prepared ₃ ) If it is desired to stabilize the target molecule, other buffers of similar ionic strength (containing metal ions, etc.) may also be used;

(2) Adding 1.5mL (150 μ L per well of microporous plate) of the above solution into each plate (well), and repeatedly rotating until the surface is completely wet;

(3) Shaking slightly at 4 deg.C in a humidifying container (such as sealable plastic box lined with wet paper towel), incubating overnight, and storing flat plate in the container at 4 deg.C;

the next day

(4) Selecting ER2738 monoclonal (plate paved when measuring bacteriophage titer) in 10mL LB liquid culture medium, if amplifying eluted bacteriophage on the same day, also inoculating ER2738 in 20mL LB liquid culture medium, using 250mL triangular flask, shaking culture at 37 deg.C;

(5) Pouring out the coating liquid in each plate, inverting the plate, forcibly patting and throwing the plate on a clean paper towel to remove residual solution, filling the sealing liquid in each plate (or hole), and acting for at least 1h at 4 ℃;

(6) Spin wash plate 6 times, spin each time to wash the bottom and edges of the plate or well, pour off buffer, shake-off upside down on dry paper towel to remove residual solution (or use an automatic plate washer);

(7) 4X 10 dilutions were made in 1mL (100. Mu.L in microwell plates) of TBST buffer ¹⁰ The phage (i.e., 10. Mu.L of the original library) was then added to the coated plate and gently shaken at room temperature for 10-60min;

(8) Dumping to remove the unbound phage, inverting the plate, and patting on a clean paper towel to remove residual solution;

(9) Washing the plate with TBST buffer solution 10 times according to the method described in 6, and changing a clean paper towel each time to avoid cross contamination;

(10) According to the intermolecular interactions studied, the bound phage were eluted with 1mL (100. Mu.L for microwell plates) of appropriate elution buffer, the known ligand for the target molecule was dissolved in TBS solution at a concentration of 0.1-1mM or the bound phage were competitively eluted from the immobilized target molecule with free target solution (-100. Mu.g/mL in TBS), gently shaken at room temperature for 10-60min, and the eluate was aspirated into another clean microfuge tube; non-specific buffers such as 0.2M Glycine-HCl (pH 2.2), 1mg/mL BSA can also be used to separate the bound molecules: gently shaking for >10min, and sucking the eluate into another clean microcentrifuge tube, and neutralizing the eluate with 150 μ L (15 μ L for microperforations) of 1M Tris-HCl (pH9.1);

(11) The titers of the small (. About.1. Mu.L) fractions were determined as described above for the conventional M13 protocol, and plaques from the first or second run of eluate titer determinations were sequenced as required as follows: the remaining eluate may be stored overnight at 4 ℃ if necessary, expanded the next day, at which time ER2738 may be cultured overnight in LB-Tet medium, culture 1;

(12) Amplification of the remaining eluate: adding the eluate into 20mL ER2738 culture (the thallus is in the prophase of logarithm), and culturing at 37 ℃ for 4.5h by shaking vigorously;

(13) The culture was transferred to a centrifuge tube and then centrifuged at 10,000 rpm at 4 ℃ for 10min. Transferring the supernatant into another centrifugal tube, and centrifuging;

(14) Transferring the upper 80% of the supernatant into a fresh tube, adding 1/6 volume of PEG/NaCl, and allowing the phage to precipitate at 4 ℃ for at least 60min overnight;

the third day

(15) Centrifuging PEG at 4 ℃ at 10,000 rpm for 15min, pouring out the supernatant, centrifuging for a short time, and sucking out the residual supernatant;

(16) Resuspending the precipitate in 1mL TBS, transferring the suspension into a microcentrifuge tube, and centrifuging at 4 ℃ for 5min to precipitate residual cells;

(17) Transferring the supernatant into another fresh microfuge tube, precipitating with 1/6 volume of PEG/NaCl, incubating on ice for 15-60min, centrifuging at 4 deg.C for 10min, discarding supernatant, centrifuging for a short time, and removing residual supernatant with micropipette;

(18) The pellet was resuspended in 200. Mu.L TBS,0.02% NaN ₃ Centrifuging for 1min, precipitating any residual insoluble substances, and transferring the supernatant into a fresh tube, wherein the supernatant is the eluate after amplification;

(19) Titrating the amplified eluate with LB/IPTG/Xgal plate according to the above conventional M13 method, and storing at 4 deg.C;

(20) Coating a plate or hole for the second round of elutriation;

the fourth and fifth days

(21) The number of blue spots on the plate was counted to determine the titer, which was usedValue calculation corresponds to 1-2 x 10 ¹¹ The amount of pfu added; if the titer is too low, the next rounds of panning may be performed down to 10 ⁹ Testing the phage addition amount of pfu;

(22) And (3) carrying out a second round of panning: the eluate obtained by the first panning and amplification is 1-2X 10 ¹¹ Repeating steps 4-18 for the amount of phage in pfu, increasing the concentration of Tween to 0.5% (v/v) in the washing step;

(23) The titer of the eluate obtained from the second round of panning after amplification was determined on LB/IPTG/Xgal plates;

(24) Coating a plate or a hole for a third round of elutriation;

day six

(25) Performing a third panning: 2X 10 of the eluate amplified by the second panning ¹¹ The phage amount of pfu repeats steps 4-11, with the washing step again using 0.5% (v/v) Tween;

(26) The titers of the eluates from the third round of panning were determined on LB/IPTG/Xgal plates without amplification, and the eluates from the third round were not necessarily amplified unless a fourth round of panning was performed, and plaques obtained from the titer determination were used for sequencing: as long as the plate culture time is not longer than 18h, the culture time is too long, the loss is easy to occur, and the rest eluates are stored at 4 ℃;

(27) One ER2738 monoclonal was selected and cultured overnight in LB-Tet medium.

