CN113527435A - Novel polypeptide for specifically identifying prostate cancer cells, derivatives and application thereof - Google Patents
Novel polypeptide for specifically identifying prostate cancer cells, derivatives and application thereof Download PDFInfo
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Abstract
The invention discloses a novel polypeptide for specifically identifying prostate cancer cells, a derivative and an application thereof, wherein the polypeptide is obtained by screening a phage-polypeptide library, can be specifically bound with tumor cells, has high binding efficiency, can be used for preparing tumor antigen binding peptide-engineered immune cells by replacing a traditional single-chain antibody as an extracellular recognition area, can be used for aiming at tumors with undefined antigens, and has the advantages of remarkably shortening the time for screening the extracellular recognition area, higher binding efficiency of a screened polypeptide ligand and the tumor cells and good application prospect.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a novel polypeptide for specifically identifying prostate cancer cells, a derivative thereof and application thereof.
Background
At present, tumors become the second leading cause of death of cardiovascular diseases in the world, the number of tumor patients increases year by year, the current treatment methods for tumors mainly comprise surgical treatment, radiotherapy, chemotherapy, targeted drug therapy and the like, the traditional tumor treatment methods focus on the local part of a tumor focus and kill tumor cells on the physical and chemical levels, wherein the conventional surgical treatment mainly adopts a surgical excision mode, but the tumor cells are difficult to be excised through the surgical excision, so that the patients are easy to relapse after the surgery; radiotherapy and chemotherapy can kill normal human cells while killing tumor cells, thus causing great damage to patients; although the targeted drug therapy can reduce the adverse reaction of the drug to a certain extent, the drug resistance of the tumor is easy to generate, and further the tumor relapse is caused. With the continuous development of molecular biology and tumor biology, tumor immunotherapy is produced, and becomes another effective treatment method following surgery treatment, radiotherapy, chemotherapy and targeted drug treatment, and is different from the traditional tumor treatment method, the tumor immunotherapy does not directly act on focus, but achieves the purpose of controlling and killing tumors by enhancing the immune system of a human body, and compared with the traditional tumor treatment method, the tumor immunotherapy has the advantages of small side effect, strong specificity, wide tumor killing spectrum, low recurrence rate and the like.
Tumor immunotherapy falls into two categories: the principle of the method is that T cells or NK cells modified by Chimeric antigen receptors (CAR-NK) can specifically recognize tumor-related antigens on the surface of tumor cells, so that the targeting, killing activity and persistence of effector T cells or NK cells are higher than those of immune cells which are conventionally applied, and the local tumor immunosuppression microenvironment can be overcome and the host immune tolerance state can be broken. The CAR is a core structure of CAR-T and CAR-NK, and endows cells with the capability of specifically recognizing tumor-associated antigens on the surface of tumor cells, and is mainly composed of three functional domains, namely an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain is composed of a Single-chain variable fragment (scFv) of a monoclonal antibody which is responsible for recognizing and binding the antigens and a section of Hinge region (Hinge) for connection; the intracellular domain is composed of a Costimulatory Signaling domain (Costimulatory domain) and a Signaling domain (Signaling domain).
Current CAR cell therapies rely on specific Tumor Associated Antigen (TAA) defined Tumor types, if the Tumor associated antigen of the Tumor is not defined or identified, single chain antibody sequences of the relevant specific recognition cannot be prepared based on the Tumor associated antigen, and then specific CARs capable of effectively recognizing Tumor cells and CAR-T or CAR-NK cells specifically recognizing and killing Tumor cells cannot be constructed. In order to solve the technical problems in the prior art, the present invention aims to provide a method for screening a sequence of an extracellular recognition region of TABP-EIC (Tumor Antigen Binding Peptide-engineered Immune Cell) by using a phage-polypeptide library, wherein a polypeptide ligand obtained by the method can replace a traditional single-chain antibody to prepare the TABP-EIC Cell. To date, no reports have been made concerning the use of the novel polypeptides of the present invention in immunotherapy.
Disclosure of Invention
In order to solve the above problems in the art, the present invention provides a novel polypeptide specifically recognizing prostate cancer cells, a derivative thereof, and an application thereof, wherein the polypeptide is obtained by screening a phage-polypeptide library, can specifically bind to tumor cells, and has high binding efficiency.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect, the present invention provides a novel polypeptide that specifically recognizes tumor cells.
Further, the polypeptide comprises a polypeptide of an amino acid sequence shown as SEQ ID NO. 1, or an active fragment and an analogue thereof;
preferably, the amino acid sequence of the active fragment and the amino acid sequence of the analogue have at least 95 percent of identity with the amino acid sequence shown as SEQ ID NO. 1;
preferably, the tumor cells are prostate cancer cells;
more preferably, the amino acid sequence of the polypeptide is shown as SEQ ID NO 1;
most preferably, the nucleotide sequence of the polypeptide is shown as SEQ ID NO 12.
In a second aspect, the invention provides a tumor antigen binding peptide.
