WO2023284471A1 - 对前列腺癌细胞特异性识别的新型多肽及其衍生物与应用 - Google Patents
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- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
Definitions
- the invention belongs to the technical field of biomedicine, and specifically relates to a novel polypeptide specifically recognizing prostate cancer cells, its derivatives and applications.
- the current treatment methods for tumors mainly include surgery, radiotherapy, chemotherapy and targeted drug therapy.
- These traditional tumor treatment methods Focus on the local tumor lesion and kill tumor cells at the physical and chemical levels.
- conventional surgical treatment mainly adopts surgical resection, but surgical resection is difficult to remove tumor cells, so patients are prone to recurrence after surgery; radiotherapy and While chemotherapy kills tumor cells, it also kills normal human cells, causing great harm to patients; although targeted drug therapy can reduce the adverse reactions of drugs to a certain extent, it is prone to tumor drug resistance, and thus lead to tumor recurrence.
- tumor immunotherapy has emerged as the times require, and has become another effective treatment method after surgery, radiotherapy, chemotherapy, and targeted drug therapy. It is different from traditional tumor treatment methods. Tumor immunotherapy does not directly act on the lesion, but achieves the purpose of controlling and killing tumors by enhancing the human immune system. Compared with traditional tumor treatment methods, tumor immunotherapy has less side effects, strong specificity, and a tumor-killing spectrum. Wide, low recurrence rate and other advantages.
- Tumor immunotherapy is divided into two categories: one is immune checkpoint inhibitors, and the other is cellular immunotherapy.
- chimeric antigen receptor modification T cells Chimeric antigen receptor modification T cells, CAR-T
- chimeric antigen receptor modification NK cells Chimeric antigen receptor modification NK cells, CAR-NK
- CAR-T Chimeric antigen receptor modification T cells
- CAR-NK Chimeric antigen receptor modification NK cells
- CAR is the core structure of CAR-T and CAR-NK, endowing cells with the ability to specifically recognize tumor-associated antigens on the surface of tumor cells.
- CAR is mainly composed of three functional domains, namely extracellular domain, transmembrane domain and The intracellular domain and the extracellular domain are composed of a single-chain variable fragment (Single-chain variable fragment, scFv) of a monoclonal antibody responsible for recognizing and binding to an antigen and a connecting hinge region (Hinge); the intracellular structure
- the domain consists of costimulatory domain (Costimulatory domain) and signal transduction domain (Signaling domain).
- TAA tumor associated antigen
- TAA tumor associated antigen
- the object of the present invention is to provide a phage-polypeptide library for screening TABP-EIC (Tumor Antigen Binding Peptide-Engineering Immune Cell, Tumor Antigen Binding Peptide-Engineering Immune Cell) extracellular recognition region
- TABP-EIC Tumor Antigen Binding Peptide-Engineering Immune Cell
- the sequence method, the polypeptide ligand obtained by this method can replace the traditional single-chain antibody for the preparation of TABP-EIC cells.
- the preparation method of the present invention can target different antigens. For clear tumors, the time for screening extracellular recognition regions is significantly shortened, and the binding efficiency of the screened polypeptide ligands to tumor cells is higher. So far, there is no relevant report on the application of the novel polypeptide described in the present invention in immunotherapy.
- the object of the present invention is to provide a novel polypeptide specifically recognizing prostate cancer cells and its derivatives and applications.
- the polypeptide is obtained by screening a phage-polypeptide library and can specifically recognize Combining with tumor cells and with high binding efficiency, the preparation method of the present invention can significantly shorten the time for screening extracellular recognition regions for tumors with unclear antigens, and the binding efficiency of the screened polypeptide ligands to tumor cells is higher.
- the first aspect of the present invention provides a novel polypeptide that specifically recognizes tumor cells.
- polypeptide includes a polypeptide of the amino acid sequence shown in SEQ ID NO: 1, or an active fragment or analog thereof;
- amino acid sequence of the active fragment and analog has at least 95% identity with the amino acid sequence shown in SEQ ID NO:1;
- the tumor cells are prostate cancer cells
- amino acid sequence of the polypeptide is shown in SEQ ID NO:1;
- nucleotide sequence of the polypeptide is shown in SEQ ID NO:12.
- a second aspect of the present invention provides a tumor antigen binding peptide.
- the tumor antigen binding peptide includes an extracellular signaling domain containing an antigen recognition region, a transmembrane domain connected to the extracellular signaling domain, and an intracellular signaling domain connected to the transmembrane domain;
- the extracellular signaling domain includes an antigen recognition region
- the antigen recognition region is obtained by concatenating one or more polypeptides described in the first aspect of the present invention through a linker;
- amino acid sequence of the polypeptide is shown in SEQ ID NO:1;
- amino acid sequence of the linker is as shown in SEQ ID NO:4;
- the antigen recognition region is obtained by connecting three polypeptides according to the first aspect of the present invention in series through a linker;
- amino acid sequence of the antigen recognition region is shown in SEQ ID NO:5;
- nucleotide sequence of the antigen recognition region is shown in SEQ ID NO:6;
- the extracellular signaling domain also includes a hinge region
- the hinge region includes the hinge region of any one or more of the following molecules: CD3 ⁇ , CD4, CD5, CD8, CD8 ⁇ , PD1, CD16, CD22, CD28, CD33, CD37, CD45, CD134, CD137, CD152 , CD154;
- the hinge region is a hinge region of a CD8 ⁇ molecule
- amino acid sequence of the hinge region of the CD8 ⁇ molecule is as shown in SEQ ID NO:7;
- the extracellular signaling domain also includes a signal peptide
- the signal peptide is derived from the following molecules: ⁇ chain and ⁇ chain of T cell receptor, CD3 ⁇ , CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD8 ⁇ , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, immunoglobulin heavy chain, immunoglobulin light chain;
- the signal peptide is derived from CD8 ⁇ ;
- amino acid sequence of the signal peptide is shown in SEQ ID NO: 8;
- the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of the ⁇ , ⁇ or ⁇ chain of a T cell receptor, GITR, ICOS, D45, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD8 ⁇ , CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, PD1, 2B4;
- the transmembrane domain is the transmembrane region of 2B4;
- amino acid sequence of the transmembrane region of said 2B4 is shown in SEQ ID NO:9;
- said intracellular signaling domain comprises a signal transduction domain
- the signal transduction domain comprises a functional signal transduction domain selected from the following proteins: CD3 ⁇ , FcR ⁇ , FcR ⁇ , CDS, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD66d, CD79a, CD79b, NKG2D ;
- the signal transduction domain is a functional signal transduction domain of NKG2D;
- amino acid sequence of the functional signal transduction domain of the NKG2D is shown in SEQ ID NO: 10;
- the intracellular signaling domain also includes a co-stimulatory signaling domain
- the co-stimulatory signal domain comprises a functional signal transduction domain selected from the following proteins: 4-1BB (CD137), HVEM, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA- 1 (CD11a/CD18), ICOS (CD278), CARD11, 2B4;
- the co-stimulatory signal domain is a functional signal transduction domain of 2B4;
- amino acid sequence of the functional signal transduction domain of the 2B4 is shown in SEQ ID NO: 11;
- the tumor antigen-binding peptide is sequentially connected by a signal peptide, an antigen recognition region, a hinge region, a transmembrane domain, a co-stimulatory signal domain, and a signal transduction domain;
- the tumor antigen-binding peptide includes the amino acid sequence described in any of the following:
- the amino acid sequence of the tumor antigen-binding peptide is shown in SEQ ID NO:2.
- a third aspect of the invention provides a conjugate.
- conjugate includes the polypeptide described in the first aspect of the present invention.