4. Results of the experiment

The experimental result shows that the amino acid sequence of the polypeptide WTN specifically combined with PSMA obtained by screening is WTNHQHSKVRE (SEQ ID NO: 1).

Example 2 validation of specificity of WTN Polypeptides

1. Experimental methods

The WTN polypeptide is respectively subjected to immunofluorescence detection with an Lncap cell line with higher PSMA expression level and a PC3 cell line with low PSMA expression level, and the used fluorescence marker is FITC (namely FITC-labeled WTN polypeptide).

2. Results of the experiment

Fluorescence detection of WTN polypeptide and Lncap cell line is shown in FIG. 1, and fluorescence detection of WTN polypeptide and PC3 cell line is shown in FIG. 2; in FIG. 1, the center of the dot is the nucleus and the light color around the dot is the fluorescence exhibited by the binding of WTN to the cell surface antigen PSMA; only the cell nucleus is stained in fig. 2, and the cell surface fluorescence marked by WTN is only shown in fig. 1, which proves that the binding of WTN and the cell surface antigen PSMA has high specificity.

Example 3 cell line according to the present invention, and culture method and construction method thereof

1. Construction method of TABP-EIC-WTN

(1) Preparation of TABP-EIC-WTN cells

The NK cells used in the experiment are all obtained by Peripheral Blood Mononuclear Cell (PBMC) amplification.

(2) Construction of tumor antigen-binding peptide expression vector

The tumor antigen binding peptide structure (complete structure: WTN polypeptide region-CD 8 alpha hinge region-2B 4 transmembrane domain-2B 4 costimulation domain-NKG 2D primary signal conduction domain, nucleic acid sequence shown in SEQ ID NO: 12) sequences are obtained by gene synthesis (general purpose organism), and the expression vector is pLenti-EF1a-Backbone (NN) (adddge # 27961). The restriction sites of the structure of the tumor antigen-binding peptide are BsiWI and EcoRI (i.e., the sequence shown in SEQ ID NO:12 replaces the sequence between the restriction sites BsiWI-EcoRI). After the tumor antigen binding peptide structure is inserted, the carrier is called TABP-EIC-WTN skeleton carrier, and the carrier can express the tumor antigen binding peptide with the amino acid sequence of SEQ ID NO. 11.

(3) Lentiviral packages

TABP-EIC-WTN backbone vector and helper vector pMD2.G (addgen # 12259), pMDLg/pRRE (addgene # 12251), pRSV-Rev (addgene # 12253) were mixed at a ratio of 10. Supernatants were collected 48 hours and 72 hours after transfection and were purified and concentrated to obtain lentiviruses.

(4) Lentiviral transduction

Mixing the concentrated lentivirus with NK cells at a concentration of 200. Mu.L/100 ten thousand cells of the purified lentivirus, and subjecting the mixture to 5% CO in a 37 ℃ incubator ₂ Conditioned culture, 24 hours laterAnd (4) completely changing the liquid.

(5) Amplification of TABP-EIC-WTN cells: and (3) normally culturing and amplifying TABP-EIC cells obtained after lentivirus infection.

(6) Detection of tumor antigen binding peptide expression efficiency of TABP-EIC-WTN cells: it is 100% positive for single clone.

2. Methods of culturing other cell lines

(1) C4-2 cells: human CRPC cell line expressing PSMA (prostate cancer cell line), C4-2 cells were cultured in RPMI-1640 medium (Servicebio) containing 10% fetal bovine serum FBS (Biological Industries) and 1% penicillin/streptomycin (Hyclone);

in order to conveniently observe the tumor inhibition condition, a C4-2 cell line which stably expresses GFP is established by a single cell cloning method and is hereinafter referred to as C4-2 GFP.

(2) NK92 cells: human malignant non-Hodgkin lymphoma natural killer cell lines, NK92 and TABP-EIC-WTN cells were cultured in recombinant human IL-2 (SL Pharm) and 1% penicillin-streptomycin alpha MEM medium (Gibco) supplemented with 20% FBS, 0.2mM myo-inositol (Sigma), 0.1mM beta-mercaptoethanol (PAN-Biotech), 0.02mM folate (Sigma), 200U/mL.

(3) 293T cells: human embryonic kidney cell lines, from American type culture Collection ATCC,293T cells in DMEM medium (Hyclone) or opti-MEM (Gibco);

(4) PCa cells (prostate cancer cells): prostate tissue from one CRPC patient undergoing radical prostatectomy.

The cell culture of the present invention was carried out at 37 ℃ and 5% CO ₂ Is cultured in a humid environment.

Example 4 upregulation of PD-L1 expression on C4-2 cells cocultured with TABP-EIC-WTN cells dependent on IFN-. Gamma.

C4-2 GFP cells and TABP-EIC-WTN cells were co-cultured and flow-assayed at 2, 6, 12 and 24 hours, and the results are shown in FIG. 3: and PD-L1 expressed by C4-2 cells is detected, and the result is shown in figure 3: the Mean Fluorescence Intensity (MFI) of PD-L1 in C4-2 cells and the percentage of PD-L1 expressing C4-2 cells increased significantly with time.

