CN113527433B - Polypeptide and application thereof in diagnosis and treatment of prostate cancer - Google Patents
Polypeptide and application thereof in diagnosis and treatment of prostate cancer Download PDFInfo
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Abstract
The invention discloses a novel polypeptide and application thereof in diagnosing and treating prostate cancer, wherein a phage-polypeptide library is used for screening extracellular recognition region sequences of TABP-EIC cells, the screened polypeptide ligand can replace a traditional single-chain antibody for preparing the TABP-EIC cells, compared with the traditional method for preparing the TABP-EIC cells, the inventive preparation method obviously shortens the screening time of the extracellular recognition region sequences of the TABP-EIC cells, the screened polypeptide ligand has higher binding efficiency with related tumor specific antigens, and can screen the recognition polypeptide ligand aiming at tumors with undefined antigens, thereby providing a new idea for immunotherapy of tumors and having extremely high application value in cell therapy of tumors.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide and application thereof in diagnosis and treatment of prostate cancer.
Background
Prostate Cancer (PCa), which is one of the common malignant tumors of the male and occurs in the Prostate, is the first disease occurrence in men, with the mortality rate being inferior to lung and colorectal cancers (Siegel RL, miller KD, jemal a Cancer Statistics, 2017[ J ]. CA Cancer J Clin,2017, 67 (1): 7-30.). In recent years, the incidence of prostate Cancer has tended to rise and younger year by year (Feng RM, zong YN, cao SM, et al Current Cancer situation in China: good or bad news from the 2018 Global Cancer Statistics. Currently, advanced prostate cancer is treated clinically by blocking androgens and their signaling pathways in patients with prostate cancer, but after 18-30 months of such androgen deprivation therapy (Androgen deprivation therapy, ADT), most patients develop castration resistant prostate cancer (castration resistant prostate cancer, CRPC) (Lo EM, balasubramanian A, pastezak AW, et al Bipolar androgen therapy in prostate cancer (update) [ J ] J set Med, 2020, 17 (5): 831-834.). The treatment of prostate cancer at CRPC stage is limited and the prognosis is poor. Immunotherapy is a brand-new treatment method, achieves the aim of killing tumor cells by regulating the immune system of a human body, and can improve the prognosis of partial prostate cancer patients to a certain extent.
In immunotherapy, chimeric antigen receptor-modified T cells (Chimeric antigen receptor modification T cells, CAR-T) have achieved remarkable clinical efficacy in the treatment of hematological malignancies such as acute and chronic lymphocytic leukemia and non-hodgkin's lymphoma (Chen SF, zhu ZP, wu YF. Clinical application challenges and countermeasures of CAR-T cell therapy in treatment of tumors [ J ]. Chinese Journal of Tumor Biotherapy, 2019, 26 (7): 802-809.), but CAR-T treatment also has problems such as off-target effects, cytokine release syndrome (Cytokine release syndrome, CRS), etc. Natural killing (Natural killer cell, NK) is of increasing interest in CAR cell therapies because it does not require prior sensitization and does not lead to graft versus host disease, and in hematologic tumors, many of the currently studied are chimeric antigen receptor modified NK cells (Chimeric antigen receptor modification NK cells, CAR-NK). In addition to hematological tumors, CAR cell therapies have also made great progress in the study of prostate cancer treatment, and the identification of prostate cancer-associated specifically recognized tumor-associated antigens (Tumor associated antigen, TAA) is the first step in the effective development of CAR-T or CAR-NK, with prostate cancer cell-expressed TAA having a prostate-specific membrane antigen (Prostate specific membrane antigen, PSMA), a prostate stem cell antigen (Prostate stem cell antigen, PSCA), a prostate-specific antigen (Prostate specific antigen, PSA), where PSMA and PSCA-targeted CAR-T or CAR-NK treatment progresses faster, which can significantly inhibit the growth of tumor cells, and to some extent delay disease progression.
However, the extracellular recognition regions used by the CAR-T or CAR-NK prepared at present are all single-chain antibodies, the development of the single-chain antibodies depends on specific recognition of tumor associated antigens, and the single-chain antibodies are obtained by conventional monoclonal antibody preparation and sequencing, and the method for preparing the CAR-T or CAR-NK has the following technical problems: the preparation period of the antibody is long; (2) The specific binding efficiency of the antibody after single-chain is generally low; (3) The corresponding CAR-T or CAR-NK cells can be prepared only by the tumor with definite surface antigen, and cannot be aimed at the tumor with undefined antigen. In view of the above, the present invention aims to provide a method for screening a phage-polypeptide library for a sequence of a TABP-EIC (Tumor Antigen Binding Peptide-Engineering Immune Cell, tumor antigen binding peptide-engineered immune cell) extracellular recognition region, wherein the polypeptide ligand obtained by the method can be used for preparing a TABP-EIC cell instead of a conventional single-chain antibody, compared with the conventional method for preparing a TABP-EIC cell, the inventive preparation method significantly shortens the screening time of the TABP-EIC extracellular recognition region sequence, the screened polypeptide ligand has higher binding efficiency with a related tumor-specific antigen, and the identified polypeptide ligand for an antigen-undefined tumor can be screened.