- said conjugate further comprises a modification moiety linked to said polypeptide
- said modifying moiety comprises a detectable label or therapeutic agent
- the detectable label comprises an enzyme, an imaging agent, a contrast agent, a fluorescent dye, a luminescent substance or biotin;
- the therapeutic agent comprises a drug or cytotoxic agent with antineoplastic activity.
- a fourth aspect of the invention provides an isolated nucleic acid molecule.
- nucleic acid molecule includes a nucleotide sequence encoding the polypeptide described in the first aspect of the present invention, and a nucleotide sequence of the tumor antigen-binding peptide described in the second aspect of the present invention;
- said nucleic acid molecule comprises a nucleic acid molecule selected from the following group:
- nucleic acid molecule encoding a polypeptide of the amino acid sequence shown in SEQ ID NO: 1, or an active fragment or analog thereof;
- 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 as a nucleotide sequence as shown in SEQ ID NO:3;
- nucleotide sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:12.
- the fifth aspect of the present invention provides a recombinant vector.
- the vector comprises the nucleic acid molecule described in the fourth aspect of the present invention.
- the vector is a recombinant vector constructed from the nucleic acid molecule described in the fourth aspect of the present invention and a DNA vector, RNA vector, plasmid, transposon vector, CRISPR/Cas9 vector, or viral vector expression vector;
- the viral vectors include lentiviral vectors, adenoviral vectors, and retroviral vectors.
- a sixth aspect of the invention provides an engineered host cell.
- the modified host cell comprises the polypeptide described in the first aspect of the present invention, the tumor antigen-binding peptide described in the second aspect of the present invention, the conjugate described in the third aspect of the present invention, and the conjugate described in the fourth aspect of the present invention.
- the host cells include Escherichia coli, yeast, insect cells, mammalian cells;
- said host cells comprise immune cells
- the immune cells include T lymphocytes, NK cells, monocytes, macrophages, dendritic cells and any combination thereof;
- the immune cells include T lymphocytes and NK cells;
- the immune cells are NK cells.
- the host cell described in the specific embodiments of the present invention is TABP-EIC (Tumor Antigen Binding Peptide-Engineering Immune Cell, Tumor Antigen Binding Peptide-Engineering Immune Cell).
- a seventh aspect of the invention provides an engineered population of host cells.
- modified host cell population comprises the modified host cells described in the sixth aspect of the present invention.
- the engineered host cell population also does not contain the polypeptide described in the first aspect of the present invention, the tumor antigen-binding peptide described in the second aspect of the present invention, the conjugate described in the third aspect of the present invention, The nucleic acid molecule described in the fourth aspect of the present invention, the host cell of the vector described in the fifth aspect of the present invention;
- said host cells comprise immune cells
- the immune cells include T lymphocytes, NK cells, monocytes, macrophages, dendritic cells and any combination thereof.
- the host cell described in the specific embodiments of the present invention is TABP-EIC (Tumor Antigen Binding Peptide-Engineering Immune Cell, Tumor Antigen Binding Peptide-Engineering Immune Cell).
- the eighth aspect of the present invention provides a composition or kit.
- composition or kit includes the polypeptide described in the first aspect of the present invention, the tumor antigen-binding peptide described in the second aspect of the present invention, the conjugate described in the third aspect of the present invention, and the conjugate described in the fourth aspect of the present invention.
- composition includes a pharmaceutical composition
- pharmaceutical composition further includes a pharmaceutically acceptable carrier and/or adjuvant.
- composition includes a cell injection containing the tumor antigen-binding peptide described in the second aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, and the nucleic acid molecule described in the fifth aspect of the present invention.
- the engineered host cells or population of engineered host cells express the tumor antigen-binding peptide according to the second aspect of the present invention.
- the ninth aspect of the present invention provides any one of the following methods:
- a screening method for polypeptides that replace single-chain antibodies for preparing tumor antigen-binding peptides comprising the following steps: taking cancer cells as positive targets and non-cancerous cells as negative targets, and using a phage-polypeptide library to screen out said polypeptide;
- the cancer cells include solid tumor tumor cells and non-solid tumor tumor cells;
- the solid tumors include prostate cancer, bladder cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, lung cancer, lung adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, colon cancer, rectal cancer, vascular Endothelioma, testicular cancer, skin cancer;
- the non-solid tumors include acute lymphoblastic leukemia, chronic lymphocytic leukemia, hematopoietic tumors, multiple myeloma, non-Hodgkin's lymphoma, acute myeloid leukemia, B-cell lymphoma, T-cell lymphoma tumor;
- the cancer cells are prostate cancer cells
- polypeptide is the polypeptide described in the first aspect of the present invention.
- a method for preparing the modified host cell described in the sixth aspect of the present invention comprising the following steps: the polypeptide described in the first aspect of the present invention, the tumor antigen described in the second aspect of the present invention.
- the binding peptide, the conjugate described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, and the vector described in the fifth aspect of the present invention are introduced into host cells to obtain the nucleic acid molecule described in the sixth aspect of the present invention.
- Modified host cells comprising the following steps: the polypeptide described in the first aspect of the present invention, the tumor antigen described in the second aspect of the present invention.
- the binding peptide, the conjugate described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, and the vector described in the fifth aspect of the present invention are introduced into host cells to obtain the nucleic acid molecule described in the sixth aspect of the present invention.
- Modified host cells comprising the following steps: the poly
- the nucleic acid molecule or vector is transfected into cells by virus, liposome, electroporation or transposon system;
- a method for producing a novel polypeptide comprising the steps of: cultivating the modified host cell population described in the sixth aspect of the present invention or the modified host cell population described in the seventh aspect of the present invention, from the cells A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 was isolated from the culture.
- the method also includes a method for diagnosing whether a subject has a tumor, the method comprising: using the polypeptide described in the first aspect of the present invention or the kit described in the eighth aspect of the present invention to detect Whether the subject sample contains tumor cells.
- the method also includes a method for preventing and/or treating tumors in a subject, the method comprising administering to a subject in need an effective amount of the modified The host cell of the present invention, the modified host cell population described in the seventh aspect of the present invention, and the composition described in the eighth aspect of the present invention.
- the tenth aspect of the present invention provides the application of any of the following aspects:
- the polypeptide described in the first aspect of the present invention, the tumor antigen-binding peptide described in the second aspect of the present invention, the conjugate described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the present invention The carrier according to the fifth aspect of the invention, the modified host cell according to the sixth aspect of the present invention, and the modified host cell population according to the seventh aspect of the present invention are used in the preparation of drugs for preventing and/or treating tumors Applications;
- the polypeptide described in the first aspect of the present invention, the tumor antigen-binding peptide described in the second aspect of the present invention, the conjugate described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the present invention The vectors described in the fifth aspect of the invention, the modified host cells described in the sixth aspect of the present invention, and the modified host cell populations described in the seventh aspect of the present invention are used in the preparation of diagnosing whether a subject has a tumor. Application in the kit;
- composition or kit described in the eighth aspect of the present invention in preventing and/or treating tumors, and in diagnosing whether a subject has a tumor;
- the tumor antigen-binding peptide is the tumor antigen-binding peptide described in the second aspect of the present invention.
- the conjugate is the conjugate described in the third aspect of the present invention.
- the nucleic acid molecule is the nucleic acid molecule described in the fourth aspect of the present invention.
- the carrier is the carrier described in the fifth aspect of the present invention.
- the tumors include solid tumors and non-solid tumors;
- the solid tumors include prostate cancer, bladder cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, lung cancer, lung adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, colon cancer, rectal cancer, vascular Endothelioma, testicular cancer, skin cancer;
- the non-solid tumors include acute lymphoblastic leukemia, chronic lymphocytic leukemia, hematopoietic tumors, multiple myeloma, non-Hodgkin's lymphoma, acute myeloid leukemia, B-cell lymphoma, T-cell lymphoma tumor;
- the tumor is prostate cancer.