TABP-EIC-WTN cells were cultured and IFN-. Gamma.secretion levels were measured at 2, 6, 12, and 24 hour time points in the culture supernatant by ELISA in the presence or absence of C4-2 GFP co-culture, and the results are shown in FIG. 4.

The results of adding IFN-. Gamma.to C4-2 cells and the expression amount of PD-L1 on C4-2 cells at different concentrations of IFN-. Gamma.are shown in FIG. 5: both the Mean Fluorescence Intensity (MFI) of PD-L1 and the percentage of C4-2 cells expressing PD-L1 increased in a concentration-dependent manner.

The results of measuring the expression level of PD-L1 on C4-2 cells co-cultured with TABP-EIC-WTN cells for 24 hours using an IFN γ blocker (IFN γ mab) are shown in FIG. 6: IFN gamma blockers completely reversed the up-regulation of PD-L1 of C4-2 co-cultured with TABP-EIC-WTN cells.

The above experimental results indicate that the up-regulation of PD-L1 expression on C4-2 cells co-cultured with TABP-EIC-WTN cells depends on IFN-. Gamma..

Example 5 upregulation of PD-L1 expression on TABP-EIC-WTN cells cocultured with C4-2 cells dependent on direct cell contact

TABP-EIC-WTN cells and C4-2 cells were co-cultured, and the expression levels of PD-L1 and PD-1 of TABP-EIC-WTN were measured at 2, 6, 12, and 24 hours, and the results are shown in FIG. 7 and FIG. 8, respectively. The results show that: the percentage of TABP-EIC-WTN that is PD-L1 or PD-1 positive is significantly upregulated over time.

The flow-through assay of the expression level of PD-L1 on TABP-EIC-WTN cells stimulated with different concentrations of IFN-. Gamma.is shown in FIG. 9. After IFN-gamma stimulation, the expression level of PD-L1 of TABP-EIC-WTN cells is not changed significantly.

Example 6 analysis of transcriptional Difference between Co-culture and Single culture of TABP-EIC-WTN/C4-2 cells

Total RNA was extracted from the samples using TRIzol (Invitrogen). DNA digestion was performed after RNA extraction by DNaseI. RNA quality was determined by examining A260/A280 using a NanodropTM OneC spectrophotometer (Thermo Fisher Scientific Inc). RNA integrity was confirmed by 1.5% agarose gel electrophoresis. Finally, qualified RNA was quantified by Qubit3.0 and QubitTM RNA Broad Range Assay kit (Life Technologies).

Using KC-Digital TM Stranded mRNA Library Prep Kit for

(Seqhealth Technology Co., ltd.) 2. Mu.g of total RNA was used for RNA sequencing library preparation. Library products corresponding to 200-500bps were enriched, quantified and finally sequenced on Illumina Novaseq 6000.

Raw sequencing data was first filtered by trimmatic (version 0.36) and clear Reads were further processed using internal scripts to eliminate the repetitive bias introduced in library preparation and sequencing. Briefly, clean reads are first clustered according to UMI sequences, where reads with the same UMI sequence are grouped into the same cluster, resulting in 65, 536 clusters. Reads in the same cluster are compared to each other by pairwise alignment, and reads with more than 95% sequence identity are then extracted into new sub-clusters. After the sub-clusters are generated, multiple sequence alignments are performed to obtain one consensus sequence for each sub-cluster. After these steps, any errors and deviations introduced by PCR amplification or sequencing are eliminated.

They were mapped to Homo _ sapiens. Grch38 (ftp:// ftp. Ensembl. Org/pub/release-87/fasta/Homo _ sapiens/dnas /) wisdom reference genome data using STAR software (version 2.5.3a). Reads mapped to exon regions of each gene were calculated by signature counting (Suclean-1.5.1 bioconductor) followed by calculation of RPKM. Genes differentially expressed between groups were identified using the edgeR package (version 3.12.1). FDR corrected p-value cutoff was 0.05 and fold change cutoff was 2 for statistical significance of gene expression differences.

Both Gene Ontology (GO) analysis and KEGG enrichment analysis of differentially expressed genes were performed with KOBAS software (version: 2.1.1) to correct the P-cut to 0.05 to determine statistically significant enrichment. Alternative splicing events were detected by using rMATS (version 3.2.5) with an FDR value cut-off of 0.05 and an absolute Δ ψ of 0.05.

The volcano pattern of deregulated genes between co-cultured TABP-EIC-WTN and separately cultured TABP-EIC-WTN cells is shown in FIG. 13A, and the volcano pattern of deregulated genes between co-cultured C4-2 and separately cultured C4-2 cells is shown in FIG. 13B. In fig. 13, differentially expressed genes with fold change greater than 2.0 and P <0.05 were color-labeled. P values were calculated using a two-sided unpaired student t-test.

The KEGG pathway enrichment analysis for up-regulated genes in co-culture in fig. 13A is shown in fig. 14A, and down-regulated genes in fig. 14B. The KEGG pathway enrichment analysis of the up-regulated genes in the co-culture in FIG. 13B is shown in FIG. 14C. In fig. 14, the color of the circle points represents the enrichment factor and the size represents the number of inputs per KEGG entry. The horizontal axis represents the importance of enrichment. The longitudinal axis represents the enriched KEGG pathway.

FIG. 15A shows the measurement of the NKG2D expression level on TABP-EIC-WTN cells. The measurement of MICA/B expression level on C4-2 cells is shown in FIG. 15B. FIG. 15 shows the detection of marker on the cell surface (determination of the expression level of the corresponding marker).

