CN113527434A

CN113527434A - WTN polypeptide and application thereof in detection of prostate cancer

Info

Publication number: CN113527434A
Application number: CN202110797547.9A
Authority: CN
Inventors: 尹乐; 顾雨春
Original assignee: Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
Priority date: 2021-07-14
Filing date: 2021-07-14
Publication date: 2021-10-22
Also published as: CN114315976A; CN114560910A; CN114315977A; CN114835781A; CN114315976B; CN114835781B

Abstract

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

Description

WTN polypeptide and application thereof in detection of prostate cancer

Technical Field

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

Background

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

The tumor cell therapy has attracted great attention due to its advantages of specific targeting, remarkable effect, few side effects and the like, gradually becomes an important means in the comprehensive treatment of tumors, is called as 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 result in 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 consisted of an extracellular-binding domain-single chain antibody (scFV), a transmembrane domain (TM), and a signaling domain-immunoreceptor tyrosine-activation motif (ITAM), where the chimeric antigen receptor portions were 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 4-1BB), where the chimeric antigen receptor portions are linked as follows: scFv-TM-CD28-ITAM or scFv-TM-CD 137-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 portions of the chimeric antigen receptor are linked as follows: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM further improves the survival cycle of CAR-T in vivo and its anti-tumor effect. 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. The WTN polypeptide can be used for detecting a cancer marker PSMA after being combined with a detectable marker, and the 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, sulfydryl, 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 of the polypeptide backbone and amidation of the C-terminal 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 attached 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 transmembrane domain comprises a transmembrane domain of a protein comprising: 2B4 gene, the α, β or ζ chain of a T cell receptor, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 and CD 154.

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

Preferably, the co-stimulatory domain comprises functional signaling domains of 2B4, CD3 ζ, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278) and 4-1BB (CD 137).

Preferably, the primary signaling domain comprises a signaling region of one or any combination of proteins of CD 3-zeta, fcepsilon RI gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66 d;

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

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 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.

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

Preferably, the DNA molecule sequence for encoding WTN polypeptide is shown as SEQ ID NO. 2.

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

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-neo CMV.

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

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-CMV, pShuttle-IRES-GFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC1, pBHGE3, pAMCS, pARC-pHelperr, pA-LacZ, pAV 0.1-CMV, ppLKO-CMV, pLKO-1-1.0.1-CMV, pLKO-1-pLKO-1.1-GFP, pLKO-1.1.1-pLKO, pLKO-1.1-GFP, pLKO-1.1.1.1, pLKO.1-puro-CMV-TagYFP, pLKO.l-puro-CMV-TagFP, pLKO.1-puro-CMV-TagFP635, pLKO. -puro-UbC-TurboGFP, pLKO.l-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLPl, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-minshenna, pcDNAl, 2/V5-lacGW/Z, pLenti 6.2/N-Lumio/CSV 5-DEST, pGIL-3896.2/Luiso-LumiV 5/LumiV.

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 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 is 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 a common preparation, and also can be prepared into a sustained release preparation, a controlled release preparation, a targeting preparation and various microparticle 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 153 Sm.

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

Preferably, the fluorescent label 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 the WTN polypeptide, the DNA molecule of the polypeptide complex, and the vector into the cell.

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

In a tenth aspect, the invention provides a method of producing the WTN polypeptide described above, comprising culturing the host cell described above in a culture environment suitable for large scale 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 of detecting PSMA, 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, bacteria, 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.

In a twelfth aspect, the invention provides a kit for detecting PSMA, the kit comprising a conjugate of the WTN polypeptide described 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 WTN polypeptide and the DNA molecule of the polypeptide complex in the preparation of the vector.

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 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 a polypeptide complex, the aforementioned vector, the aforementioned host cell, or the aforementioned pharmaceutical composition for the preparation 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.

Drawings

FIG. 1 shows the immunofluorescence results of Lncap cell lines with higher expression of polypeptides WTN and PSMA.

FIG. 2 shows the immunofluorescence results of PC3 cell line with low expression of polypeptide WTN and PSMA.

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 modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification 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 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 ¹³pfu/mL, stored in TBS solution with 50% Glycerol, complexity 2.7X 10⁹Transforming the cells; -28gIII sequencing primers: 5 '-HOGTATGGGATTTTTGCTAAACAAC-3', 100pmol, 1 pmol/. 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)/fhaA 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 methods

Day one

The panning experiments were performed on 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, and the other medium-sized wells adjusted accordingly, but in each case the same number of phage was added: 1.5X 10¹¹A virus seed;

(1) A100. mu.g/mL solution of the target molecule (NaHCO dissolved in 0.1M, pH 8.6) was prepared₃) 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 arranged with wet paper towel), incubating overnight, and storing the 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, patting and throwing the plate on a clean paper towel forcibly to remove residual solution, filling sealing liquid in each plate (or hole), and acting at 4 ℃ for at least 1 h;

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

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

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

(9) wash the plate 10 times with TBST buffer as described in 6, change 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 the 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 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 shake for >10min, the eluate is aspirated into another clean microcentrifuge tube, and the eluate is neutralized with 150. mu.L (15. mu.L for microwells) 1M Tris-HCl (pH 9.1);

(11) the titers of the small amounts (. about.1. mu.L) of the eluates were determined as described above in the conventional M13 procedure, and plaques from the first or second round of eluate titer determination were sequenced as needed as follows: if necessary, the remaining eluate may be stored at 4 ℃ overnight and expanded the next day, in which case ER2738 may be cultured overnight in LB-Tet medium, the next day the culture 1:100 is diluted in 20mL LB (contained in a 250mL Erlenmeyer flask), the unexpanded eluate is added, and the culture is vigorously shaken at 37 ℃ for 4.5h, and step 13 is continued;