Further, the tumor antigen binding peptide comprises an extracellular signaling domain comprising an antigen recognition region, a transmembrane domain linked to the extracellular signaling domain, an intracellular signaling domain linked to the transmembrane domain;
preferably, the extracellular signaling domain comprises an antigen recognition region;
more preferably, the antigen recognition region is obtained by linking one or more polypeptides of the first aspect of the present invention in tandem by a linker;
most preferably, the amino acid sequence of the polypeptide is shown as SEQ ID NO 1;
most preferably, the amino acid sequence of the linker is shown as SEQ ID NO. 4;
most preferably, the antigen recognition region is formed by linking three polypeptides of the first aspect of the invention in tandem by a linker;
most preferably, the amino acid sequence of the antigen recognition region is shown as SEQ ID NO. 5;
most preferably, the nucleotide sequence of the antigen recognition region is shown as SEQ ID NO 6;
preferably, the extracellular signaling domain further comprises a hinge region;
more preferably, the hinge region comprises a hinge region of any one or more of the following molecules: CD3 epsilon, CD4, CD5, CD8, CD8 alpha, PD1, CD16, CD22, CD28, CD33, CD37, CD45, CD134, CD137, CD152, CD 154;
Most preferably, the hinge region is that of a CD8 a molecule;
most preferably, the amino acid sequence of the hinge region of the CD8 a molecule is set forth in SEQ ID NO. 7;
preferably, the extracellular signaling domain further comprises a signal peptide;
more preferably, the signal peptide is derived from the following molecule: the α and β chains of the T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD8 α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, immunoglobulin heavy chain, immunoglobulin light chain;
most preferably, the signal peptide is derived from CD8 a;
most preferably, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 8;
preferably, the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of: the α, β or ζ chain of a T cell receptor, GITR, ICOS, D45, CD3 ∈ CD3 ζ, CD4, CD5, CD8, CD8 α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, PD1, 2B 4;
more preferably, the transmembrane domain is the transmembrane region of 2B 4;
most preferably, the amino acid sequence of the transmembrane region of 2B4 is shown in SEQ ID NO 9;
preferably, the intracellular signaling domain comprises a signaling domain;
More preferably, the signal transduction domain comprises a functional signal transduction domain selected from the group consisting of: CD3 ζ, FcR γ, FcR β, CDs, CD3 ∈, CD3 γ, CD3 δ, CD5, CD22, CD66d, CD79a, CD79b, NKG 2D;
most preferably, the signaling domain is a functional signaling domain of NKG 2D;
most preferably, the amino acid sequence of the functional signaling domain of NKG2D is shown in SEQ ID NO. 10;
preferably, the intracellular signaling domain further comprises a costimulatory signaling domain;
more preferably, the co-stimulatory signaling domain comprises a functional signaling domain selected from the group consisting of: 4-1BB (CD137), HVEM, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), CARD11, 2B 4;
most preferably, the co-stimulatory signaling domain is a functional signaling domain of 2B 4;
most preferably, the amino acid sequence of the functional signaling domain of 2B4 is shown in SEQ ID NO. 11;
most preferably, the tumor antigen binding peptide is formed by connecting a signal peptide, an antigen recognition region, a hinge region, a transmembrane domain, a costimulatory signal domain and a signal transduction domain in sequence;
Most preferably, the tumor antigen binding peptide comprises the amino acid sequence of any one of:
(1) an amino acid sequence shown as SEQ ID NO. 2;
(2) an amino acid sequence having substitution, deletion or addition of one or several amino acids compared with the amino acid sequence shown as SEQ ID NO. 2;
(3) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO. 2;
most preferably, the amino acid sequence of the tumor antigen binding peptide is shown in SEQ ID NO 2.
In a third aspect of the invention, there is provided a conjugate.
Further, the conjugate comprises a polypeptide according to the first aspect of the invention;
preferably, the conjugate further comprises a modifying moiety linked to the polypeptide;
more preferably, the modifying moiety comprises a detectable label or therapeutic agent;
most preferably, the detectable label comprises an enzyme, an imaging agent, a contrast agent, a fluorescent dye, a luminescent substance, or biotin;
most preferably, the therapeutic agent comprises a drug or cytotoxic agent having anti-tumor activity.
In a fourth aspect, the invention provides an isolated nucleic acid molecule.
Further, the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of the first aspect of the invention, the tumor antigen binding peptide of the second aspect of the invention;
preferably, the nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of:
(1) a nucleic acid molecule encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 1, or an active fragment or analog thereof;
(2) a nucleic acid molecule encoding a tumor antigen-binding peptide according to the second aspect of the invention;
(3) a nucleic acid molecule complementary to (1) or (2);
(4) a nucleic acid molecule having at least 70% identity to (1) or (2) or (3);
more preferably, the nucleic acid molecule is a nucleic acid molecule encoding an amino acid sequence as shown in SEQ ID NO. 1, a nucleic acid molecule encoding a nucleotide sequence as shown in SEQ ID NO. 3;
most preferably, the nucleotide sequence of the nucleic acid molecule encoding the amino acid sequence shown as SEQ ID NO. 1 is shown as SEQ ID NO. 12.
In a fifth aspect of the invention, a recombinant vector is provided.