Example 7 Signaling pathways involved in PD-L1 expression in the Co-culture of TABP-EIC-WTN and C4-2 cells

TABP-EIC-WTN were cultured alone or in co-culture with C4-2 cells (E: T = 1).

FIGS. 16-17 are assays for protein levels, and Western blot analyses of PD-L1 (FIGS. 16A, 17A), p-PI3K (FIGS. 16A, 17B), p-AKT (FIGS. 16A, 17C), and p-mTOR (FIGS. 16A, 17D) in co-cultured TABP-EIC-WTN, respectively, are shown as the labels in the figures. TABP-EIC-WTN, blocking TABP-EIC-WTN, NK92, co-culturing NK92, blocking NK92 cells.

NKG2D blockers (NKG 2D mabs) were added to TABP-EIC-WTN or NK92 cells and incubated at 37 ℃ for 1 hour prior to co-culture with C4-2 cells. NKG2D blockers inhibit the activation of the PI3K/AKT/mTOR pathway in NK92 cells but not TABP-EIC-WTN cells.

Western blot analysis of IFN γ -mediated JAK/STAT activator members included p-JAK1 (FIGS. 16B, 17E), p-JAK2 (FIGS. 16B, 17F), and p-STAT1 (FIGS. 16B, 17G) in C4-2 cells co-cultured with TABP-EIC-WTN cells compared to C4-2 cells without any treatment.

The above experimental results indicate that the signal pathway involved in PD-L1 expression is involved in the co-culture of TABP-EIC-WTN and C4-2 cells.

Example 8 detection of the cytotoxic Activity of TABP-EIC-WTN cells treated with atezolizumab or nivolumab

The results of the measurement of the luminescence intensity of TABP-EIC-WTN co-cultured with C4-2 GFP cells are shown in FIG. 18: bioluminescence intensity (BLI) of C4-2 GFP cells co-cultured with TABP-EIC-WTN when treated with atezolizumab or nivolumab at concentrations of 10, 20, 40. Mu.g/mL.

The CCK-8 test result shows that atezolizumab (20 mug/mL) remarkably enhances the inhibition rate of TABP-EIC-WTN on the prostate cancer cells, while nivolumab (20 mug/mL) slightly enhances the inhibition rate of TABP-EIC-WTN on the prostate cancer cells. E: t is 1:1. 5: the results at1 are shown in fig. 19A and 19B, respectively. Shows that the combination of TABP-EIC-WTN and PD-L1 inhibitor can effectively treat prostatic cancer.

The effect of Atezolizumab or nivolumab on IFN-. Gamma.secretion from TABP-EIC-WTN cells was examined at hours 2 and 6 of the addition of Atezolizumab or nivolumab using ELISA, and the results are shown in FIG. 20: atezolizumab (10, 20, 40. Mu.g/mL) increased IFN-. Gamma.secretion from TABP-EIC-WTN cells incubated with C4-2 cells.

TABP-EIC-WTN cells were cultured at 37 ℃ in a 1:1 for 20 hours with C4-2 cells, TABP-EIC-WTN cells were then harvested and treated with atezolizumab (20 μ g/mL) or nivolumab (20 μ g/mL), and then the expression of CD107a in the TABP-EIC-WTN cells induced by C4-2 cells in the presence of atezolizumab or nivolumab was examined using flow cytometry, with the results shown in figure 21, indicating that activated NK cells produce cytotoxic effects primarily through two cell signaling pathways, one involving perforin and granzyme B and the other involving target cell death ligands, including TRAIL and FASL. In the presence of atezolizumab, the expression of CD107a in TABP-EIC-WTN cells was significantly increased in an ADCC-dependent manner. In addition, the results of FIGS. 19-21 indicate that atezolizumab is significantly superior in efficacy to nivolumab.

Example 9 antitumor Effect of TABP-EIC-WTN with atezolizumab or nivolumab on C4-2 cells in the Presence or absence of CD8+ T cells in vivo

1. Preparation of DC-CIK Co-cultured with C4-2 cells

Peripheral Blood Mononuclear Cells (PBMC) or lymphocytes (PBL) were isolated from a donated blood sample from normal healthy subjects by Ficoll-Paque (TBD science), followed by centrifugation at a 460g density gradient for 40 minutes, washed twice with saline and cultured in DC adherent medium consisting of X-VIVO (Lonza) and 5% FBS (Gibco). After 1 hour incubation, adherent PBMCs (monocytes) were collected for DC culture and suspended PBLs were collected for Cytokine Induced Killer (CIK) cell culture (day 0).

Adherent monocytes were cultured in DC growth medium consisting of X-VIVO (Lonza) and further containing 5% FBS and 2U/mL DC culture factor (Novoprotein) at 37 ℃ in CO ₂ Culture in the incubator, then half of the DC growth medium was changed on day 3, DC maturation factor (Novoprotein) was added on day 6 (final concentration of 2U/mL), and final harvest was performed on day 8.

For CIK cultures, suspended PBLs were adjusted to a density of 1.5X 10 ⁶ And cultured in CIK activation medium containing KBM551 (corning), 3% FBS, 50ng/mL anti-CD 3 antibody (Beijing Tianlian Biotech), 1000U/mL IFN-. Gamma. (Beijing Tianlian Biotech), 100U/mL IL-1. Alpha. (Beijing Tianlong Biotech), and 500U/mL IL-2 (SL Pharm). Cell cultures were supplemented with CIK propagation medium containing KBM551, 3% FBS and 500U/mL IL-2 on days 3, 4 and 6, during which the cell density was maintained at 1.5X 10 ⁶ More than one cell/mL.