Disclosure of Invention
The invention aims to provide a novel polypeptide and application thereof in diagnosing and treating prostate cancer, the polypeptide is obtained by screening a phage-polypeptide library, tumor specific antigens can be specifically combined, the combination efficiency is high, the screened polypeptide can replace the traditional single-chain antibody to be used for preparing TABP-EIC cells, the pioneering preparation method obviously shortens the screening time of the extracellular recognition region sequence of the TABP-EIC cells, the combination efficiency of screened polypeptide ligand and relevant tumor specific antigens is higher, and the recognition polypeptide ligand aiming at the tumor with undefined antigens can be screened.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect, the invention provides a novel polypeptide that targets PSMA.
Further, the polypeptide comprises a polypeptide with an amino acid sequence shown as SEQ ID NO. 1, or an active fragment or analogue thereof;
preferably, the amino acid sequence of the active fragment, analogue has at least 95% identity to the amino acid sequence shown as SEQ ID NO. 1;
more preferably, the nucleotide sequence of the polypeptide is shown as SEQ ID NO. 2.
In a second aspect, the invention provides a fusion polypeptide.
Further, the fusion polypeptide comprises at least two subunits in any order;
preferably, one of the subunits is specific for PSMA;
preferably, the other subunit or subunits are specific for any one or more antigens from the group: PSMA, PSCA, PSA, PAP, MSLN, CD19, CD20, CD22, CD27, CD28, CD38, CD45, CD56, CD81, CD117, CD138, CD200, BCMA;
more preferably, the subunit comprises a polypeptide having binding specificity for PSMA;
most preferably, the polypeptide is a polypeptide according to the first aspect of the invention.
In a third aspect the invention provides a polypeptide derivative.
Further, the polypeptide derivatives include conjugates comprising the polypeptides of the first aspect of the invention, conjugates comprising the fusion polypeptides of the second aspect of the invention, tumor antigen binding peptides comprising the polypeptides of the first aspect of the invention, tumor antigen binding peptides comprising the fusion polypeptides of the second aspect of the invention;
preferably, the conjugate comprising the polypeptide of the first aspect of the invention, the conjugate comprising the fusion polypeptide of the second aspect of the invention comprises a product obtained by conventional modification of the amino-or carboxyl-terminal side chain groups of the polypeptide and/or fusion polypeptide, or by ligation of a tag for detection or purification of the polypeptide, or by conjugation with other substances;
more preferably, the conventional modification comprises amination, hydroxylation, carboxylation, carbonylation, amidation, alkylation, phosphorylation, glycosylation, cyclization, biotinylation, acetylation, esterification, fluorophore modification, polyethylene glycol PEG modification, immobilization modification;
more preferably, the tag comprises His6, GST, EGFP, MBP, nus, HA, c-Myc, profinityeXact, igG, FLAG;
more preferably, the other substance comprises a polypeptide, a protein, a drug;
most preferably, the protein comprises serum albumin, the polypeptide comprises an Fc region, a signal peptide, a polypeptide label, and the drug comprises a bisphosphonate, an iridoid;
preferably, the tumor antigen binding peptide comprising the polypeptide of the first aspect of the invention, the tumor antigen binding peptide comprising the fusion polypeptide of the second aspect of the invention comprises an extracellular signal domain comprising an antigen recognition region, a transmembrane domain linked to the extracellular signal domain, an intracellular signal domain linked to the transmembrane domain;
more preferably, the extracellular signal domain comprises a polypeptide;
most preferably, the polypeptide specifically binds antigen PSMA, PSCA, PSA, PAP, MSLN, CD, CD20, CD22, CD27, CD28, CD38, CD45, CD56, CD81, CD117, CD138, CD200, BCMA;
most preferably, the polypeptide specifically binds to the antigen PSMA;
most preferably, the polypeptide is a polypeptide according to the first aspect of the invention, a fusion polypeptide according to the second aspect of the invention;
more preferably, the extracellular signal domain further comprises a hinge region;
most preferably, the hinge region comprises a hinge region of any one or more of the following molecules: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, CD154;
more preferably, the transmembrane domain comprises a transmembrane domain of any one or more of the following molecules: CD3 epsilon, CD8, CD9, CD28, CD45, CD4, CD5, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS;
more preferably, the intracellular signaling domain comprises a signaling domain of any one or more of the following molecules: cd3ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, CD66d; and/or a costimulatory domain comprising a costimulatory molecule comprising the CD27, 4-1BB, CD30, CD40, ICOS, CD28, OX40, NKG2C, B-H3, fc receptor-related gamma chain;
more preferably, the tumor antigen binding peptide further comprises a signal peptide derived from: the α chain, β chain, CD28, CD3 epsilon, CD35 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR of the T cell receptor.
In a fourth aspect, the invention provides a nucleic acid molecule.