- the present invention uses a phage-polypeptide library to screen out tumor-specific recognition peptides with tumor cell lines and negative control cell lines, and uses the polypeptides instead of traditional single-chain antibodies to prepare TABP-EIC cells.
- the method for preparing TABP-EIC cells according to the present invention has the following outstanding advantages: it can significantly shorten the time for screening the extracellular recognition region sequence of TABP-EIC cells for tumors with unclear antigens. Time, the binding efficiency of the screened polypeptide ligands to tumor cells is higher;
- the present invention provides a novel polypeptide that can specifically bind prostate cancer cells, can be applied to the detection of prostate cancer patients, and can replace traditional single-chain antibodies for the preparation of TABP-EIC, and Applying the TABP-EIC to the immunotherapy of prostate cancer, the present invention provides a new research idea for the immunotherapy of tumors, and solves the current problem in the field that can only target tumor types with clear tumor-associated antigens, screening extracellular
- the application prospect is good due to problems such as long time for identifying sequence and low binding efficiency.
- Figure 1 shows the results of immunofluorescence of polypeptide GTL in the prostate cancer cell line Lncap
- Figure 2 shows the results of immunofluorescence of polypeptide GTL in the prostate cancer cell line PC3;
- Figure 3 shows the results of immunofluorescence of polypeptide GTL in human kidney epithelial cell line 293T;
- Figure 4 shows the results of treating the prostate cancer mouse model for different days with TABP-EIC-GTL, wherein,
- Figure A 14 days after intraperitoneal injection of Lncap cells into tumor, control group (normal saline),
- Figure B 14 days after intraperitoneal injection of Lncap cells into tumor, TABP-EIC-GTL group
- Figure C after intraperitoneal injection of Lncap cells into tumor 28 days, control group (normal saline)
- picture D TABP-EIC-GTL group 28 days after intraperitoneal injection of Lncap cells into tumors
- picture E 42 days after intraperitoneal injection of Lncap cells into tumors
- Panel F TABP-EIC-GTL group 42 days after intraperitoneal injection of Lncap cells into tumors.
- the Ph.D.-12 phage display peptide library kit used in the examples of the present invention was purchased from New England Biolabs (NEB), catalog number is E8110SC, PC3 cells (CRL-1435), Lncap cells (CRL-1740 ), 293T cells (CRL-3216) were purchased from ATCC.
- Random dodecapeptide phage display library 100 ⁇ L, 1.5 ⁇ 10 13 pfu/mL, stored in TBS solution containing 50% glycerol, complexity ⁇ 2.7 ⁇ 10 9 transformants; -28gIII sequencing primer: 5'-HOGTATGGGATTTTGCTAAACAAC- 3', 100pmol, 1pmol/ ⁇ L; -96gIII sequencing primer: 5'-HOCCCTCATAGTTAGCGTAACG-3', 100pmol/ ⁇ L, 1pmol/ ⁇ L; E.coliER2738 host strain F'lacIq ⁇ (lacZ)M15proA+B+zzf::Tn10( TetR)/fhuA2 supE thi ⁇ (lac-proAB) ⁇ (hsdMS-mcrB)5(rk–mk–McrBC—): This strain is provided in the form of bacterial culture containing 50% glycerol, non-competent cells, stored in - 70°C; Streptavidin, ly
- the present invention adopts Ph.D.-12 phage display peptide library kit, takes Lncap and PC3 cells as positive targets, and 293T cells as negative targets, and screens specific polypeptide GTL that can specifically recognize prostate cancer cell lines.
- the method is specific as follows:
- panning experiments are performed in a single sterilized polystyrene petri dish, 12 or 24-well plate, or 96-well microplate, and each target molecule is at least To coat one plate (or one well), the amount given in the following method is the amount for a 60 ⁇ 15mm petri dish, and the amount for a microplate is in parentheses, and the amount for other medium-sized wells should be adjusted accordingly, but each In all cases, the number of phages added is the same: 1.5 ⁇ 10 11 virions;
- ER2738 monoclonal (the plate laid during the phage titer determination) in 10mL LB liquid medium. If the eluted phage is amplified on the same day, ER2738 can also be inoculated in 20mL LB liquid medium, and use 250mL triangular bottle, cultured with vigorous shaking at 37°C;
- Prostate cancer cell line PC3 (CRL-1435), prostate cancer cell line Lncap (CRL-1740), and human kidney epithelial cell line 293T (CRL-3216) were purchased from ATCC.
- FITC fluorescein isothiocyanate
- the experimental results show that the polypeptide GTL screened in Example 1 can specifically bind to a certain protein on the cell membrane surface of the prostate cancer cell line Lncap and PC3 (see Figure 1 and Figure 2), but not to the non-prostate cancer cell line 293T cell membrane Protein binding on the surface (see Figure 3), that is, GTL can specifically recognize a certain membrane protein unique to prostate cancer cells, which further shows that the novel polypeptide GTL of the present invention has better specific binding ability to prostate cancer cells. ability.
- NK cells used in the experiments involved in this patent are obtained from the expansion of peripheral blood mononuclear cells (PBMC).
- PBMC peripheral blood mononuclear cells
- tumor antigen-binding peptides are all obtained from gene synthesis (General Biology), and the expression vector is pLenti-EF1a-Backbone (NN) (addgene#27961);
- BsiWI and EcoRI are the enzyme cutting sites for tumor antigen binding peptide structure insertion.
- TABP-EIC backbone vector and helper vector pMD2.G (addgen #12259), pMDLg/pRRE (addgene #12251), pRSV-Rev (addgene #12253) at a ratio of 10:5:3:2, 20 ⁇ g plasmid per 10mL Transfection system, transfect 293T cells. The supernatant was collected 48 hours and 72 hours after transfection, and the lentivirus was obtained after purification and concentration.
- the concentrated lentivirus was mixed with NK cells at 200 ⁇ L of purified lentivirus per 1 million cells, and then cultured in a 37°C incubator with 5% CO 2 , and the medium was completely changed after 24 hours.
- the TABP-EIC cells obtained after lentivirus infection were cultured and expanded normally.
- TABP-EIC cells On the seventh day after TABP-EIC cells were infected with lentivirus, some cells were taken to extract the genome, and RT-PCR was performed.
- select TABP-EIC-GTL cells with infection efficiency exceeding 20% for subsequent experimental verification and prepare
- the amino acid sequence of the obtained tumor antigen-binding peptide is shown in SEQ ID NO: 2
- the nucleotide sequence is shown in SEQ ID NO: 3.
- the GTL sequence in the tumor antigen-binding peptide structure can have multiple repetitions. In the implementation of the present invention In the example there are 3 replicates.
- mice were purchased from Victoria Lihua Company, and they were 8-week-old NOD strain male mice; the prostate cancer cell line Lncap (CRL-1740) was purchased from ATCC Company.
- Prostate cancer mouse tumor model was established by intraperitoneal injection of prostate cancer cells Lncap
- mice A total of 6 healthy mice were randomly divided into 2 groups, 3 in each group, the 2 groups were respectively the control group and the TABP-EIC-GTL cell group, and 5 million cells containing green fluorescent protein (GFP) were injected intraperitoneally in all mice.
- GFP green fluorescent protein
- Lncap-GFP labeled prostate cancer cells
- mice in the control group and the TABP-EIC-GTL cell group were subjected to experimental treatment respectively
- mice in all groups were given the following administrations:
- Control group intraperitoneally perfuse 1000 ⁇ L of normal saline to mice in the control group;
- TABP-EIC-GTL group intraperitoneally perfuse 1000 ⁇ L of TABP-EIC-GTL cell suspension, 5 ⁇ 10 6 cells/mL saline per mouse in TABP-EIC-GTL group;
- mice were subjected to in vivo fluorescence imaging to observe the tumors in the mice and record the tumor size in the mice.