On day 8, harvest was about 4X 10 ⁷ DC and 1X 10 ⁸ CIK, centrifuged, and then mixed in 150mL CIK proliferation medium. The DC-CIK mixture was then added to a seed containing 1X 10 ⁷ In T75 flasks of C4-2 adherent cells, and at 5% CO ₂ Co-culturing at 37 ℃ in a humid environment.

After 24 hours of co-cultivation, the suspended DC-CIK mixture was harvested and designated "co-cultivated CIK (labeled as cocultured CIK" on the figure).

2. Construction and culture of model mouse

Male NOD/SCID mice at 5 weeks of age were purchased from Tokyo Vittal river laboratory animal technology, inc. and housed in a national cancer center molecular oncology national key laboratory animal facility without specific pathogens. All experimental procedures were approved by the ethical committee of our hospital and were performed according to the principles of experimental animal care (NIH publication volume 25, revision 28 th 1996). Mice were inoculated subcutaneously in the upper abdomen with 2X 10 in 200. Mu.L PBS ⁶ C4-2 cells. The tumor volume reached 100-200mm on day 7 ³ Treatment was started and C4-2 vaccinated mice were randomized into seven groups:

(i) In the Control (Control) group,

(ii) The TABP-EIC-WTN group,

(iii) TABP-EIC-WTN + nivolumab group,

(iv) TABP-EIC-WTN + atezolizumab group,

(v) TABP-EIC-WTN + co-cultured CIK (cultured CIK) group,

(vi) TABP-EIC-WTN + nivolumab + CIK group co-cultured,

(vii) TABP-EIC-WTN + atezolizumab + group of co-cultured CIK.

The PD-L1 antibody atezolizumab (GlpBio, GC 32704) (20 mg/kg) or PD-1 antibody nivolumab (GlpBio, GC 34218) (10 mg/kg) or control PBS tail vein injection was administered on days 7, 9, 11, 13, 15 post-tumor inoculation for a total of 5 doses, while TABP-EIC-WTN treatment was 5X 10 injections on days 8, 10, 12, 14, 16 post-PD-L1/PD-1 antibody treatment ⁶ TABP-EIC-WTN, for co-intravenous injection of 5 doses.

On day 17, CIK was co-cultured by i.v. injection based on TABP-EIC-WTN treatment, with or without the addition of PD-L1/PD-1 antibody.

Tumor volume calculation formula: l x W ² L and W represent the longest and shortest diameters measured by calipers on days 7, 10, 14, 16, 18, 20. Also by using

Spectral CT detection of bioluminescence intensity (BLI) assessment of tumor size before and after treatment, e.g. in tumor cellsBLI was measured and expressed as radiometric (p/sec/cm 2/sr) as described on days 7, 14, 21 after cell implantation.

All mice were sacrificed on day 21, tumors were collected, photographed, weighed and collected for histological examination.

3. Results of the experiment

The tumor sizes (n = 3-4) of the C4-2 GFP cell implanted mice in the above groups were measured on days 7, 14, and 21, and the results are shown in fig. 22. Tumor size was counted separately for mice treated with and without co-cultured CIK, tumor volume was counted for each group treated with and without co-cultured CIK as shown in fig. 25A, and tumor volume was counted for each group treated with and without co-cultured CIK as shown in fig. 25B.

A physical map of the excised tumor is shown in fig. 26A, a statistical analysis of tumor weight is shown in fig. 26B, values on the graph are expressed as mean ± SD, representing P <0.05; NS represents no significant difference.

To analyze the tumor-inhibiting effect of co-cultured CIK, the tumor-inhibiting effect was measured with or without co-cultured CIK, and quantitative BLI test was performed on the control group, TABP-EIC-WTN treated group, TABP-EIC-WTN + nivolumab treated group, TABP-EIC-WTN + atezolizumab treated group on days 7, 14, and 21, and the results are shown in FIG. 23A. Quantitative BLI tests were performed on the control group, TABP-EIC-WTN treated group, TABP-EIC-WTN + co-culture CIK treated group, TABP-EIC-WTN + nivolumab + co-culture CIK treated group, TABP-EIC-WTN + atezolizumab + co-culture CIK treated group, and the results are shown in FIG. 23B.

Immunohistochemical staining of tumor cells, fixing of necrotic tissue with 10% neutral buffered formalin, paraffin embedding, cutting into 4 μm thick sections, staining with Hematoxylin and Eosin (HE) for light microscopy. The necrotic area was stained red under an optical microscope with no intact cellular structures. View2 viewer software (Hamamatsu Photonics, shizuoka, japan) was used to quantify the percentage of red necrotic areas relative to the entire area, with large blood vessels excluded from the image analysis described above. As a result, as shown in FIG. 24A, significant tumor necrosis was observed in the TABP-EIC-WTN-treated group, and the addition of atezolizumab significantly increased the severity of tumor necrosis in the TABP-EIC-WTN-treated group, as shown in FIG. 24B as a statistical result.

The above experiment results show that TABP-EIC-WTN and/or atezolizumab and/or co-cultured CIK combination can effectively treat tumors, namely TABP-EIC-WTN and atezolizumab can effectively treat tumors in combination; the TABP-EIC-WTN can effectively treat tumors by combining with the co-cultured CIK; the combination of TABP-EIC-WTN and atezolizumab and the co-cultured CIK can effectively treat tumors.