Further, the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of the first aspect of the invention, a nucleotide sequence encoding the fusion polypeptide of the second aspect of the invention;
preferably, the nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of:
(a) A nucleic acid molecule encoding a polypeptide having the amino acid sequence shown in SEQ ID NO. 1, or an active fragment or analogue thereof;
(b) A nucleic acid molecule encoding a fusion polypeptide according to the second aspect of the invention;
(c) A nucleic acid molecule complementary to (a) or (b);
(d) A nucleic acid molecule having at least 70% identity to (a) or (b) or (c);
more preferably, the nucleic acid molecule is a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 1, a nucleic acid molecule encoding the fusion polypeptide according to the second aspect of the present invention;
most preferably, the nucleotide sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 1 is shown in SEQ ID NO. 2.
In a fifth aspect the invention provides a carrier.
Further, the vector comprises a nucleic acid molecule according to the fourth aspect of the invention;
preferably, the vector is a recombinant vector constructed by the nucleic acid molecule according to the fourth aspect of the present invention and a plasmid, a viral vector or a carrier expression vector;
more preferably, the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a gamma retrovirus vector, a lentivirus vector.
In a sixth aspect, the invention provides an engineered host cell.
Further, the engineered host cell comprises a polypeptide according to the first aspect of the invention, a fusion polypeptide according to the second aspect of the invention, a polypeptide derivative according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention;
preferably, the host cell comprises an immune cell;
more preferably, the immune cells include monocytes, macrophages, dendritic cells, megakaryocytes, granulocytes, T lymphocytes, B lymphocytes, NK cells.
Further, in particular embodiments of the invention the host cell is referred to as a TABP-EIC (Tumor Antigen Binding Peptide-Engineering Immune Cell, tumor antigen binding peptide-engineered immune cell).
A seventh aspect of the invention provides a pharmaceutical composition.
Further, the pharmaceutical composition comprises a polypeptide according to the first aspect of the invention, a fusion polypeptide according to the second aspect of the invention, a polypeptide derivative according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention;
preferably, the pharmaceutical composition may further comprise pharmaceutically acceptable carriers and/or excipients.
Further, the pharmaceutically acceptable carrier and/or excipients are described in detail in Remington's Pharmaceutical Sciences.
In an eighth aspect of the invention, a kit is provided.
Further, the kit comprises a polypeptide according to the first aspect of the invention, a fusion polypeptide according to the second aspect of the invention, a polypeptide derivative according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention.
A ninth aspect of the invention provides a method of any one of:
(1) A method of screening for a polypeptide according to the first aspect of the invention, the method comprising the steps of: screening out the polypeptide of the first aspect of the invention using a phage-polypeptide library with PSMA as a target;
(2) A method of making an engineered host cell according to the sixth aspect of the invention, the method comprising the steps of: introducing a polypeptide according to the first aspect of the invention, a fusion polypeptide according to the second aspect of the invention, a polypeptide derivative according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention into a host cell to obtain an engineered host cell according to the sixth aspect of the invention;
preferably, the nucleic acid molecule or vector is introduced into the host cell by means including conjugation, transformation, transfection, transduction, electroporation;
(3) A method for non-diagnostic and non-therapeutic destination detection of PSMA in a sample, the method comprising the steps of: contacting the polypeptide of the first aspect of the invention and/or the fusion polypeptide of the second aspect of the invention with a sample, and detecting a reaction of the polypeptide and/or fusion polypeptide with PSMA in the sample;
preferably, the reaction is detected by fluorescence, enzymatic coloration, chemiluminescence;
preferably, the method further comprises detecting the content of PSMA in the sample;
preferably, the sample is selected from one or more of the following group: tissue, blood, plasma, serum, urine, saliva, ascites fluid, circulating cells, circulating tumor cells, histological preparations;
(4) A method of producing a polypeptide, the method comprising the steps of: culturing the engineered host cell of the sixth aspect of the invention, and isolating the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO. 1 from the cell culture.
Further, the method further comprises a method of treating a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition according to the seventh aspect of the invention.
Further, the method further comprises a method of diagnosing whether a subject has a PSMA-expressing tumor, the method comprising detecting the amount of PSMA in a sample from which the subject is derived using the polypeptide of the first aspect of the invention and/or the fusion polypeptide of the second aspect of the invention;
preferably, the method further comprises comparing the amount of PSMA in the subject-derived sample to the amount of PSMA in a known standard or reference, and determining if the level of PSMA in the subject-derived sample falls within a level of PSMA associated with a tumor;
preferably, the method further comprises detecting the level of PSMA in the subject-derived sample with a kit according to the eighth aspect of the invention, thereby determining whether the subject has a tumor;
preferably, the sample is selected from one or more of the following group: tissue, blood, plasma, serum, urine, saliva, ascites fluid, circulating cells, circulating tumor cells, histological preparations;
preferably, the tumor comprises a solid tumor, a non-solid tumor;
more preferably, the solid tumor comprises prostate cancer, bladder cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, lung adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, colon cancer, rectal cancer, vascular endothelial tumor, testicular cancer, skin cancer;
more preferably, the non-solid tumor comprises acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hematopoietic tumor, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma;
most preferably, the tumor is prostate cancer.