- the experimental results showed that, compared with the control group mice infused with saline, the average volume of tumors in the mice in the experimental group infused with TABP-EIC-GTL cells was significantly smaller on the 28th day and 42nd day after tumor formation, The growth of the tumor in the body is obviously inhibited, which shows that the TABP-EIC-GTL cells prepared by the invention have a significant inhibitory effect on the tumor, and have a good therapeutic effect on prostate cancer.
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Abstract
涉及对前列腺癌细胞特异性识别的新型多肽及其衍生物与应用，所述多肽经噬菌体-多肽文库筛选得到，能够特异性地结合肿瘤细胞，且结合效率高，可替代传统的单链抗体作为胞外识别区用于制备嵌合抗原受体修饰的免疫细胞中，所述制备方法能够针对抗原不明确的肿瘤，显著缩短筛选胞外识别区的时间，且筛选出的多肽配体与肿瘤细胞的结合效率更高，具有很好的应用前景。
Description
本发明属于生物医学技术领域，具体而言，涉及一种对前列腺癌细胞特异性识别的新型多肽及其衍生物与应用。
目前，肿瘤已成为世界上继心血管疾病的第二大死因，肿瘤患者人数逐年增加，当前针对肿瘤的治疗方法主要包括手术治疗、放疗、化疗和靶向药物治疗等，这些传统的肿瘤治疗方法聚焦于肿瘤病灶局部，在物理和化学层面上杀灭肿瘤细胞，其中，常规的手术治疗主要采用手术切除的方式，但是手术切除难将肿瘤细胞切除干净，因此患者术后容易出现复发；放疗和化疗在杀死肿瘤细胞的同时也会杀死正常的人体细胞，对患者造成较大的伤害；靶向药物治疗虽然在一定程度上能够减轻药物的不良反应，但是易产生肿瘤耐药性，进而导致肿瘤复发。随着分子生物学和肿瘤生物学的不断发展，肿瘤免疫疗法应运而生，成为继手术治疗、放疗、化疗、靶向药物治疗后的另一种有效治疗方法，不同于传统的肿瘤治疗方法，肿瘤免疫疗法并非直接作用于病灶，而是通过增强人体免疫系统达到控制和杀灭肿瘤的目的，相较于传统的肿瘤治疗方法而言，肿瘤免疫疗法具有副作用小、特异性强、杀瘤谱广、低复发率等优点。
肿瘤免疫疗法分为两类：一类是免疫检查点抑制剂，另一类是细胞免疫疗法，在细胞免疫疗法中，嵌合抗原受体修饰T细胞(Chimeric antigen receptor modification T cells，CAR-T)和嵌合抗原受体修饰NK细胞(Chimeric antigen receptor modification NK cells，CAR-NK)免疫疗法是目前研究进展最迅速、应用前景较好的两种方法，其原理在于经嵌合抗原受体(Chimeric antigen receptor，CAR)修饰的T细胞或NK细胞，可以特异性地识别肿瘤细胞表面的肿瘤相关抗原，使效应T细胞或NK细胞的靶向性、杀伤活性和持久性均较常规应用的免疫细胞高，并可克服肿瘤局部免疫抑制微环境并打破宿主免疫耐受状态。CAR是CAR-T和CAR-NK的核心结构，赋予细胞特异性识别中肿瘤细胞表面肿瘤相关抗原的能力，CAR主要由三个功能结构域构成，分别是胞外结构域、跨膜结构域和胞内结构域，胞外结构域由负责识别并结合抗原的单克隆抗体的单链可变片段(Single-chain variable fragment，scFv)及一段起连接作用的铰链区(Hinge)构成；胞内结构域由共刺激信号结构域(Costimulatory domain)和信号转导结构域(Signaling domain)构成。
当前的CAR细胞疗法依赖于特异性的肿瘤相关抗原(Tumor associated antigen，TAA)明确的肿瘤类型，若所述肿瘤的肿瘤相关抗原不明确或未经鉴定，则无法基于肿瘤相关抗原制备相关特异性识别的单链抗体序列，进而也就无法构建能够有效地识别肿瘤细胞的特异性CAR，以及特异性识别并杀伤肿瘤细胞的CAR-T或CAR-NK细胞，此外，单链抗体来源于常规的单抗制备并测序获得，这种常规方法得到的抗体的制备周期长、抗体单链化后特异性结合效率普遍较低、无法针对于抗原不明确肿瘤。为了解决上述本领域存在的技术问题，本发明的目的在于提供一种噬菌体-多肽文库筛选TABP-EIC(Tumor Antigen Binding Peptide-Engineering Immune Cell，肿瘤抗原结合肽-工程化免疫细胞)胞外识别区序列 的方法，该方法得到的多肽配体可替代传统的单链抗体用于制备TABP-EIC细胞，相对于传统的制备TABP-EIC细胞的方法而言，本发明所述制备方法能够针对抗原不明确的肿瘤，显著缩短筛选胞外识别区的时间，且筛选出的多肽配体与肿瘤细胞的结合效率更高。迄今为止，未见将本发明所述的新型多肽应用于免疫治疗中的相关报道。
发明内容
为了解决本领域存在的上述问题，本发明的目的在于提供了一种对前列腺癌细胞特异性识别的新型多肽及其衍生物与应用，所述多肽经噬菌体-多肽文库筛选得到，能够特异性地结合肿瘤细胞，且结合效率高，本发明所述制备方法能够针对抗原不明确的肿瘤，显著缩短筛选胞外识别区的时间，且筛选出的多肽配体与肿瘤细胞的结合效率更高。
本发明的上述目的通过以下技术方案得以实现：
本发明的第一方面提供了一种特异性识别肿瘤细胞的新型多肽。
进一步，所述多肽包括如SEQ ID NO:1所示的氨基酸序列的多肽、或其活性片段、类似物；
优选地，所述活性片段、类似物的氨基酸序列具有与如SEQ ID NO:1所示的氨基酸序列至少95％的相同性；
优选地，所述肿瘤细胞为前列腺癌细胞；
更优选地，所述多肽的氨基酸序列如SEQ ID NO:1所示；
最优选地，所述多肽的核苷酸序列如SEQ ID NO:12所示。
本发明的第二方面提供了一种肿瘤抗原结合肽。
进一步，所述肿瘤抗原结合肽包括含有抗原识别区的胞外信号结构域、连接至所述胞外信号结构域的跨膜结构域、连接至所述跨膜结构域的胞内信号结构域；
优选地，所述胞外信号结构域包括抗原识别区；
更优选地，所述抗原识别区由一个或多个本发明第一方面所述的多肽通过linker串联得到；
最优选地，所述多肽的氨基酸序列如SEQ ID NO:1所示；
最优选地，所述linker的氨基酸序列如SEQ ID NO:4所示；
最优选地，所述抗原识别区由三个本发明第一方面所述的多肽通过linker串联得到；
最优选地，所述抗原识别区的氨基酸序列如SEQ ID NO:5所示；
最优选地，所述抗原识别区的核苷酸序列如SEQ ID NO:6所示；
优选地，所述胞外信号结构域还包括铰链区；