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

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

(14) transferring the upper 80% of the supernatant to 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 deg.C and 10,000rpm for 15min, discarding supernatant, centrifuging for a short time, and removing residual supernatant;

(16) the precipitate was resuspended in 1mL TBS, the suspension was transferred to a microcentrifuge tube and centrifuged at 4 ℃ for 5min to pellet the residual cells;

(17) transferring the supernatant into another fresh microfuge tube, reprecipitating 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% NaN₃Centrifuging 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 plates according to the conventional M13 method, and storing at 4 ℃;

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

the fourth and fifth days

(21) The titer was determined by counting the number of blue spots on the plate and this value was used to calculate a titer corresponding to 1-2X 10¹¹The amount of pfu added; if the titer is too low, the next rounds of panning may be performed down to 10⁹Testing 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 10¹¹Repeating 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 panning¹¹The 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 WTNHYHSSVRYG (SEQ ID NO: 1).

Example 2 validation of specificity of WTN Polypeptides

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).

Fluorescence measurements of WTN polypeptide and Lncap cell line are shown in FIG. 1, and fluorescence measurements of WTN polypeptide and PC3 cell line are 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. In FIG. 2 there is only staining of the nuclei. The cell surface fluorescence labeled by WTN appears only in FIG. 1, which proves that the binding of WTN and cell surface antigen PSMA has high specificity.

Sequence listing

<110> Shino Rev medicine science and technology (New zone of Zhuhai horizontal organ) Co., Ltd

<120> WTN polypeptides and their use in detecting prostate cancer

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Claims

1. A WTN polypeptide specifically bound with PSMA, characterized in that the WTN polypeptide is a polypeptide having more than 90% homology with the polypeptide shown in SEQ ID NO. 1;

Preferably, the WTN polypeptide is substituted, deleted or added at one or more of the 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th, 7 th, 8 th, 9 th, 10 th, 11 th and 12 th positions 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 further includes a product obtained by chemically modifying one or more sites of amino, carboxyl, sulfhydryl, phenolic hydroxyl, imidazolyl, guanidino, indolyl and methylthio at the tail end of the main chain or side chain of the polypeptide shown in SEQ ID NO. 1.

2. A polypeptide complex comprising peptide bonds joining domains at the amino-terminus and/or carboxy-terminus of the WTN polypeptide of claim 1;

preferably, the domain is composed of amino acids;

preferably, the signalling domain comprises a co-stimulatory domain and/or a primary signalling domain;

preferably, the polypeptide complex is a tumor antigen binding peptide;

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;

3. A DNA molecule encoding the WTN polypeptide of claim 1 or the polypeptide complex of claim 2;

4. A vector comprising the DNA molecule of claim 3;

preferably, the vector is an expression vector;

5. A host cell comprising one or more of the polypeptide of claim 1, the polypeptide complex of claim 2, the DNA molecule of claim 3, the vector of claim 4;

preferably, the host cell includes prokaryotic cells and eukaryotic cells;

preferably, the prokaryotic cell is a bacterial cell;

preferably, the eukaryotic cell is a mammalian cell, an insect cell, a plant cell, a fungal 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 cell is an NK cell or a T cell.

6. A pharmaceutical composition comprising one or more of the polypeptide of claim 1, the polypeptide complex of claim 2, the DNA molecule of claim 3, the vector of claim 4, or the host cell of claim 5;

preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients and/or additives;

7. 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 covalent linkage is a non-peptide linkage;

preferably, the detectable label is a fluorescent molecule.

Preferably, the fluorescent molecule is FITC.

8. A method, comprising any one of:

1) a method of making the host cell of claim 5, comprising the step of introducing into the cell one or more of a DNA molecule encoding the polypeptide of claim 1, a DNA molecule encoding the polypeptide complex of claim 2, a DNA molecule of claim 3, a vector of claim 4;

Preferably, the method for introducing the cells includes a heat shock method, calcium phosphate precipitation, transfection method, particle bombardment method, microinjection method, electroporation method;

2) a method of making the WTN polypeptide of claim 1, the method comprising culturing the host cell of claim 5 under culture conditions suitable for large scale expression of the protein;

preferably, the method further comprises the steps of extracting and purifying the polypeptide;

3) a method of detecting PSMA, comprising the step of contacting the conjugate of claim 7 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.

9. A kit for detecting PSMA, comprising a conjugate of a WTN polypeptide of claim 7;

10. An application, comprising any one of:

1) use of the polypeptide of claim 1, the polypeptide complex of claim 2, the DNA molecule of claim 3, the vector of claim 4, or the host cell of claim 5 in the preparation of the pharmaceutical composition of claim 6;

2) Use of the polypeptide of claim 1, the polypeptide complex of claim 2, the DNA molecule of claim 3, or the vector of claim 4 for the preparation of the host cell of claim 5;

3) use of the DNA molecule of claim 3 for the preparation of the vector of claim 4;

4) use of the polypeptide of claim 1, the polypeptide complex of claim 2, the DNA molecule of claim 3, the vector of claim 4, the host cell of claim 5, or the pharmaceutical composition of claim 6 for the preparation of a medicament for the treatment of cancer;

preferably, the cancer is prostate cancer;

5) use of a conjugate of the polypeptide of claim 7 for the preparation of a kit for the diagnosis of cancer;

preferably, the cancer is prostate cancer;

preferably, the diagnosis is detected PSMA;

preferably, the kit is the kit of claim 9.