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 of the fourth aspect of the invention and a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector and a viral vector expression vector;
More preferably, the viral vector comprises a lentiviral vector, an adenoviral vector, a retroviral 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 tumor antigen binding peptide according to the second aspect of the invention, a conjugate 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 escherichia coli, yeast, insect cells, mammalian cells;
preferably, the host cell comprises an immune cell;
more preferably, the immune cells comprise T lymphocytes, NK cells, monocytes, macrophages, dendritic cells and any combination thereof;
most preferably, the immune cells include T lymphocytes, NK cells;
most preferably, the immune cell is an NK cell.
Further, the host Cell described in the embodiments of the present invention is TABP-EIC (Tumor Antigen Binding Peptide-engineered Immune Cell).
In a seventh aspect of the invention, there is provided a population of engineered host cells.
Further, the population of engineered host cells comprises the engineered host cells of the sixth aspect of the invention;
preferably, the population of engineered host cells further comprises host cells that do not comprise a polypeptide of the first aspect of the invention, a tumor antigen binding peptide of the second aspect of the invention, a conjugate of the third aspect of the invention, a nucleic acid molecule of the fourth aspect of the invention, a vector of the fifth aspect of the invention;
more preferably, the host cell comprises an immune cell;
most preferably, the immune cells include T lymphocytes, NK cells, monocytes, macrophages, dendritic cells and any combination thereof.
Further, the host Cell described in the embodiments of the present invention is TABP-EIC (Tumor Antigen Binding Peptide-engineered Immune Cell).
In an eighth aspect of the invention there is provided a composition or kit.
Further, the composition or kit comprises a polypeptide according to the first aspect of the invention, a tumor antigen-binding peptide according to the second aspect of the invention, a conjugate 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 population of engineered host cells according to the seventh aspect of the invention.
Further, the composition comprises a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or an auxiliary material.
Further, the composition comprises a cell injection comprising the tumor antigen-binding peptide of the second aspect of the invention, the nucleic acid molecule of the fourth aspect of the invention, the vector of the fifth aspect of the invention, the engineered host cell of the sixth aspect of the invention, the population of engineered host cells of the seventh aspect of the invention;
preferably, the engineered host cell, population of engineered host cells expresses a tumor antigen binding peptide according to the second aspect of the invention.
A ninth aspect of the present invention provides any one of the following methods:
(1) a screening method for preparing a polypeptide of a tumor antigen-binding peptide instead of a single-chain antibody, the method comprising the steps of: screening the polypeptide by using a phage-polypeptide library by using cancer cells as positive targets and non-cancer cells as negative targets;
preferably, the cancer cells comprise solid tumor cells, non-solid tumor cells;
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 lymphocytic leukemia, chronic lymphocytic leukemia, hematopoietic tumors, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma;
most preferably, the cancer cells are prostate cancer cells;
preferably, the polypeptide is a polypeptide according to the first aspect of the invention;
(2) a method of making an engineered host cell according to the sixth aspect of the invention, the method comprising the steps of: introducing the polypeptide of the first aspect of the invention, the tumor antigen binding peptide of the second aspect of the invention, the conjugate of the third aspect of the invention, the nucleic acid molecule of the fourth aspect of the invention, the vector of the fifth aspect of the invention into a host cell to obtain an engineered host cell of the sixth aspect of the invention;
preferably, the nucleic acid molecule or vector is transfected into a cell by a virus, liposome, electrotransfection or transposon system;
(3) a method of producing a novel polypeptide, said method comprising the steps of: culturing the engineered host cell of the sixth aspect of the invention or the population of engineered host cells of the seventh aspect of the invention and isolating the polypeptide comprising the amino acid sequence set forth in SEQ ID NO 1 from the cell culture.
Further, the method also includes a method for diagnosing whether a subject has a tumor in a patient, the method comprising: detecting whether a sample from said subject contains tumor cells using a polypeptide according to the first aspect of the invention or a kit according to the eighth aspect of the invention.
Further, the method also includes a method for preventing and/or treating a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the engineered host cell of the sixth aspect of the invention, the population of engineered host cells of the seventh aspect of the invention, the composition of the eighth aspect of the invention.