A tenth aspect of the invention provides the use of any one of the following:
(1) The application of the polypeptide in the first aspect of the invention in preparing fusion polypeptide, polypeptide derivative, nucleic acid molecule and carrier;
(2) Application of fusion polypeptide in the second aspect of the invention in preparation of polypeptide derivatives, nucleic acid molecules and vectors;
(3) The use of a polypeptide derivative according to the third aspect of the invention in the preparation of a nucleic acid molecule, a vector;
(4) The use of a nucleic acid molecule according to the fourth aspect of the invention for the preparation of a vector;
(5) Use of a polypeptide according to the first aspect of the invention, a fusion polypeptide according to the second aspect of the invention, a polypeptide derivative according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention for the preparation of an immune cell for tumour immunotherapy;
(6) Use of a polypeptide according to the first aspect of the invention, a fusion polypeptide according to the second aspect of the invention, a polypeptide derivative according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention for the preparation of a medicament for the prevention and/or treatment of a tumor;
(7) Use of a polypeptide according to the first aspect of the invention, a fusion polypeptide according to the second aspect of the invention, a polypeptide derivative according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention for the preparation of a kit for detecting PSMA and/or for diagnosing a tumor;
(8) Use of an engineered host cell according to the sixth aspect of the invention for the prevention and/or treatment of a tumor;
(9) The use of a pharmaceutical composition according to the seventh aspect of the invention for the prevention and/or treatment of tumors;
(10) The kit according to the eighth aspect of the invention is used for detecting PSMA and/or diagnosing tumors;
preferably, the detecting PSMA and/or diagnosing a tumor comprises detecting the level or level of PSMA in a test sample, diagnosing whether the subject has a tumor;
preferably, the fusion polypeptide is a fusion polypeptide according to the second aspect of the invention;
preferably, the polypeptide derivative is a polypeptide derivative according to the third aspect of the invention;
preferably, the nucleic acid molecule is a nucleic acid molecule according to the fourth aspect of the invention;
preferably, the carrier is a carrier according to the fifth aspect of the invention;
preferably, the tumor comprises a solid tumor, a non-solid tumor;
more preferably, the solid tumor comprises prostate cancer, bladder cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, lung adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, colon cancer, rectal cancer, vascular endothelial tumor, testicular cancer, skin cancer;
more preferably, the non-solid tumor comprises acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hematopoietic tumor, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma;
most preferably, the tumor is prostate cancer.
The invention has the advantages and beneficial effects that:
(1) According to the invention, the specific polypeptide obtained by screening the phage-polypeptide library is used for preparing the TABP-EIC cell for the first time instead of the traditional single-chain antibody, compared with the traditional method for preparing the TABP-EIC cell, the pioneering preparation method obviously shortens the screening time of the extracellular recognition region sequence of the TABP-EIC cell, the binding efficiency of the screened polypeptide ligand and the related tumor specific antigen is higher, and the recognition polypeptide ligand aiming at the tumor with undefined antigen can be screened;
(2) The invention provides a novel polypeptide, which can specifically bind with a prostate cancer related tumor antigen PSMA, has the advantages of high antigen binding efficiency, short screening time and the like, can be applied to detection of the prostate cancer related tumor antigen PSMA, can replace a traditional single-chain antibody to prepare a TABP-EIC cell, and is further used for immunotherapy of the prostate cancer;
(3) The method for preparing the TABP-EIC cells by using the peptide ligand to replace the single-chain antibody provides a new thought for the immunotherapy of tumors, and has extremely high application value in the immunocyte therapy of tumors.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the immunofluorescence results of polypeptide HFK in Lncap cell line with higher PSMA expression level;
FIG. 2 shows the immunofluorescence results of polypeptide HFK in PC3 cell lines with low PSMA expression.
Detailed Description
The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.
EXAMPLE 1 screening for PSMA as a target to obtain polypeptide HFK that specifically binds PSMA
1. Experimental materials
The ph.d. -12 phage display peptide library kit used in the examples of the present invention was purchased from New England Biolabs (NEB) company under the catalog number E8110SC.
2. Ph.D. -12 phage display peptide library kit composition
Random dodecapeptide phage display library: 100. mu L, 1.5X10) 13 pfu/mL, stored in TBS solution containing 50% glycerol, complexity of 2.7X10 9 A plurality of transformants; -28 gIII sequencing primer: 5'-HOGTATGGGATTTTGCTAAACAAC-3',100 pmol,1 pmol/. Mu.L; -96 gIII sequencing primer: 5 '-HOCCCTACATTAGTTAGGTAACG-3', 100 pmol/. Mu.L, 1 pmol/. Mu.L; coli ER2738 host strain F' lacIqΔ (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 cell culture containing 50% glycerol, and non-competent cells are stored at-70 ℃; streptavidin, freeze-dried powder 1.5 mg; biotin: 10 mM 100. Mu.L.