更优选地，所述铰链区包括以下任一种或多种分子的铰链区：CD3ε、CD4、CD5、CD8、CD8α、PD1、CD16、CD22、CD28、CD33、CD37、CD45、CD134、CD137、CD152、CD154；
最优选地，所述铰链区为CD8α分子的铰链区；
最优选地，所述CD8α分子的铰链区的氨基酸序列如SEQ ID NO:7所示；
优选地，所述胞外信号结构域还包括信号肽；
更优选地，所述信号肽来源于以下分子：T细胞受体的α链及β链、CD3ζ、CD28、CD3ε、CD45、CD4、CD5、CD8、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、 ICOS、CD154、GITR、免疫球蛋白重链、免疫球蛋白轻链；
最优选地，所述信号肽来源于CD8α；
最优选地，所述信号肽的氨基酸序列如SEQ ID NO:8所示；
优选地，所述跨膜结构域包括选自下列蛋白质的跨膜区：T细胞受体的α、β或ζ链、GITR、ICOS、D45、CD3ε、CD3ζ、CD4、CD5、CD8、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD123、CD134、CD137、CD154、PD1、2B4；
更优选地，所述跨膜结构域为2B4的跨膜区；
最优选地，所述2B4的跨膜区的氨基酸序列如SEQ ID NO:9所示；
优选地，所述胞内信号结构域包括信号转导结构域；
更优选地，所述信号转导结构域包括选自下列蛋白质的功能性信号转导结构域：CD3ζ、FcRγ、FcRβ、CDS、CD3ε、CD3γ、CD3δ、CD5、CD22、CD66d、CD79a、CD79b、NKG2D；
最优选地，所述信号转导结构域为NKG2D的功能性信号转导结构域；
最优选地，所述NKG2D的功能性信号转导结构域的氨基酸序列如SEQ ID NO:10所示；
优选地，所述胞内信号结构域还包括共刺激信号结构域；
更优选地，所述共刺激信号结构域包括选自下列蛋白质的功能性信号转导结构域：4-1BB(CD137)、HVEM、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、CARD11、2B4；
最优选地，所述共刺激信号结构域为2B4的功能性信号转导结构域；
最优选地，所述2B4的功能性信号转导结构域的氨基酸序列如SEQ ID NO:11所示；
最优选地，所述肿瘤抗原结合肽由信号肽、抗原识别区、铰链区、跨膜结构域、共刺激信号结构域、信号转导结构域依次连接而成；
最优选地，所述肿瘤抗原结合肽包括以下任一项所述的氨基酸序列：
(1)如SEQ ID NO:2所示的氨基酸序列；
(2)与如SEQ ID NO:2所示的氨基酸序列相比，具有一个或几个氨基酸的置换、缺失或添加的氨基酸序列；
(3)与如SEQ ID NO:2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的相同性的氨基酸序列；
最优选地，所述肿瘤抗原结合肽的氨基酸序列如SEQ ID NO:2所示。
本发明的第三方面提供了一种缀合物。
进一步，所述缀合物包括本发明第一方面所述的多肽；
优选地，所述缀合物还包括连接所述多肽的修饰部分；
更优选地，所述修饰部分包括可检测的标记或治疗剂；
最优选地，所述可检测的标记包括酶、成像剂、造影剂、荧光染料、发光物质或生物素；
最优选地，所述治疗剂包括具有抗肿瘤活性的药物或细胞毒剂。
本发明的第四方面提供了一种分离的核酸分子。
进一步，所述核酸分子包括编码本发明第一方面所述的多肽的核苷酸序列、本发 明第二方面所述的肿瘤抗原结合肽的核苷酸序列；
优选地，所述核酸分子包括选自下列组中的核酸分子：
(1)编码如SEQ ID NO:1所示的氨基酸序列的多肽、或其活性片段、类似物的核酸分子；
(2)编码本发明第二方面所述的肿瘤抗原结合肽的核酸分子；
(3)与(1)或(2)互补的核酸分子；
(4)与(1)或(2)或(3)有至少70％相同性的核酸分子；
更优选地，所述核酸分子为编码如SEQ ID NO:1所示氨基酸序列的核酸分子、如SEQ ID NO:3所示核苷酸序列的核酸分子；
最优选地，编码如SEQ ID NO:1所示氨基酸序列的核酸分子的核苷酸序列如SEQ ID NO:12所示。
本发明的第五方面提供了一种重组载体。
进一步，所述载体包含本发明第四方面所述的核酸分子；
优选地，所述载体为本发明第四方面所述的核酸分子与DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体、病毒载体表达载体构建而成的重组载体；
更优选地，所述病毒载体包括慢病毒载体、腺病毒载体、逆转录病毒载体。
本发明的第六方面提供了一种经改造的宿主细胞。
进一步，所述经改造的宿主细胞包含本发明第一方面所述的多肽、本发明第二方面所述的肿瘤抗原结合肽、本发明第三方面所述的缀合物、本发明第四方面所述的核酸分子、本发明第五方面所述的载体；
优选地，所述宿主细胞包括大肠杆菌、酵母、昆虫细胞、哺乳动物细胞；
优选地，所述宿主细胞包括免疫细胞；
更优选地，所述免疫细胞包括T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突状细胞及其任意组合；
最优选地，所述免疫细胞包括T淋巴细胞、NK细胞；
最优选地，所述免疫细胞为NK细胞。
进一步，本发明具体实施例中所述的宿主细胞为TABP-EIC(Tumor Antigen Binding Peptide-Engineering Immune Cell，肿瘤抗原结合肽-工程化免疫细胞)。
本发明的第七方面提供了一种经改造的宿主细胞群体。
进一步，所述经改造的宿主细胞群体包含本发明第六方面所述的经改造的宿主细胞；
优选地，所述经改造的宿主细胞群体还包含不包含本发明第一方面所述的多肽、本发明第二方面所述的肿瘤抗原结合肽、本发明第三方面所述的缀合物、本发明第四方面所述的核酸分子、本发明第五方面所述的载体的宿主细胞；
更优选地，所述宿主细胞包括免疫细胞；
最优选地，所述免疫细胞包括T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突状细胞及其任意组合。
进一步，本发明具体实施例中所述的宿主细胞为TABP-EIC(Tumor Antigen Binding Peptide-Engineering Immune Cell，肿瘤抗原结合肽-工程化免疫细胞)。
本发明的第八方面提供了一种组合物或试剂盒。
进一步，所述组合物或试剂盒包括本发明第一方面所述的多肽、本发明第二方面所述的肿瘤抗原结合肽、本发明第三方面所述的缀合物、本发明第四方面所述的核酸分子、本发明第五方面所述的载体、本发明第六方面所述的经改造的宿主细胞、本发明第七方面所述的经改造的宿主细胞群体。
进一步，所述组合物包括药物组合物，所述药物组合物还包括药学上可接受的载体和/或辅料。
进一步，所述组合物包括一种细胞注射液，所述细胞注射液中含有本发明第二方面所述的肿瘤抗原结合肽、本发明第四方面所述的核酸分子、本发明第五方面所述的载体、本发明第六方面所述的经改造的宿主细胞、本发明第七方面所述的经改造的宿主细胞群体；