A tenth aspect of the invention provides the use of any one of the following aspects:
(1) the polypeptide of the first aspect of the invention is applied to the preparation of tumor antigen binding peptides, conjugates, nucleic acid molecules and carriers;
(2) the use of a tumor antigen binding peptide according to the second aspect of the invention in the preparation of a conjugate, a nucleic acid molecule, a vector;
(3) the use of a conjugate according to the third aspect of the invention for the preparation of a nucleic acid molecule, a vector;
(4) use of a nucleic acid molecule according to the fourth aspect of the invention in the preparation of a vector;
(5) Use of a polypeptide according to the first aspect of the invention, a tumor antigen-binding peptide according to the second aspect of the invention, a conjugate 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 the immunotherapy of a tumor;
(6) use of a polypeptide according to the first aspect of the invention, a tumor antigen binding peptide according to the second aspect of the invention, a conjugate 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 population of immune cells for use in the immunotherapy of tumors;
(7) use of a polypeptide according to the first aspect of the invention, a tumor antigen-binding peptide according to the second aspect of the invention, a conjugate 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 population of engineered host cells according to the seventh aspect of the invention for the preparation of a medicament for the prevention and/or treatment of a tumor;
(8) Use of a polypeptide according to the first aspect of the invention, a tumor antigen-binding peptide according to the second aspect of the invention, a conjugate 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 population of engineered host cells according to the seventh aspect of the invention in the preparation of a kit for diagnosing whether a subject has a tumor;
(9) the polypeptide replaces single-chain antibody to prepare tumor antigen binding peptide, immune cells modified by the tumor antigen binding peptide and drugs for preventing and/or treating tumors;
(10) use of the engineered host cell according to the sixth aspect of the invention for the prevention and/or treatment of a tumour;
(11) use of a population of engineered host cells according to the seventh aspect of the invention for the prevention and/or treatment of a tumour;
(12) the composition or the kit according to the eighth aspect of the present invention is used for preventing and/or treating tumors, diagnosing whether a subject has a tumor;
preferably, the tumor antigen binding peptide is a tumor antigen binding peptide according to the second aspect of the invention;
Preferably, the conjugate is a conjugate 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 vector is a vector 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 lymphocytic leukemia, chronic lymphocytic leukemia, hematopoietic tumors, 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) the invention adopts a phage-polypeptide library, uses a tumor cell strain and a negative control cell strain to screen out a tumor specific recognition peptide segment, uses the polypeptide to replace a traditional single-chain antibody to prepare the TABP-EIC cell, and compared with the traditional method for preparing the TABP-EIC cell, the method for preparing the TABP-EIC cell has the following outstanding advantages: the method can obviously shorten the time for screening the TABP-EIC extracellular identification region sequence aiming at the tumor with undefined antigen, and the screened polypeptide ligand has higher binding efficiency with the tumor cell;
(2) The invention provides a novel polypeptide, which can be specifically combined with prostate cancer cells, can be applied to detection of prostate cancer patients, can replace the traditional single-chain antibody to prepare TABP-EIC, and can be applied to immunotherapy of prostate cancer.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows a graph of immunofluorescence results obtained with the polypeptide GTL in the prostate cancer cell line Lncap;
FIG. 2 shows a graph of immunofluorescence results obtained with polypeptide GTL in prostate cancer cell line PC 3;
FIG. 3 shows a graph of immunofluorescence results obtained with the polypeptide GTL in human renal epithelial cell line 293T;
FIG. 4 is a graph showing the results of a mouse model of prostate cancer treated with TABP-EIC-GTL for different days, wherein,
a, picture A: lncap cells were injected intraperitoneally 14 days after tumorigenesis, control group (saline), B panel: 14 days after the Incap cells were intraperitoneally injected into tumors, TABP-EIC-GTL group, panel C: lncap cells were injected intraperitoneally 28 days after tumorigenesis, control group (saline), D-graph: 28 days after the Incap cells were intraperitoneally injected into tumors, TABP-EIC-GTL group, FIG. E: lncap cells were injected intraperitoneally for 42 days after tumorigenesis, control group (saline), F panel: 42 days after the Lncap cells are injected into the abdominal cavity to form tumors, and a TABP-EIC-GTL group.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Example 1 screening of specific polypeptide GTL that specifically recognizes prostate cancer cell line
1. Experimental Material
The Ph.D. -12 phage display peptide library kit used in the examples of the present invention was purchased from New England Biolabs (NEB) Inc., catalog No. E8110SC, PC3 cell (CRL-1435), Lncap cell (CRL-1740), 293T cell (CRL-3216) from ATCC Inc.
2. Ph.D. -12 phage display peptide library kit composition
Random dodecapeptide phage display library: 100 μ L, 1.5X 1013pfu/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
The invention adopts a Ph.D. -12 phage display peptide library kit, uses Lncap and PC3 cells as positive targets and 293T cells as negative targets, and screens specific polypeptide GTL capable of specifically recognizing prostate cancer cell strains, and the method specifically comprises the following steps:
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 buffer10The 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 several rounds of panning may be usedDown 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 specific polypeptide GTL which is obtained by screening and specifically recognizes the prostate cancer cell strain is GTLLNPYPFSWGY (SEQ ID NO: 1).
Example 2 immunofluorescence techniques to identify the affinity and specificity of polypeptide GTL for prostate cancer cells
1. Experimental Material
Prostate cancer cell line PC3(CRL-1435), prostate cancer cell line Lncap (CRL-1740), and human renal epithelial cell line 293T (CRL-3216) were purchased from ATCC.