3. Experimental method
The invention adopts a Ph.D. -12 phage display peptide library kit to screen out polypeptide HFK which specifically binds to PSMA, and the method comprises the following steps:
first day
The number and types of target molecules on which the library panning was performed simultaneously, as required, were varied, panning experiments were performed in a single sterilized polystyrene dish, 12 or 24 well plate, 96 well microtiter plate, each target molecule coated with at least one plate (or one well), the amounts given in the following method were 60 x 15 mm dishes, microplates in brackets, and other medium-sized wells adjusted accordingly, but in each case the number of phages added was the same: 1.5X10 11 Each virus seed;
(1) 100. Mu.g/mL of target molecule solution (dissolved in 0.1M NaHCO 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 be used;
(2) 1.5. 1.5 mL (150. Mu.L per well of microplate) of the above solution was added per plate (well), and the rotation was repeated until the surface was completely wetted;
(3) Slightly shaking at 4deg.C in a humidifying container (such as sealable plastic box with wet paper towel), incubating overnight, and storing the plate at 4deg.C in the container;
the next day
(4) ER2738 monoclonal (plate paved during phage titer determination) is selected and placed in 10 mL of LB liquid medium, if eluted phage is amplified on the same day, ER2738 can also be inoculated in 20 mL of LB liquid medium, and the phage is subjected to severe shake culture at 37 ℃ by using a 250 mL triangular flask;
(5) Pouring out the coating liquid in each plate, inverting the plates, and forcefully beating and throwing on clean paper towels to remove residual solution, wherein each plate (or hole) is filled with sealing liquid, and the action is at least 1 h at 4 ℃;
(6) The plate was washed 6 times, each time rotated so that the bottom and edges of the plate or well were washed, the buffer was decanted, and the plate was inverted and whipped over a clean paper towel to remove residual solution (or using an automatic plate washer);
(7) Dilution with 1 mL (microplates with 100. Mu.L) TBST buffer 4X 10 10 (i.e., 10. Mu.L of the original library) and then added to the coated plate, gently shaken at room temperature for 10-60 min;
(8) Pouring to remove unbound phage, and inverting the plate to remove residual solution by beating on a clean paper towel;
(9) Washing the plate 10 times with TBST buffer solution 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 is eluted with 1 mL (microplates then with 100. Mu.L of a suitable elution buffer), the known ligand of the target molecule is dissolved in TBS solution at a concentration of 0.1-1 mM or the bound phage is competitively eluted from the immobilized target molecule with a free target molecule solution (100. Mu.g/mL in TBS), gently shaken at room temperature for 10-60 min, and the eluate is aspirated into another clean microcentrifuge tube; bound molecules can also be isolated with a non-specific buffer such as 0.2M Glycine-HCl (pH 2.2), 1 mg/mL BSA: gently shaking for >10 min, sucking the eluate into another clean microcentrifuge tube, and neutralizing the eluate with 150 μl (15 μl for microperforations) of 1M Tris-HCl (pH 9.1);
(11) The titer of a small amount (1. Mu.L) of eluate was determined by the procedure described in the conventional M13 method, and plaques obtained from the first or second round of eluate titer determination were sequenced, if necessary, as follows: if necessary, the remaining eluate may be stored overnight at 4deg.C for the next day for amplification, at which time ER2738 may be grown overnight in LB-Tet medium, and the next day culture 1:100 may be diluted in 20 mL LB (in 250 mL Erlenmeyer flask), unamplified eluate may be added, and grown under vigorous shaking at 37deg.C for 4.5 h, continuing step 13;
(12) Amplifying the remaining eluate: the eluate was added to 20 mL of ER2738 culture (cells in log phase), and cultured with vigorous shaking at 37℃for 4.5. 4.5 h;
(13) The culture was transferred to a centrifuge tube and then centrifuged at 10,000 rpm at 4℃for 10 min. Transferring the supernatant into another centrifuge tube, and centrifuging again;
(14) Transferring 80% of the upper part of the supernatant into a fresh tube, adding 1/6 volume of PEG/NaCl, and allowing phage to precipitate at 4deg.C for at least 60 min overnight;
third day
(15) Centrifuging PEG precipitation at 4deg.C and 10,000 rpm for 15 min, removing supernatant, centrifuging briefly, and sucking off residual supernatant;
(16) The sediment is resuspended in 1 mL TBS, the suspension is transferred into a microcentrifuge tube, and the residual cells are precipitated by centrifugation for 5 min at 4 ℃;
(17) Transferring the supernatant into another fresh microcentrifuge tube, reprecipitating with 1/6 volume of PEG/NaCl, incubating on ice for 15-60 min, centrifuging at 4deg.C for 10 min, discarding supernatant, centrifuging briefly, and removing residual supernatant with micropipette;
(18) The pellet was resuspended in 200. Mu.L TBS,0.02% NaN 3 Centrifuging for 1 min, precipitating any residual insoluble substances, transferring the supernatant into a fresh tube, and obtaining the amplified eluate;
(19) Titrating the amplified eluate with LB/IPTG/Xgal plates according to the conventional M13 method described above, and storing at 4 ℃;
(20) Coating a plate or well in preparation for a second round of panning;
fourth and fifth day
(21) The number of blue spots on the plate was counted to determine the titer, and this value was used to calculate the corresponding 1-2X 10 11 The addition amount of pfu; if the titer is too low, the next rounds of panning can be as low as 10 9 phage addition to pfu was tested;
(22) A second round of panning was performed: 1-2×10 of the eluate amplified by the first round of panning 11 phage amount of pfu step 4-18 was repeated, and Tween concentration was increased to 0.5% (v/v) in the washing step;
(23) Titer after amplification of the eluate from the second round of panning was determined on LB/IPTG/Xgal plates;
(24) Coating a plate or well in preparation for a third round of panning;
sixth day
(25) A third round of panning was performed: 2X 10 of the eluate amplified by the second round of panning 11 phage amount of pfu steps 4-11 were repeated, again with 0.5% (v/v) Tween in the washing step;
(26) Titer was determined on LB/IPTG/Xgal plates without amplification of the eluate from the third round of panning, and plaques obtained from the titer determination were sequenced without further amplification of the eluate unless the fourth round of panning was performed: as long as the culture time of the flat plate is not more than 18 h, the culture time is too long, the deletion is easy to occur, and the rest of the eluate is stored at 4 ℃;
(27) One ER2738 clone was selected and cultured overnight in LB-Tet medium.