优选地，所述经改造的宿主细胞、经改造的宿主细胞群体表达本发明第二方面所述的肿瘤抗原结合肽。
本发明的第九方面提供了如下任一种方法：
(1)一种替代单链抗体用于制备肿瘤抗原结合肽的多肽的筛选方法，所述方法包括如下步骤：以癌细胞为阳性靶标，非癌细胞为阴性靶标，采用噬菌体-多肽文库筛选出所述多肽；
优选地，所述癌细胞包括实体瘤肿瘤细胞、非实体瘤肿瘤细胞；
更优选地，所述实体瘤包括前列腺癌、膀胱癌、肾癌、肝癌、乳腺癌、卵巢癌、肺癌、肺腺癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、直肠癌、血管内皮瘤、睾丸癌、皮肤癌；
更优选地，所述非实体瘤包括急性淋巴细胞白血病、慢性淋巴细胞白血病、造血系肿瘤、多发性骨髓瘤、非霍奇金氏淋巴瘤、急性髓性白血病、B细胞淋巴瘤、T细胞淋巴瘤；
最优选地，所述癌细胞为前列腺癌细胞；
优选地，所述多肽为本发明第一方面所述的多肽；
(2)一种制备本发明第六方面所述的经改造的宿主细胞的方法，所述方法包括如下步骤：将本发明第一方面所述的多肽、本发明第二方面所述的肿瘤抗原结合肽、本发明第三方面所述的缀合物、本发明第四方面所述的核酸分子、本发明第五方面所述的载体引入到宿主细胞中，得到本发明第六方面所述的经改造的宿主细胞；
优选地，所述核酸分子或载体通过病毒、脂质体、电转染或转座子系统转染至细胞中；
(3)一种生产新型多肽的方法，所述方法包括如下步骤：培养本发明第六方面所述的经改造的宿主细胞或本发明第七方面所述的经改造的宿主细胞群体，从细胞培养物中分离出包含如SEQ ID NO:1所示的氨基酸序列的多肽。
进一步，所述方法还包括一种用于诊断受试者是否患者肿瘤的方法，所述方法包括：使用本发明第一方面所述的多肽或本发明第八方面所述的试剂盒检测来自于所述受试者样本中是否含有肿瘤细胞。
进一步，所述方法还包括一种用于在受试者中预防和/或治疗肿瘤的方法，所述方法包括向有需要的受试者施用有效量的本发明第六方面所述的经改造的宿主细胞、本发明第七方面所述的经改造的宿主细胞群体、本发明第八方面所述的组合物。
本发明的第十方面提供了如下任一方面的应用：
(1)本发明第一方面所述的多肽在制备肿瘤抗原结合肽、缀合物、核酸分子、载体中的应用；
(2)本发明第二方面所述的肿瘤抗原结合肽在制备缀合物、核酸分子、载体中的应用；
(3)本发明第三方面所述的缀合物在制备核酸分子、载体中的应用；
(4)本发明第四方面所述的核酸分子在制备载体中的应用；
(5)本发明第一方面所述的多肽、本发明第二方面所述的肿瘤抗原结合肽、本发明第三方面所述的缀合物、本发明第四方面所述的核酸分子、本发明第五方面所述的载体在制备用于肿瘤免疫治疗的免疫细胞中的应用；
(6)本发明第一方面所述的多肽、本发明第二方面所述的肿瘤抗原结合肽、本发明第三方面所述的缀合物、本发明第四方面所述的核酸分子、本发明第五方面所述的载体、本发明第六方面所述的经改造的宿主细胞在制备用于肿瘤免疫治疗的免疫细胞群体中的应用；
(7)本发明第一方面所述的多肽、本发明第二方面所述的肿瘤抗原结合肽、本发明第三方面所述的缀合物、本发明第四方面所述的核酸分子、本发明第五方面所述的载体、本发明第六方面所述的经改造的宿主细胞、本发明第七方面所述的经改造的宿主细胞群体在制备用于预防和/或治疗肿瘤的药物中的应用；
(8)本发明第一方面所述的多肽、本发明第二方面所述的肿瘤抗原结合肽、本发明第三方面所述的缀合物、本发明第四方面所述的核酸分子、本发明第五方面所述的载体、本发明第六方面所述的经改造的宿主细胞、本发明第七方面所述的经改造的宿主细胞群体在制备用于诊断受试者是否患有肿瘤的试剂盒中的应用；
(9)多肽替代单链抗体在制备肿瘤抗原结合肽、肿瘤抗原结合肽修饰的免疫细胞、预防和/或治疗肿瘤的药物中的应用；
(10)本发明第六方面所述的经改造的宿主细胞在预防和/或治疗肿瘤中的应用；
(11)本发明第七方面所述的经改造的宿主细胞群体在预防和/或治疗肿瘤中的应用；
(12)本发明第八方面所述的组合物或试剂盒分别在预防和/或治疗肿瘤中的应用、在诊断受试者是否患有肿瘤中的应用；
优选地，所述肿瘤抗原结合肽为本发明第二方面所述的肿瘤抗原结合肽；
优选地，所述缀合物为本发明第三方面所述的缀合物；
优选地，所述核酸分子为本发明第四方面所述的核酸分子；
优选地，所述载体为本发明第五方面所述的载体；
优选地，所述肿瘤包括实体瘤、非实体瘤；
更优选地，所述实体瘤包括前列腺癌、膀胱癌、肾癌、肝癌、乳腺癌、卵巢癌、肺癌、肺腺癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、直肠癌、血管内皮瘤、睾丸癌、皮肤癌；
更优选地，所述非实体瘤包括急性淋巴细胞白血病、慢性淋巴细胞白血病、造血系肿瘤、多发性骨髓瘤、非霍奇金氏淋巴瘤、急性髓性白血病、B细胞淋巴瘤、T细胞淋巴瘤；
最优选地，所述肿瘤为前列腺癌。
本发明的优点和有益效果：
(1)本发明采用噬菌体-多肽文库，以肿瘤细胞株和阴性对照细胞株筛选出肿瘤特异性识别肽段，将所述多肽替代传统的单链抗体用于制备TABP-EIC细胞，相较于传统的制备TABP-EIC细胞的方法而言，本发明所述的制备TABP-EIC细胞的方法具有如下突出的优势：可针对抗原不明确肿瘤，显著缩短筛选TABP-EIC细胞胞外识别区序列的时间，筛选出的多肽配体与肿瘤细胞的结合效率更高；
(2)本发明提供了一种新型多肽，所述多肽能够特异性地结合前列腺癌细胞，能够应用于对前列腺癌患者的检测，且能够替代传统的单链抗体用于制备TABP-EIC，并将所述TABP-EIC应用于前列腺癌的免疫治疗中，本发明为肿瘤的免疫治疗提供了新的研究思路，解决了目前本领域面临的只能针对肿瘤相关抗原明确的肿瘤类型、筛选胞外识别区序列的时间长、结合效率不高等问题，应用前景较好。
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示多肽GTL在前列腺癌细胞系Lncap中所得的免疫荧光结果图；
图2显示多肽GTL在前列腺癌细胞系PC3中所得的免疫荧光结果图；
图3显示多肽GTL在人肾上皮细胞株293T中所得的免疫荧光结果图；
图4显示用TABP-EIC-GTL治疗不同天数的前列腺癌小鼠模型的结果图，其中，
A图：Lncap细胞腹腔注射成瘤后14天、对照组(生理盐水)，B图：Lncap细胞腹腔注射成瘤后14天、TABP-EIC-GTL组，C图：Lncap细胞腹腔注射成瘤后28天、对照组(生理盐水)，D图：Lncap细胞腹腔注射成瘤后28天、TABP-EIC-GTL组，E图：Lncap细胞腹腔注射成瘤后42天、对照组(生理盐水)，F图：Lncap细胞腹腔注射成瘤后42天、TABP-EIC-GTL组。
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1筛选特异性识别前列腺癌细胞株的特异性多肽GTL
1、实验材料
本发明实施例中所使用的Ph.D.-12噬菌体展示肽库试剂盒购自于New England Biolabs(NEB)公司，目录号为E8110SC，PC3细胞(CRL-1435)、Lncap细胞(CRL-1740)、293T细胞(CRL-3216)购自于ATCC公司。