2. Experimental methods
(1) Fixing
Fixing the cells in a newly prepared 4% paraformaldehyde solution in a culture plate for 15 minutes, cleaning the sample by using DPBS, and carefully operating to avoid the cells from falling off the glass slide;
(2) Cleaning of
DPBS washes three times, each time for 5 minutes;
(3) sealing of
Blocking with 2% BSA at 4 deg.C for 30 min, washing with DPBS twice, each for 5 min;
(4) polypeptide incubation
GTL polypeptide (final concentration 1. mu.g/mL) labeled with FITC (fluorescein isothiocyanate) was added and incubated overnight at 4 ℃ in a humidity chamber in the dark;
(5) cleaning of
DPBS washes three times, each time for 5 minutes;
(6) sealing sheet and observation
Adding 0.5 μ g/mL DAPI (prepared by PBS) for dyeing for 10 min, or using other nuclear dyeing dye;
wash three times with PBS, remove excess DAPI, add 20 μ L of mounting medium, mount under microscope, note the decay of fluorescence intensity with light stimulus, and take pictures as soon as possible.
3. Results of the experiment
The experimental result shows that the polypeptide GTL obtained by screening in example 1 can be specifically bound with a certain protein on the cell membrane surface of a prostate cancer cell line Lncap and PC3 (see figures 1 and 2), but not bound with a protein on the cell membrane surface of a non-prostate cancer cell line 293T (see figure 3), i.e., the GTL can specifically identify a certain specific membrane protein of a prostate cancer cell, and further shows that the novel polypeptide GTL has better specific binding capacity with the prostate cancer cell.
EXAMPLE 3 preparation of TABP-EIC (TABP-EIC-GTL) cells Using polypeptide GTL as the extracellular recognition region
1. NK cells
The NK cells used in the experiments described in this patent were all obtained by amplification from Peripheral Blood Mononuclear Cells (PBMC).
2. Construction of tumor antigen-binding peptide expression vector
The structural sequences of the tumor antigen binding peptide are obtained by gene synthesis (general organisms), and the expression vector is pLenti-EF1a-Backbone (NN) (addendum # 27961);
the restriction sites of the tumor antigen binding peptide structure insertion are BsiWI and EcoRI.
3. Lentiviral packaging
TABP-EIC backbone vector and auxiliary vector pMD2.G (addgen #12259), pMDLg/pRRE (addgen #12251) and pRSV-Rev (addgen #12253) were mixed at a ratio of 10:5:3:2, and 293T cells were transfected with 20. mu.g of plasmid per 10mL of transfection system. Supernatants were collected 48 hours and 72 hours after transfection, purified and concentrated to obtain lentiviruses.
4. Lentiviral transduction
Mixing the concentrated lentivirus with NK cells, purifying the lentivirus at 200 μ L per 100 ten thousand cells, and placing the mixture in an incubator at 37 ℃ with 5% CO2Culturing under the condition, and completely replacing the culture solution after 24 hours.
5. Amplification of TABP-EIC-GTL cells
And (3) normally culturing and amplifying TABP-EIC cells obtained after lentivirus infection.
6. Detection of TABP-EIC-GTL cell tumor antigen binding peptide expression efficiency
And (4) taking part of cells to extract genome and carrying out RT-PCR detection on the seventh day after the TABP-EIC cell lentivirus is infected.
7. Results of the experiment
According to the RT-PCR result, selecting TABP-EIC-GTL cells with infection efficiency more than 20% for subsequent experimental verification according to the formula of infection efficiency (%). 63.21-6.36 × Delta CT (detection group CT-control group CT), wherein the amino acid sequence of the prepared tumor antigen binding peptide is shown as SEQ ID NO:2, the nucleotide sequence is shown as SEQ ID NO:3, and the GTL sequence in the structure of the tumor antigen binding peptide can have a plurality of repetitions, and in the embodiment of the invention, the number of the repetitions is 3.
Example 4 validation of the killing Effect of TABP-EIC-GTL cells on in vivo tumors
1. Experimental Material
Mice were purchased from vintongli, inc, as male mice of 8 week old NOD strain; the prostate cancer cell line Lncap (CRL-1740) was purchased from ATCC.
2. Experimental methods
(1) Prostate cancer cell Lncap intraperitoneal injection construction of prostate cancer mouse tumor model
Dividing 6 healthy mice into 2 groups randomly, wherein each group comprises 3 mice, the 2 groups comprise a control group and a TABP-EIC-GTL cell group respectively, injecting 500 million prostate cancer cells (Lncap-GFP) containing Green Fluorescent Protein (GFP) marks into the abdominal cavity of all the mice, and feeding the mice normally for 14 days;
(2) Mice in the control group and TABP-EIC-GTL cell group were treated in the experiment
On day 14 of tumorigenesis, all groups of mice were subjected to in vivo fluorescence imaging, and the growth of tumors in the mice was recorded, and after fluorescence imaging, each group of mice was administered as follows:
control group: injecting 1000 mu L of normal saline into the abdominal cavities of the mice of the control group respectively;
TABP-EIC-GTL group: mice in TABP-EIC-GTL group were separately perfused intraperitoneally with 1000. mu.L, 5X 10. mu.L of TABP-EIC-GTL cell suspension6Individual cells/mL physiological saline/individual;
peritoneal perfusion was performed once a week for 3 weeks, and the mice were subjected to in vivo fluorescence imaging on days 14, 28, and 42, and the tumor status in the mice was observed and the tumor size in the mice was recorded.