Example 10 composition and functional characterization of Co-cultured CIK

CD3, CD8 and CD56 expression in co-cultured CIK was detected using flow cytometry, where cells were stained with the following antibodies: CD3-APC, CD8-FITC, CD56-PerCP from BD Bioscience; the results of the statistical analysis of the detection results are shown in fig. 27.

Fluorescence imaging assays were performed on day 5 and day 10 in control, CIK treated and co-cultured CIK treated mice, and BLI measurements and statistical analysis (n = 3) of C4-2 GFP engraftment tumor activity are shown in figure 28. Measurements were taken on days 5, 10, and 20 and tumor volumes were calculated, and the results are shown in fig. 29.

The experimental results show that the CIK and the co-cultured CIK have certain treatment effects on tumors, and the co-cultured CIK prepared by the invention has better treatment effect and is obviously superior to a control group and a CIK group.

Example 11 Atezolizumab enhances the cytotoxicity of TABP-EIC-WTN in vitro on CRPC cells from a prostate cancer patient

One prostate cancer patient was randomly selected and their serum testosterone and PSA levels after ADT were recorded, with the PSA level rising continuously above nadir from 5 months 2021 at the post ADT testosterone castration level as shown in fig. 30A and fig. 30B, respectively.

On 26/5/2021, the patient successfully received robot-assisted radical prostatectomy (RARP) and bilateral pelvic lymphadenectomy under general anesthesia. A portion of the fresh specimens was sent to the laboratory for primary culture of PCa cells (prostate cancer cells) within 2 hours after removal from the body. The procedure was as follows: CRPC tissue was rinsed several times with sterile saline and cut into 3mm thick pieces. These pieces of tissue were then digested with 0.1% collagenase I (Sigma) in alpha-MEM (Corning) for 2 hours at 37 ℃ in a shaking incubator. The pieces were washed 3 times with PBS and centrifuged at 300g for 5 minutes at low speed to remove collagenase I. A10 cm cell culture dish was pre-coated with FBS (Gibco).

Inoculating the tissue on a FBS-free culture dish at 37 deg.C with 5% CO ₂ After 24 hours of incubation in the humidified incubator of (1), 10mL of α -MEM was added and 10% of FBS was added to the culture dish for further cell culture.

The tumor sections were HE stained (x 100) and the staining results are shown in fig. 31A, and fig. 31B is an optical micrograph of primary prostate cancer cells (x 200) at day 14.

After PCa (prostate cancer cells) and TABP-EIC-WTN cells were incubated for 24 hours, the expression of PD-L1 on PCa was detected by flow measurement, and the results are shown in FIG. 32A and FIG. 32B, which demonstrates that Atezolizumab enhances the cytotoxicity of TABP-EIC-WTN on CRPC cells.

The CCK-8 assay analyzes the tumor-inhibiting effect of TABP-EIC-WTN cells upon addition of atezolizumab/nivolumab (E: T = 1), and statistics are shown in fig. 32C, and the results show that atezolizumab (20 μ g/mL) significantly enhances the inhibition rate of TABP-EIC-WTN (E: T = 1.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications will fall within the scope of the claims of the present invention.

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<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgat 135

<210> 5

<211> 35

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gln Asp Cys Gln Asn Ala His Gln Glu Phe Arg Phe Trp Pro Phe Leu

1 5 10 15

Val Ile Ile Val Ile Leu Ser Ala Leu Phe Leu Gly Thr Leu Ala Cys

20 25 30

Phe Cys Val

35

<210> 6

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

caggactgtc agaatgccca tcaggaattc agattttggc cgtttttggt gatcatcgtg 60

attctaagcg cactgttcct tggcaccctt gcctgcttct gtgtg 105

<210> 7

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu

1 5 10 15

Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg Asn

20 25 30

His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr Ser

35 40 45

Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala Tyr

50 55 60

Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg Lys

65 70 75 80

Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly

85 90 95

Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu

100 105 110

Leu Glu Asn Phe Asp Val Tyr Ser

115 120

<210> 8

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tggaggagaa agaggaagga gaagcagtca gagaccagtc ccaaggaatt tttgacaatt 60

tacgaagatg tcaaggatct gaaaaccagg agaaatcacg agcaggagca gacttttcct 120

ggagggggga gcaccatcta ctctatgatc cagtcccagt cttctgctcc cacgtcacaa 180

gaaccagcat atacattata ttcattaatt cagccttcca ggaagtctgg ttccaggaag 240

aggaaccaca gcccttcctt caatagcact atctatgaag tgattggaaa gagtcaacct 300

aaagcccaga accctgctcg attgagccgc aaagagctgg agaactttga tgtttattcc 360

<210> 9

<211> 51

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser

1 5 10 15

Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr

20 25 30

Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu

35 40 45

Asn Ala Ser

50

<210> 10

<211> 153

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggggtgga ttcgtggtcg gaggtctcga cacagctggg agatgagtga atttcataat 60

tataacttgg atctgaagaa gagtgatttt tcaacacgat ggcaaaagca aagatgtcca 120

gtagtcaaaa gcaaatgtag agaaaatgca tct 153

<210> 11

<211> 316

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Trp Thr Asn His His Gln His Ser Lys Val Arg