2、Ph.D.-12噬菌体展示肽库试剂盒组成
随机十二肽噬菌体展示文库：100μL，1.5×10
13pfu/mL，贮存于含50％甘油的TBS溶液中，复杂度～2.7×10
9个转化子；-28gIII测序引物：5’-HOGTATGGGATTTTGCTAAACAAC-3’，100pmol，1pmol/μL；-96gIII测序引物：5’-HOCCCTCATAGTTAGCGTAACG-3’，100pmol/μL，1pmol/μL；E.coliER2738宿主菌F’lacIqΔ(lacZ)M15proA+B+zzf::Tn10(TetR)/fhuA2 supE thiΔ(lac-proAB)Δ(hsdMS-mcrB)5(rk–mk–McrBC–)：该菌株以含50％甘油的菌体培养物形式提供，非感受态细胞，贮存于-70℃；链霉亲和素Streptavidin，冻干粉1.5mg；生物素：10mM 100μL。
3、实验方法
本发明采用Ph.D.-12噬菌体展示肽库试剂盒，以Lncap、PC3细胞为阳性靶标，293T细胞为阴性靶标，筛选可以特异性识别前列腺癌细胞株的特异性多肽GTL，所述方法具体如下：
第一天
根据需要同时在其上进行文库淘选的靶分子的数量和种类不同，淘选试验在单个灭菌聚苯乙烯培养皿、12或24孔板、96孔微量板中进行，每种靶分子至少包被一个板(或一个孔)，下述方法中给出的量是60×15mm培养皿的用量，括号中为微孔板的用量，其他中等尺寸的孔相应地调整此用量，但每种情况下，加入的噬菌体数量都是相同的：1.5×10
11个病毒子；
(1)准备100μg/mL的靶分子溶液(溶于0.1M pH 8.6的NaHCO
3)，若需要稳定靶分子，也可使用其他相似离子强度的缓冲液(含金属离子等)；
(2)每板(孔)加入1.5mL(微孔板每孔150μL)上述溶液，反复旋转直到表面完全湿润；
(3)在增湿容器(如：排列有湿纸巾的可封口塑料盒)中4℃轻微震荡，孵育过夜，平板在此容器中4℃贮存备用；
第二天
(4)挑ER2738单克隆(噬菌体滴度测定时铺的板)于10mL LB液体培养基中，如果同一天扩增洗脱的噬菌体，也可将ER2738接种于20mL LB液体培养基，用250mL三角瓶，37℃剧烈震荡培养；
(5)倒掉每板中的包被液，板倒置在干净的纸巾上用力拍甩以除去残余溶液，每板(或孔)加满封阻液，4℃作用至少1h；
(6)速洗板6次，每次均旋转以使板或孔的底部及边缘均被洗到，倾去缓冲液，倒置在干净纸巾上拍甩以除去残余溶液(或使用自动洗板机)；
(7)用1mL(微孔板则用100μL)的TBST缓冲液稀释4×10
10的噬菌体(即10μL的原始文库)，然后加到已包被好的板上，室温温和摇动10-60min；
(8)倾倒除去未结合噬菌体，倒置板在干净的纸巾上拍甩除去残余溶液；
(9)按6中所述方法用TBST缓冲液洗板10次，每次换一干净纸巾以避免交叉污染；
(10)根据所研究的分子间相互作用，用1mL(微孔板则用100μL)适当的洗脱缓冲液洗脱结合的噬菌体，将靶分子的已知配体以0.1-1mM的浓度溶于TBS溶液中或者用游离靶分子溶液(～100μg/mL溶于TBS中)从固定靶分子上将结合的噬菌体竞争性洗脱下来，室温温和摇动10-60min，将洗脱液吸入另一干净微量离心管中；也可用非特异性缓冲液如0.2M Glycine-HCl(pH 2.2)，1mg/mL BSA来分离已结合的分子：温和摇动>10min，洗脱液吸入另一干净微量离心管中，然后再用150μL(微量孔则用15μL)1M Tris-HCl(pH 9.1)中和上述洗脱液；
(11)按上述常规M13方法中的程序测定少量(～1μL)洗脱物的滴度，如需要，可对 第一或第二轮洗脱物滴度测定所得的噬菌斑进行测序，方法见下述：必要时可将剩余洗脱物4℃贮存过夜，第二天扩增，这时，可将ER2738在LB-Tet培养基中过夜培养，第二天将培养物1:100稀释于20mL LB中(用250mL三角瓶盛装)，加入未扩增洗脱物，37℃剧烈摇动培养4.5h，继续第13步；
(12)扩增剩余洗脱物：将洗脱物加入到20mL ER2738培养物中(菌体处于对数前期)，37℃剧烈摇动培养4.5h；
(13)将培养物转入一离心管中，然后，4℃10,000rpm离心10min。上清液转入另一离心管中，再离心；
(14)将上清的上部80％转入一新鲜管中，加入1/6体积的PEG/NaCl，让噬菌体4℃沉淀至少60min，过夜；
第三天
(15)4℃10,000rpm离心PEG沉淀15min，倒掉上清液，再短暂离心，吸去残留上清液；
(16)沉淀物重悬于1mL TBS中，悬液转入微量离心管中，4℃离心5min使残余细胞沉淀；
(17)上清转入另一新鲜微量离心管，用1/6体积的PEG/NaCl再沉淀，冰上孵育15-60min，4℃离心10min，弃上清，再短暂离心，用微量移液器吸去残余上清；
(18)沉淀物重悬于200μL TBS，0.02％NaN
3中，离心1min，沉淀任何残余的不溶物，上清转入新鲜管中，此即为扩增后的洗脱物；
(19)根据上述常规M13方法用LB/IPTG/Xgal平板滴定扩增后的洗脱物，4℃贮存；
(20)再包被一个板或孔准备第二轮淘选时用；
第四和第五天
(21)计数板上蓝斑数确定滴度，用这个值来计算相应于1-2×10
11pfu的加入量；若滴度太低，接下来的几轮淘选可用低至10
9pfu的噬菌体加入量进行试验；
(22)进行第二轮淘选：用第一轮淘选扩增的洗脱物中1-2×10
11pfu的噬菌体量重复步骤4-18，在清洗步骤中将Tween的浓度增至0.5％(v/v)；
(23)在LB/IPTG/Xgal平板上测定第二轮淘选所得洗脱物扩增后的滴度；
(24)再包被一个板或孔准备第三轮淘选时用；
第六天
(25)进行第三轮淘选：用第二轮淘选扩增的洗脱物中2×10
11pfu的噬菌体量重复步骤4-11，清洗步骤中同样用0.5％(v/v)的Tween；
(26)在LB/IPTG/Xgal平板上测定第三轮淘选所得洗脱物未扩增时的滴度，第三轮洗脱物不必再扩增，除非还要进行第四轮淘选，滴度测定时得到的噬菌斑可做测序用：只要注意平板培养时间不要超过18h，培养时间过长容易出现缺失，其余洗脱物4℃贮存；
(27)挑一ER2738单克隆于LB-Tet培养基中培养过夜。
4、实验结果
实验结果显示，筛选得到的特异性识别前列腺癌细胞株的特异性多肽GTL的氨基酸序列为GTLLNPYPFSWGY(SEQ ID NO:1)。
实施例2免疫荧光技术鉴定多肽GTL对前列腺癌细胞的亲和力和特异性
1、实验材料
前列腺癌细胞株PC3(CRL-1435)、前列腺癌细胞株Lncap(CRL-1740)、人肾上皮细胞株293T(CRL-3216)购自于ATCC公司。
2、实验方法
(1)固定
用新配制的4％多聚甲醛溶液在培养板中固定细胞15分钟，DPBS清洗样品，小心操作避免细胞从玻片上脱落；
(2)清洗
DPBS洗三遍，每次5分钟；
(3)封闭
以2％BSA在4℃温度条件下封闭30分钟，DPBS洗两次，每次5分钟；
(4)多肽孵育
加入使用FITC(异硫氰酸荧光素)标记的GTL多肽(终浓度1μg/mL)，黑暗条件下在湿度箱内4℃过夜孵育；