3. Results of the experiment
The experimental result shows that compared with a control group mouse which is perfused with normal saline in the abdominal cavity, the average volume of the in vivo tumor of the experimental group mouse which is perfused with TABP-EIC-GTL cell in the abdominal cavity is obviously smaller after 28 th and 42 th days of the tumor formation, and the growth of the in vivo tumor is obviously inhibited, which indicates that the TABP-EIC-GTL cell prepared by the invention has obvious inhibition effect on the tumor and has better treatment effect on the prostatic cancer.
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, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
<110> Shino Rev medicine science and technology (New zone of Zhuhai horizontal organ) Co., Ltd
<120> novel polypeptide specifically recognized for prostate cancer cells, derivatives and applications thereof
<141> 2021-07-13
<160> 12
<170> SIPOSequenceListing 1.0
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Gly Thr Leu Leu Asn Pro Tyr Pro Phe Ser Trp Gly Tyr
1 5 10
<210> 2
<211> 319
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
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 Gly Thr Leu Leu Asn Pro Tyr Pro Phe Ser Trp
20 25 30
Gly Tyr Gly Gly Gly Ser Gly Thr Leu Leu Asn Pro Tyr Pro Phe Ser
35 40 45
Trp Gly Tyr Gly Gly Gly Ser Gly Thr Leu Leu Asn Pro Tyr Pro Phe
50 55 60
Ser Trp Gly Tyr Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
65 70 75 80
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
85 90 95
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
100 105 110
Asp Gln Asp Cys Gln Asn Ala His Gln Glu Phe Arg Phe Trp Pro Phe
115 120 125
Leu Val Ile Ile Val Ile Leu Ser Ala Leu Phe Leu Gly Thr Leu Ala
130 135 140
Cys Phe Cys Val Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr
145 150 155 160
Ser Pro Lys Glu Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys
165 170 175
Thr Arg Arg Asn His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser
180 185 190
Thr Ile Tyr Ser Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln
195 200 205
Glu Pro Ala Tyr Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser
210 215 220
Gly Ser Arg Lys Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr
225 230 235 240
Glu Val Ile Gly Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu
245 250 255
Ser Arg Lys Glu Leu Glu Asn Phe Asp Val Tyr Ser Met Gly Trp Ile
260 265 270
Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn
275 280 285
Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys
290 295 300
Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser
305 310 315
<210> 3
<211> 957
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atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgggcaccc tgctgaaccc ctaccccttc agctggggct acggcggcgg cagcggcacc 120
ctgctgaacc cctacccctt cagctggggc tacggcggcg gcagcggcac cctgctgaac 180
ccctacccct tcagctgggg ctacaccact accccagcac cgaggccacc caccccggct 240
cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt 300
ggggccgtgc atacccgggg tcttgacttc gcctgcgatc aggactgtca gaatgcccat 360
caggaattca gattttggcc gtttttggtg atcatcgtga ttctaagcgc actgttcctt 420
ggcacccttg cctgcttctg tgtgtggagg agaaagagga aggagaagca gtcagagact 480
agtcccaagg aatttttgac aatttacgaa gatgtcaagg atctgaaaac caggagaaat 540
cacgagcagg agcagacttt tcctggaggg gggagcacca tctactctat gatccagtcc 600
cagtcttctg ctcccacgtc acaagaacca gcatatacat tatattcatt aattcagcct 660
tccaggaagt ctggttccag gaagaggaac cacagccctt ccttcaatag cactatctat 720
gaagtgattg gaaagagtca acctaaagcc cagaaccctg ctcgattgag ccgcaaagag 780
ctggagaact ttgatgttta ttccatgggg tggattcgtg gtcggaggtc acgacacagc 840
tgggagatga gtgaatttca taattataac ttggatctga agaagagtga tttttcaaca 900
cgatggcaaa agcaaagatg tccagtagtc aaaagcaaat gtagagaaaa tgcatct 957
<210> 4
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gly Gly Ser
1
<210> 5
<211> 47
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Thr Leu Leu Asn Pro Tyr Pro Phe Ser Trp Gly Tyr Gly Gly Gly
1 5 10 15
Ser Gly Thr Leu Leu Asn Pro Tyr Pro Phe Ser Trp Gly Tyr Gly Gly
20 25 30
Gly Ser Gly Thr Leu Leu Asn Pro Tyr Pro Phe Ser Trp Gly Tyr
35 40 45
<210> 6
<211> 141
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ggcaccctgc tgaaccccta ccccttcagc tggggctacg gcggcggcag cggcaccctg 60
ctgaacccct accccttcag ctggggctac ggcggcggca gcggcaccct gctgaacccc 120
taccccttca gctggggcta c 141
<210> 7
<211> 45
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 8
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
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
20
<210> 9
<211> 35
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
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> 10
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
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> 11
<211> 51
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
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> 12
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ggcaccctgc tgaaccccta ccccttcagc tggggctac 39
Claims (10)
1. A novel polypeptide for specifically recognizing tumor cells, which comprises a polypeptide of an amino acid sequence shown as SEQ ID NO. 1, or an active fragment or an analogue thereof;
Preferably, the amino acid sequence of the active fragment and the amino acid sequence of the analogue have at least 95 percent of identity with the amino acid sequence shown as SEQ ID NO. 1;
preferably, the tumor cells are prostate cancer cells.