4. Experimental results
Experimental results show that the amino acid sequence of the polypeptide HFK which specifically binds to PSMA is HFKAPHTVRLKS (SEQ ID NO: 1), and the nucleotide sequence is CACTTCAAGGCCCCCCACACCGTGAGACTGAAGAGC (SEQ ID NO: 2).
Example 2 immunofluorescence techniques to identify the affinity of polypeptide HFK for prostate cancer cells
1. Experimental materials
The prostate cancer cell line PC3 cell (CRL-1435) and the prostate cancer cell line Lncap cell (CRL-1740) were purchased from ATCC company.
2. Experimental method
(1) Fixing
Fixing cells in a culture plate with a newly prepared 4% paraformaldehyde solution for 15 minutes, and cleaning the sample by DPBS (digital video broadcasting) with careful operation to avoid the cells from falling off the glass slide;
(2) Cleaning
DPBS is washed three times for 5 minutes each time;
(3) Closure
Blocking with 2% BSA at 4deg.C for 30 min, and DPBS washing twice for 5 min each;
(4) Polypeptide incubation
HFK polypeptide (final concentration 1. Mu.g/mL) labeled with FITC (fluorescein isothiocyanate) was added and incubated overnight in a humidity chamber at 4deg.C in dark conditions;
(5) Cleaning
DPBS is washed three times for 5 minutes each time;
(6) Sealing sheet and observation
Adding 0.5 mug/mL DAPI (prepared by PBS) for dyeing for 10 minutes, and other nuclear dyeing dyes can be adopted;
the sample was washed three times with PBS, excess DAPI was removed, 20. Mu.L of caplet was added for caplet sealing, and observed under a microscope, taking care that the fluorescence intensity decayed with light stimulation, and photographed as soon as possible.
3. Experimental results
Experimental results show that immunofluorescence results obtained in the Lncap cell line with higher PSMA expression level and the PC3 cell line with low PSMA expression level obtained by screening in the example 1 are respectively shown in fig. 1 and 2, wherein blue is a cell nucleus stain, green is that the polypeptide HFK is combined with a cell surface antigen PSMA after being marked by green fluorescent dye, and the results show that the screened polypeptide HFK can specifically identify a PSMA positive prostate cancer cell line (Lncap cell line).
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Sequence listing
<110> Novague medical science and technology (Zhuhai Violin New district) Limited
<120> a novel polypeptide and its use in diagnosis and treatment of prostate cancer
<141> 2021-07-13
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
His Phe Lys Ala Pro His Thr Val Arg Leu Lys Ser
1 5 10
<210> 2
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
cacttcaagg ccccccacac cgtgagactg aagagc 36
Claims (58)
1. A PSMA targeting polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.
2. A fusion polypeptide comprising at least two subunits in any order, wherein one subunit is a polypeptide having binding specificity for PSMA;
the polypeptide is the polypeptide of claim 1.
3. The fusion polypeptide of claim 2, wherein the other one or more subunits are specific for any one or more antigens from the group: PSMA, PSCA, PSA, PAP, MSLN, CD19, CD20, CD22, CD27, CD28, CD38, CD45, CD56, CD81, CD117, CD138, CD200, BCMA.
4. A polypeptide derivative comprising a conjugate comprising the polypeptide of claim 1, a conjugate comprising the fusion polypeptide of claim 2 or 3, a tumor antigen binding peptide comprising the polypeptide of claim 1, and a tumor antigen binding peptide comprising the fusion polypeptide of claim 2 or 3.
5. The polypeptide derivative according to claim 4, wherein the conjugate comprising the polypeptide of claim 1, the conjugate comprising the fusion polypeptide of claim 2 or 3 comprises a product obtained by conventional modification of the amino acid side chain group, amino terminus or carboxyl terminus of the polypeptide and/or the fusion polypeptide, or a product obtained by ligation of a tag for detection or purification of the polypeptide, or a product obtained by binding with other substances.