20 25 30

Glu Gly Gly Gly Ser Trp Thr Asn His His Gln His Ser Lys Val Arg

35 40 45

Glu Gly Gly Gly Ser Trp Thr Asn His His Gln His Ser Lys Val Arg

50 55 60

Glu Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

65 70 75 80

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

85 90 95

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

100 105 110

Cys Gln Asn Ala His Gln Glu Phe Arg Phe Trp Pro Phe Leu Val Ile

115 120 125

Ile Val Ile Leu Ser Ala Leu Phe Leu Gly Thr Leu Ala Cys Phe Cys

130 135 140

Val Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys

145 150 155 160

Glu Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg

165 170 175

Asn His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr

180 185 190

Ser Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala

195 200 205

Tyr Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg

210 215 220

Lys Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile

225 230 235 240

Gly Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys

245 250 255

Glu Leu Glu Asn Phe Asp Val Tyr Ser Met Gly Trp Ile Arg Gly Arg

260 265 270

Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn Tyr Asn Leu

275 280 285

Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys Gln Arg Cys

290 295 300

Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser

305 310 315

<210> 12

<211> 951

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgtggacca accaccacca gcacagcaag gtgagagagg gcggcggcag ctggaccaac 120

caccaccagc acagcaaggt gagagagggc ggcggcagct ggaccaacca ccaccagcac 180

agcaaggtga gagagaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 240

gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 300

catacccggg gtcttgactt cgcctgcgat caggactgtc agaatgccca tcaggaattc 360

agattttggc cgtttttggt gatcatcgtg attctaagcg cactgttcct tggcaccctt 420

gcctgcttct gtgtgtggag gagaaagagg aaggagaagc agtcagagac cagtcccaag 480

gaatttttga caatttacga agatgtcaag gatctgaaaa ccaggagaaa tcacgagcag 540

gagcagactt ttcctggagg ggggagcacc atctactcta tgatccagtc ccagtcttct 600

gctcccacgt cacaagaacc agcatataca ttatattcat taattcagcc ttccaggaag 660

tctggttcca ggaagaggaa ccacagccct tccttcaata gcactatcta tgaagtgatt 720

ggaaagagtc aacctaaagc ccagaaccct gctcgattga gccgcaaaga gctggagaac 780

tttgatgttt attccatggg gtggattcgt ggtcggaggt ctcgacacag ctgggagatg 840

agtgaatttc ataattataa cttggatctg aagaagagtg atttttcaac acgatggcaa 900

aagcaaagat gtccagtagt caaaagcaaa tgtagagaaa atgcatctta a 951

Claims (66)

1. A WTN polypeptide specifically bound to PSMA, characterized in that the WTN polypeptide is a polypeptide represented by SEQ ID NO. 1.

2. The WTN polypeptide of claim 1, further comprising a chemically modified product at one or more of amino, carboxyl, imidazolyl, guanidino, indolyl groups at the terminus of the polypeptide backbone or side chain set forth in SEQ ID NO. 1.

3. A polypeptide complex consisting of peptide-bonded domains at the amino-terminus and/or carboxy-terminus of the WTN polypeptide of claim 1.

4. The polypeptide complex of claim 3 wherein the domain consists of amino acids.

5. The polypeptide complex of claim 4 wherein the domains comprise a hinge region, a transmembrane domain, and a signaling domain.

6. The polypeptide complex of claim 5 wherein the signaling domain comprises a co-stimulatory domain and/or a primary signaling domain.

7. The polypeptide complex of claim 3 which is a tumor antigen binding peptide.

8. The polypeptide complex according to claim 7, wherein the tumor antigen binding peptide consists of WTN polypeptide region-hinge region-transmembrane domain-costimulatory domain-primary signaling domain or WTN polypeptide region-hinge region-transmembrane domain-primary signaling domain.

9. The polypeptide complex of claim 8, wherein the WTN polypeptide region comprises a plurality of WTN polypeptide repeats and a linker between the plurality of WTN polypeptide repeats.

10. The polypeptide complex of claim 5 or 8, wherein the hinge region comprises a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, or an ICOS hinge region.

11. The polypeptide complex of claim 5 or 8, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: 2B4 gene expression protein, the α, β or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CD154.

12. The polypeptide complex of claim 6 or 8 wherein the co-stimulatory domain comprises a functional signaling domain of 2B4, CD3 ζ, OX40, CD2, CD27, CD28, CDs, ICAM-1, LFA-1, ICOS, or 4-1 BB.

13. The polypeptide complex of claim 6 or 8, wherein the primary signaling domain comprises signaling regions of one or any combination of proteins of NKG2D primary signaling, CD 3-zeta, fcsri gamma, fcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD 66D.

14. The polypeptide complex of claim 10, wherein the hinge region is a CD8 a hinge region.

15. The polypeptide complex of claim 14, wherein the amino acid sequence of the CD8 a hinge region is set forth in SEQ ID NO 3.

16. The polypeptide complex of claim 14, wherein the nucleic acid sequence encoding the CD8 a hinge region is set forth in SEQ ID NO 4.

17. The polypeptide complex of claim 11 wherein the transmembrane domain is the transmembrane domain of 2B 4.

18. The polypeptide complex of claim 17, wherein the transmembrane domain of 2B4 has the amino acid sequence of SEQ ID No. 5.

19. The polypeptide complex of claim 17, wherein the transmembrane domain of 2B4 has a nucleic acid sequence as set forth in SEQ ID No. 6.

20. The polypeptide complex of claim 12 wherein the co-stimulatory domain is a functional signaling domain of 2B 4.

21. The polypeptide complex of claim 20, wherein the functional signaling domain of 2B4 has the amino acid sequence set forth in SEQ ID No. 7.

22. The polypeptide complex of claim 20, wherein the functional signaling domain of 2B4 encodes a nucleic acid sequence as set forth in SEQ ID NO 8.