2. A tumor antigen binding peptide comprising an extracellular signaling domain comprising an antigen recognition region, a transmembrane domain linked to the extracellular signaling domain, an intracellular signaling domain linked to the transmembrane domain;
preferably, the extracellular signaling domain comprises an antigen recognition region;
more preferably, the antigen recognition region is obtained by linking one or more polypeptides of claim 1 in tandem by a linker;
most preferably, the amino acid sequence of the polypeptide is shown as SEQ ID NO 1;
most preferably, the amino acid sequence of the linker is shown as SEQ ID NO. 4;
most preferably, the antigen recognition region is obtained by connecting three polypeptides of claim 1 in series by a linker;
most preferably, the amino acid sequence of the antigen recognition region is shown as SEQ ID NO. 5;
most preferably, the nucleotide sequence of the antigen recognition region is shown as SEQ ID NO 6;
preferably, the extracellular signaling domain further comprises a hinge region;
More preferably, the hinge region comprises a hinge region of any one or more of the following molecules: CD3 epsilon, CD4, CD5, CD8, CD8 alpha, PD1, CD16, CD22, CD28, CD33, CD37, CD45, CD134, CD137, CD152, CD 154;
most preferably, the hinge region is that of a CD8 a molecule;
most preferably, the amino acid sequence of the hinge region of the CD8 a molecule is set forth in SEQ ID NO. 7;
preferably, the extracellular signaling domain further comprises a signal peptide;
more preferably, the signal peptide is derived from the following molecule: the α and β chains of the T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD8 α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, immunoglobulin heavy chain, immunoglobulin light chain;
most preferably, the signal peptide is derived from CD8 a;
most preferably, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 8;
preferably, the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of: the α, β or ζ chain of a T cell receptor, GITR, ICOS, D45, CD3 ∈ CD3 ζ, CD4, CD5, CD8, CD8 α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, PD1, 2B 4;
More preferably, the transmembrane domain is the transmembrane region of 2B 4;
most preferably, the amino acid sequence of the transmembrane region of 2B4 is shown in SEQ ID NO 9;
preferably, the intracellular signaling domain comprises a signaling domain;
more preferably, the signal transduction domain comprises a functional signal transduction domain selected from the group consisting of: CD3 ζ, FcR γ, FcR β, CDs, CD3 ∈, CD3 γ, CD3 δ, CD5, CD22, CD66d, CD79a, CD79b, NKG 2D;
most preferably, the signaling domain is a functional signaling domain of NKG 2D;
most preferably, the amino acid sequence of the functional signaling domain of NKG2D is shown in SEQ ID NO. 10;
preferably, the intracellular signaling domain further comprises a costimulatory signaling domain;
more preferably, the co-stimulatory signaling domain comprises a functional signaling domain selected from the group consisting of: 4-1BB (CD137), HVEM, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), CARD11, 2B 4;
most preferably, the co-stimulatory signaling domain is a functional signaling domain of 2B 4;
most preferably, the amino acid sequence of the functional signaling domain of 2B4 is shown in SEQ ID NO. 11;
Most preferably, the tumor antigen binding peptide is formed by connecting a signal peptide, an antigen recognition region, a hinge region, a transmembrane domain, a costimulatory signal domain and a signal transduction domain in sequence;
most preferably, the tumor antigen binding peptide comprises the amino acid sequence of any one of:
(1) an amino acid sequence shown as SEQ ID NO. 2;
(2) an amino acid sequence having substitution, deletion or addition of one or several amino acids compared with the amino acid sequence shown as SEQ ID NO. 2;
(3) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO. 2;
most preferably, the amino acid sequence of the tumor antigen binding peptide is shown in SEQ ID NO 2.
3. A conjugate, comprising the polypeptide of claim 1;
preferably, the conjugate further comprises a modifying moiety linked to the polypeptide;
more preferably, the modifying moiety comprises a detectable label or therapeutic agent;
most preferably, the detectable label comprises an enzyme, an imaging agent, a contrast agent, a fluorescent dye, a luminescent substance, or biotin;
Most preferably, the therapeutic agent comprises a drug or cytotoxic agent having anti-tumor activity.
4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 1, the tumor antigen binding peptide of claim 2;
preferably, the nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of:
(1) a nucleic acid molecule encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 1, or an active fragment or analog thereof;
(2) a nucleic acid molecule encoding the tumor antigen binding peptide of claim 2;
(3) a nucleic acid molecule complementary to (1) or (2);
(4) a nucleic acid molecule having at least 70% identity to (1) or (2) or (3);
more preferably, the nucleic acid molecule is a nucleic acid molecule encoding an amino acid sequence as shown in SEQ ID NO. 1, a nucleic acid molecule encoding a nucleotide sequence as shown in SEQ ID NO. 3;
most preferably, the nucleotide sequence of the nucleic acid molecule encoding the amino acid sequence shown as SEQ ID NO. 1 is shown as SEQ ID NO. 12.