6. The polypeptide derivative of claim 5, wherein the conventional modification comprises amination, hydroxylation, carboxylation, carbonylation, amidation, alkylation, phosphorylation, glycosylation, cyclization, biotinylation, acetylation, esterification, fluorophore modification, polyethylene glycol PEG modification, immobilization modification.
7. The polypeptide derivative according to claim 5, wherein the tag comprises His6, GST, EGFP, MBP, nus, HA, c-Myc, profinityeXact, igG, FLAG.
8. The polypeptide derivative according to claim 5, wherein the other substance comprises a polypeptide, a protein, a drug.
9. The polypeptide derivative according to claim 4, wherein the tumor antigen binding peptide comprising the polypeptide of claim 1, the tumor antigen binding peptide comprising the fusion polypeptide of claim 2 or 3 comprises an extracellular signal domain comprising an antigen recognition region, a transmembrane domain linked to the extracellular signal domain, an intracellular signal domain linked to the transmembrane domain.
10. The polypeptide derivative of claim 9, wherein the extracellular signal domain comprises a polypeptide.
11. The polypeptide derivative according to claim 10, wherein the polypeptide is the polypeptide of claim 1, the fusion polypeptide of claim 2 or 3.
12. The polypeptide derivative according to claim 11, wherein the polypeptide specifically binds any one or more antigens from the group: PSMA, PSCA, PSA, PAP, MSLN, CD19, CD20, CD22, CD27, CD28, CD38, CD45, CD56, CD81, CD117, CD138, CD200, BCMA.
13. The polypeptide derivative of claim 12, wherein the polypeptide specifically binds to the antigen PSMA.
14. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 1, a nucleotide sequence encoding the fusion polypeptide of claim 2 or 3.
15. The nucleic acid molecule of claim 14, wherein the nucleotide sequence encoding the polypeptide of claim 1 is set forth in SEQ ID No. 2.
16. A vector comprising the nucleic acid molecule of claim 14.
17. The vector of claim 16, wherein the vector is a recombinant vector constructed from the nucleic acid molecule of claim 14 and a plasmid, viral vector or expression vector.
18. The vector of claim 17, wherein the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a gamma retrovirus vector, a lentivirus vector.
19. An engineered host cell comprising the polypeptide of claim 1, the fusion polypeptide of claim 2 or 3, the polypeptide derivative of claim 4, the nucleic acid molecule of claim 14, and the vector of claim 16.
20. The engineered host cell of claim 19, wherein the host cell comprises an immune cell.
21. The engineered host cell of claim 20, wherein the immune cells comprise monocytes, macrophages, dendritic cells, megakaryocytes, granulocytes, T lymphocytes, B lymphocytes, NK cells.
22. A pharmaceutical composition comprising the polypeptide of claim 1, the fusion polypeptide of claim 2 or 3, the polypeptide derivative of claim 4, the nucleic acid molecule of claim 14, the vector of claim 16, and the engineered host cell of claim 19.
23. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable carrier and/or adjuvant.
24. A kit comprising the polypeptide of claim 1, the fusion polypeptide of claim 2 or 3, the polypeptide derivative of claim 4, the nucleic acid molecule of claim 14, the vector of claim 16, and the engineered host cell of claim 19.
25. A method of making the engineered host cell of claim 19, comprising the steps of: introducing the polypeptide of claim 1, the fusion polypeptide of claim 2 or 3, the polypeptide derivative of claim 4, the nucleic acid molecule of claim 14, the vector of claim 16 into a host cell, resulting in an engineered host cell of claim 19.
26. The method of claim 25, wherein the nucleic acid molecule or vector is introduced into the host cell by conjugation, transformation, transfection, transduction, or electroporation.
27. A method for non-diagnostic and non-therapeutic destination detection of PSMA in a sample, the method comprising the steps of: contacting the polypeptide of claim 1 and/or the fusion polypeptide of claim 2 or 3 with a sample, and detecting a reaction of the polypeptide and/or fusion polypeptide with PSMA in the sample.
28. The method of claim 27, wherein the reaction is detected by fluorescence, enzymatic coloration, or chemiluminescence.
29. A method of producing a polypeptide, comprising the steps of: culturing the engineered host cell of claim 19, and isolating the polypeptide comprising the amino acid sequence set forth in SEQ ID No. 1 from the cell culture.
30. Use of the polypeptide of claim 1 for the preparation of fusion polypeptides, polypeptide derivatives, nucleic acid molecules, vectors.
31. The use according to claim 30, wherein the fusion polypeptide is a fusion polypeptide according to claim 2 or 3.
32. The use according to claim 30, wherein the polypeptide derivative is a polypeptide derivative according to claim 4.
33. The use according to claim 30, wherein the nucleic acid molecule is a nucleic acid molecule according to claim 14.
34. The use according to claim 30, wherein the carrier is the carrier of claim 16.
35. Use of the fusion polypeptide of claim 2 or 3 for the preparation of polypeptide derivatives, nucleic acid molecules, vectors.