23. The polypeptide complex of claim 13, wherein the primary signaling domain is a signaling region of NKG 2D.

24. The polypeptide complex of claim 23, wherein the amino acid sequence of the signaling region of NKG2D is set forth in SEQ ID NO. 9.

25. The polypeptide complex of claim 23, wherein the coding nucleic acid sequence of the NKG2D signaling region is set forth in SEQ ID NO. 10.

26. The polypeptide complex according to claim 8, wherein the composition of the tumor antigen binding peptide is WTN polypeptide region-hinge region-transmembrane domain-co-stimulatory domain-primary signaling domain.

27. The polypeptide complex according to claim 26, wherein the tumor antigen binding peptide consists of the WTN polypeptide region-the transmembrane domain of CD8 α hinge region-2B 4-the functional signaling domain of 2B 4-the signaling region of NKG 2D.

28. The polypeptide complex of claim 27, wherein the amino acid sequence of said tumor antigen binding peptide is set forth in SEQ ID NO. 11.

29. The polypeptide complex of claim 27, wherein the nucleic acid sequence encoding the tumor antigen binding peptide is set forth in SEQ ID No. 12.

30. A DNA molecule encoding a WTN polypeptide according to claim 1 or a polypeptide complex according to claim 3.

31. The DNA molecule according to claim 30, wherein the DNA molecule encoding a WTN polypeptide has the sequence shown in SEQ ID NO. 2.

32. The DNA molecule of claim 30, wherein the DNA molecule encoding the polypeptide complex has the sequence shown in SEQ ID NO 12.

33. A vector comprising the DNA molecule of claim 30.

34. The vector of claim 33, which is an expression vector.

35. The vector of claim 34, wherein the expression vector is a plasmid expression vector or a viral expression vector.

36. A host cell comprising one or more of the polypeptide of claim 1, the polypeptide complex of claim 3, the DNA molecule of claim 30, and the vector of claim 33.

37. The host cell of claim 36, comprising prokaryotic and eukaryotic cells.

38. The host cell of claim 37, wherein the prokaryotic cell is a bacterial cell.

39. The host cell of claim 37, wherein the eukaryotic cell is a mammalian cell, an insect cell, a plant cell, a fungal cell, or an algal cell.

40. The host cell of claim 39, wherein the mammalian cell comprises a cell of human or non-human origin.

41. The host cell of claim 40, wherein the human cell is an immune cell.

42. The host cell of claim 41, wherein the immune cell is an NK cell or a T cell.

43. The host cell of claim 42, wherein the immune cell is an NK cell.

44. The host cell of claim 43, wherein said NK cell is a peripheral blood monocyte-derived NK cell.

45. A pharmaceutical composition comprising one or more of the polypeptide of claim 1, the polypeptide complex of claim 3, the DNA molecule of claim 30, the vector of claim 33, and the host cell of claim 36.

46. The pharmaceutical composition of claim 45, further comprising a pharmaceutically acceptable excipient and/or additive.

47. The pharmaceutical composition of claim 45, wherein the pharmaceutical composition is administered in a liquid, solid, or semi-solid dosage form.

48. A conjugate of a WTN polypeptide that specifically binds to PSMA, wherein the WTN polypeptide is the polypeptide set forth in SEQ ID No. 1, and wherein a detectable label is attached to the WTN polypeptide.

49. The conjugate of claim 48, wherein the linkage is covalent linkage or physisorption.

50. The conjugate of claim 49, wherein said covalent linkage is a non-peptide linkage.

51. The conjugate of claim 48, wherein the detectable label is a fluorescent molecule.

52. The conjugate of claim 51, wherein the fluorescent molecule is FITC.

53. A method of making the host cell of claim 36, the method comprising the step of introducing into the host cell one or more of a DNA molecule encoding the polypeptide of claim 1, a DNA molecule encoding the polypeptide complex of claim 3, a DNA molecule of claim 30, a vector of claim 33.

54. The method of claim 53, wherein the method for introducing the cells comprises heat shock, calcium phosphate precipitation, particle bombardment, microinjection, or electroporation.

55. A method of making the WTN polypeptide of claim 1, the method comprising culturing the host cell of claim 36 under culture conditions suitable for large scale expression of the protein.

56. The method of claim 55, further comprising the step of extracting and purifying the polypeptide.

57. A method for detecting PSMA for non-diagnostic purposes, comprising the step of contacting the conjugate of claim 48 with a sample to be detected.

58. The method of claim 57, further comprising the step of processing the sample.

59. The method of claim 57, wherein the sample to be assayed is a sample suspected of containing PSMA.

60. A kit for detecting PSMA, comprising a conjugate of the WTN polypeptide of claim 48.

61. The kit of claim 60, further comprising one or more of a sample processing fluid, a buffer, an ionic strength modifier, a surfactant, and a preservative.

62. Use of the polypeptide of claim 1, the polypeptide complex of claim 3, the DNA molecule of claim 30, or the vector of claim 33 in the preparation of the host cell of claim 36.

63. Use of the DNA molecule of claim 30 in the preparation of the vector of claim 33.

64. Use of the polypeptide of claim 1, the polypeptide complex of claim 3, the DNA molecule of claim 30, the vector of claim 33, the host cell of claim 36, or the pharmaceutical composition of claim 45 in the manufacture of a medicament for the treatment of prostate cancer.

65. Use of a conjugate of the polypeptide of claim 48 for the preparation of a kit for the diagnosis of prostate cancer.

66. The use according to claim 65, wherein the kit is the kit according to claim 60.

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