5. A recombinant vector comprising the nucleic acid molecule of claim 4;
preferably, the vector is a recombinant vector constructed by the nucleic acid molecule of claim 4 and DNA vector, RNA vector, plasmid, transposon vector, CRISPR/Cas9 vector and virus vector expression vector;
More preferably, the viral vector comprises a lentiviral vector, an adenoviral vector, a retroviral vector.
6. An engineered host cell comprising the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the conjugate of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5;
preferably, the host cell comprises escherichia coli, yeast, insect cells, mammalian cells;
preferably, the host cell comprises an immune cell;
more preferably, the immune cells comprise T lymphocytes, NK cells, monocytes, macrophages, dendritic cells and any combination thereof;
most preferably, the immune cells include T lymphocytes, NK cells;
most preferably, the immune cell is an NK cell.
7. A population of engineered host cells, wherein the population of engineered host cells comprises the engineered host cell of claim 6;
preferably, the population of engineered host cells further comprises host cells that do not comprise the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the conjugate of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5;
More preferably, the host cell comprises an immune cell;
most preferably, the immune cells include T lymphocytes, NK cells, monocytes, macrophages, dendritic cells and any combination thereof.
8. A composition or kit comprising the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the conjugate of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the engineered host cell of claim 6, the population of engineered host cells of claim 7.
9. Any of the following methods, wherein the method comprises:
(1) a screening method for preparing a polypeptide of a tumor antigen-binding peptide instead of a single-chain antibody, comprising the steps of: screening the polypeptide by using a phage-polypeptide library by using cancer cells as positive targets and non-cancer cells as negative targets;
preferably, the tumor cells comprise solid tumor cells, non-solid tumor cells;
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 lymphocytic leukemia, chronic lymphocytic leukemia, hematopoietic tumors, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma;
most preferably, the tumor cells are prostate cancer cells;
preferably, the polypeptide is the polypeptide of claim 1;
(2) a method of making the engineered host cell of claim 6, comprising the steps of: introducing the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the conjugate of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5 into a host cell to obtain an engineered host cell of claim 6;
preferably, the nucleic acid molecule or vector is transfected into a cell by a virus, liposome, electrotransfection or transposon system;
(3) a method for producing a novel polypeptide, comprising the steps of: culturing the engineered host cell of claim 6 or the population of engineered host cells of claim 7, and isolating a polypeptide comprising the amino acid sequence set forth in SEQ ID No. 1 from the cell culture.
10. The use of any one of the following aspects, wherein said use comprises:
(1) use of the polypeptide of claim 1 for the preparation of a tumor antigen binding peptide, conjugate, nucleic acid molecule, vector;
(2) use of the tumor antigen binding peptide of claim 2 for the preparation of conjugates, nucleic acid molecules, vectors;
(3) use of the conjugate of claim 3 for the preparation of nucleic acid molecules, vectors;
(4) use of the nucleic acid molecule of claim 4 for the preparation of a vector;
(5) use of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the conjugate of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5 for the preparation of an immune cell for tumor immunotherapy;
(6) use of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the conjugate of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the engineered host cell of claim 6 for the preparation of a population of immune cells for tumor immunotherapy;
(7) use of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the conjugate of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the engineered host cell of claim 6, the population of engineered host cells of claim 7 in the manufacture of a medicament for the prevention and/or treatment of a tumor;
(8) Use of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the conjugate of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the engineered host cell of claim 6, the population of engineered host cells of claim 7 in the preparation of a kit for diagnosing whether a subject has a tumor;
(9) the polypeptide replaces single-chain antibody to prepare tumor antigen binding peptide, immune cells modified by the tumor antigen binding peptide and drugs for preventing and/or treating tumors;
(10) use of the engineered host cell of claim 6 for the prevention and/or treatment of a tumor;
(11) use of the population of engineered host cells of claim 7 for the prevention and/or treatment of a tumor;
(12) use of the composition or kit of claim 8 for the prevention and/or treatment of a tumor, for the diagnosis of whether a subject has a tumor, respectively;
preferably, the tumor antigen binding peptide is the tumor antigen binding peptide of claim 2;
preferably, the conjugate is the conjugate of claim 3;
preferably, the nucleic acid molecule is the nucleic acid molecule of claim 4;
Preferably, the vector is the vector of claim 5;
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 lymphocytic leukemia, chronic lymphocytic leukemia, hematopoietic tumors, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma;
most preferably, the tumor is prostate cancer.
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| CN202110797558.7A CN113527435B (en) | 2021-07-14 | 2021-07-14 | Novel polypeptide for specifically identifying prostate cancer cells, derivatives and application thereof |
| PCT/CN2022/098826 WO2023284471A1 (en) | 2021-07-14 | 2022-06-15 | Novel polypeptide for specific recognition of prostate cancer cell, and derivative and use thereof |
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| CN202110797558.7A CN113527435B (en) | 2021-07-14 | 2021-07-14 | Novel polypeptide for specifically identifying prostate cancer cells, derivatives and application thereof |
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Also Published As
| Publication number | Publication date |
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| WO2023284471A1 (en) | 2023-01-19 |
| CN113527435B (en) | 2022-06-07 |
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