36. The use according to claim 35, wherein the polypeptide derivative is a polypeptide derivative according to claim 4.
37. The use according to claim 35, wherein the nucleic acid molecule is a nucleic acid molecule according to claim 14.
38. The use according to claim 35, wherein the carrier is the carrier of claim 16.
39. The use of the polypeptide derivative according to claim 4 for preparing nucleic acid molecules and vectors.
40. The use according to claim 39, wherein the nucleic acid molecule is a nucleic acid molecule according to claim 14.
41. The use according to claim 39, wherein the carrier is the carrier according to claim 16.
42. Use of the nucleic acid molecule of claim 14 for the preparation of a vector.
43. The use according to claim 42, wherein the carrier is the carrier according to claim 16.
44. Use of the polypeptide of claim 1, the fusion polypeptide of claim 2 or 3, the polypeptide derivative of claim 4, the nucleic acid molecule of claim 14, the vector of claim 16 for the preparation of an immune cell for PSMA-expressing tumor immunotherapy.
45. The use of claim 44, wherein the PSMA-expressing tumor comprises a solid tumor, a non-solid tumor.
46. The use of claim 45, wherein the solid tumor comprises prostate cancer, bladder cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, lung adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, colon cancer, rectal cancer, vascular endothelial tumor, testicular cancer, skin cancer.
47. The use of claim 45, wherein the non-solid tumor comprises acute lymphoblastic leukemia, chronic lymphocytic leukemia, hematopoietic tumor, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma.
48. The use of claim 46, wherein the tumor is prostate cancer.
49. Use of the polypeptide of claim 1, the fusion polypeptide of claim 2 or 3, the polypeptide derivative of claim 4, the nucleic acid molecule of claim 14, the vector of claim 16, the engineered host cell of claim 19 in the manufacture of a medicament for the prevention and/or treatment of PSMA-expressing tumors.
50. The use of claim 49, wherein the PSMA-expressing tumor comprises a solid tumor, a non-solid tumor.
51. The use of claim 50, wherein the solid tumor comprises prostate cancer, bladder cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, lung adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, colon cancer, rectal cancer, vascular endothelial tumor, testicular cancer, skin cancer.
52. The use according to claim 50, wherein the non-solid tumor comprises acute lymphoblastic leukemia, chronic lymphocytic leukemia, hematopoietic tumor, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma.
53. The use of claim 51, wherein the tumor is prostate cancer.
54. Use of the polypeptide of claim 1, the fusion polypeptide of claim 2 or 3, the polypeptide derivative of claim 4, the nucleic acid molecule of claim 14, the vector of claim 16, the engineered host cell of claim 19 for the preparation of a kit for detecting PSMA and/or diagnosing PSMA-expressing tumors.
55. The use of claim 54, wherein the PSMA-expressing tumor comprises a solid tumor, a non-solid tumor.
56. The use of claim 55, wherein the solid tumor comprises prostate cancer, bladder cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, lung adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, colon cancer, rectal cancer, vascular endothelial tumor, testicular cancer, skin cancer.
57. The use of claim 55, wherein the non-solid tumor comprises acute lymphoblastic leukemia, chronic lymphocytic leukemia, hematopoietic tumor, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma.
58. The use of claim 56, wherein said tumor is prostate cancer.
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| CN102162006A (en) * | 2010-02-20 | 2011-08-24 | 北京金菩嘉医疗科技有限公司 | Detection agent for detecting prostate cancer and application thereof |
| CN106905415A (en) * | 2017-02-23 | 2017-06-30 | 新疆军区总医院 | The polypeptide combined with resistance cervical carcinoma cancerous cell line Hela cell membrane surface molecules |
| CN107446021A (en) * | 2017-07-26 | 2017-12-08 | 中国药科大学 | Folacin receptor alpha specific binding peptide 5 and its application |
| CN108822189A (en) * | 2018-06-26 | 2018-11-16 | 江苏大学 | A kind of targeting combines specific polypeptide and its application of lymphocytic cancer cell system |
| CN112584849A (en) * | 2018-08-24 | 2021-03-30 | 杭州优善生物科技有限公司 | Therapeutic agents comprising nucleic acids and CAR-modified immune cells and uses thereof |
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| CN102162006A (en) * | 2010-02-20 | 2011-08-24 | 北京金菩嘉医疗科技有限公司 | Detection agent for detecting prostate cancer and application thereof |
| CN106905415A (en) * | 2017-02-23 | 2017-06-30 | 新疆军区总医院 | The polypeptide combined with resistance cervical carcinoma cancerous cell line Hela cell membrane surface molecules |
| CN107446021A (en) * | 2017-07-26 | 2017-12-08 | 中国药科大学 | Folacin receptor alpha specific binding peptide 5 and its application |
| CN108822189A (en) * | 2018-06-26 | 2018-11-16 | 江苏大学 | A kind of targeting combines specific polypeptide and its application of lymphocytic cancer cell system |
| CN112584849A (en) * | 2018-08-24 | 2021-03-30 | 杭州优善生物科技有限公司 | Therapeutic agents comprising nucleic acids and CAR-modified immune cells and uses thereof |
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