(5)清洗
DPBS洗三遍，每次5分钟；
(6)封片及观察
加入0.5μg/mL DAPI(采用PBS配制)染色10分钟，也可采用其它的染核染料；
用PBS洗三遍，去除多余的DAPI，加入20μL封片剂封片，在显微镜下观察，注意荧光强度随着灯光刺激衰减，并尽快拍照。
3、实验结果
实验结果显示，实施例1中筛选得到的多肽GTL可与前列腺癌细胞株Lncap、PC3细胞膜表面的某种蛋白特异性结合(见图1和图2)，而不与非前列腺癌细胞株293T细胞膜表面的蛋白结合(见图3)，即GTL可特异性地识别前列腺癌细胞某种特有的膜蛋白，进一步表明了本发明所述的新型多肽GTL具有较好的与前列腺癌细胞特异性结合的能力。
实施例3将多肽GTL作为胞外识别区制备TABP-EIC(TABP-EIC-GTL)细胞
1、NK细胞
本专利涉及的实验中使用到的NK细胞均为来源于外周血单核细胞(PBMC)扩增获得。
2、肿瘤抗原结合肽表达载体的构建
肿瘤抗原结合肽结构序列均为基因合成获得(通用生物)，表达载体为pLenti-EF1a-Backbone(NN)(addgene#27961)；
肿瘤抗原结合肽结构插入酶切位点为BsiWI及EcoRI。
3、慢病毒包装
将TABP-EIC骨架载体及辅助载体pMD2.G(addgen#12259)、pMDLg/pRRE(addgene#12251)、pRSV-Rev(addgene#12253)按10:5:3:2比例混合，20μg质粒每10mL转染体系，转染293T细胞。转染后48小时、72小时收集上清，纯化浓缩后获得慢病毒。
4、慢病毒转导
将浓缩后的慢病毒与NK细胞按每100万细胞200μL纯化后慢病毒混合，之后置于37 ℃培养箱5％CO
2条件下培养，24小时后完全换液。
5、TABP-EIC-GTL细胞的扩增
将慢病毒感染后获得的TABP-EIC细胞正常培养扩增。
6、TABP-EIC-GTL细胞肿瘤抗原结合肽表达效率的检测
TABP-EIC细胞慢病毒感染后第七天取部分细胞提取基因组，进行RT-PCR检测。
7、实验结果
根据RT-PCR结果，按照公式感染效率(％)＝63.21-6.36×△CT(检测组CT-对照组CT)，选择感染效率超过20％的TABP-EIC-GTL细胞用于后续实验验证，制备得到的肿瘤抗原结合肽的氨基酸序列如SEQ ID NO:2所示，核苷酸序列如SEQ ID NO:3所示，肿瘤抗原结合肽结构中GTL序列可有多个重复，在本发明的实施例中为3个重复。
实施例4TABP-EIC-GTL细胞对体内肿瘤杀伤效果的验证
1、实验材料
小鼠购自于维通利华公司，为8周龄NOD品系雄鼠；前列腺癌细胞株Lncap(CRL-1740)购自于ATCC公司。
2、实验方法
(1)前列腺癌细胞Lncap腹腔注射构建前列腺癌小鼠肿瘤模型
将健康的小鼠共6只随机分为2组，每组3只，2组分别为对照组和TABP-EIC-GTL细胞组，在所有小鼠的腹腔内注射500万个含有绿色荧光蛋白(GFP)标记的前列腺癌细胞(Lncap-GFP)/只，并正常喂养14天；
(2)对对照组和TABP-EIC-GTL细胞组小鼠分别进行实验处理
成瘤第14天时，对所有组别的小鼠进行活体荧光成像，记录小鼠体内肿瘤的生长情况，荧光成像之后，分别对各组小鼠分别进行如下给药：
对照组：给对照组小鼠分别腹腔灌注生理盐水1000μL；
TABP-EIC-GTL组：给TABP-EIC-GTL组小鼠分别腹腔灌注TABP-EIC-GTL细胞悬浮液1000μL，5×10
6个细胞/mL生理盐水/只；
每周进行一次腹腔灌注，持续3周，第14天、28天、42天对小鼠进行活体荧光成像，观察小鼠体内的肿瘤情况，并记录小鼠体内的肿瘤大小。
3、实验结果
实验结果显示，与腹腔灌注生理盐水的对照组小鼠相比，腹腔灌注TABP-EIC-GTL细胞的实验组小鼠在成瘤第28天和第42天后的体内肿瘤的平均体积显著较小，体内肿瘤的生长明显受到抑制，表明了本发明制备的TABP-EIC-GTL细胞对肿瘤有显著的抑制作用，对前列腺癌具有较好的治疗效果。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。
Claims (10)
- 一种特异性识别肿瘤细胞的新型多肽，其特征在于，所述多肽包括如SEQ ID NO:1所示的氨基酸序列的多肽、或其活性片段、类似物；优选地，所述活性片段、类似物的氨基酸序列具有与如SEQ ID NO:1所示的氨基酸序列至少95％的相同性；优选地，所述肿瘤细胞为前列腺癌细胞。
- 一种肿瘤抗原结合肽，其特征在于，所述肿瘤抗原结合肽包括含有抗原识别区的胞外信号结构域、连接至所述胞外信号结构域的跨膜结构域、连接至所述跨膜结构域的胞内信号结构域；优选地，所述胞外信号结构域包括抗原识别区；更优选地，所述抗原识别区由一个或多个权利要求1所述的多肽通过linker串联得到；最优选地，所述多肽的氨基酸序列如SEQ ID NO:1所示；最优选地，所述linker的氨基酸序列如SEQ ID NO:4所示；最优选地，所述抗原识别区由三个权利要求1所述的多肽通过linker串联得到；最优选地，所述抗原识别区的氨基酸序列如SEQ ID NO:5所示；最优选地，所述抗原识别区的核苷酸序列如SEQ ID NO:6所示；优选地，所述胞外信号结构域还包括铰链区；更优选地，所述铰链区包括以下任一种或多种分子的铰链区：CD3ε、CD4、CD5、CD8、CD8α、PD1、CD16、CD22、CD28、CD33、CD37、CD45、CD134、CD137、CD152、CD154；最优选地，所述铰链区为CD8α分子的铰链区；最优选地，所述CD8α分子的铰链区的氨基酸序列如SEQ ID NO:7所示；优选地，所述胞外信号结构域还包括信号肽；更优选地，所述信号肽来源于以下分子：T细胞受体的α链及β链、CD3ζ、CD28、CD3ε、CD45、CD4、CD5、CD8、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、ICOS、CD154、GITR、免疫球蛋白重链、免疫球蛋白轻链；最优选地，所述信号肽来源于CD8α；最优选地，所述信号肽的氨基酸序列如SEQ ID NO:8所示；优选地，所述跨膜结构域包括选自下列蛋白质的跨膜区：T细胞受体的α、β或ζ链、GITR、ICOS、D45、CD3ε、CD3ζ、CD4、CD5、CD8、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD123、CD134、CD137、CD154、PD1、2B4；更优选地，所述跨膜结构域为2B4的跨膜区；最优选地，所述2B4的跨膜区的氨基酸序列如SEQ ID NO:9所示；优选地，所述胞内信号结构域包括信号转导结构域；更优选地，所述信号转导结构域包括选自下列蛋白质的功能性信号转导结构域：CD3ζ、FcRγ、FcRβ、CDS、CD3ε、CD3γ、CD3δ、CD5、CD22、CD66d、CD79a、CD79b、NKG2D；最优选地，所述信号转导结构域为NKG2D的功能性信号转导结构域；最优选地，所述NKG2D的功能性信号转导结构域的氨基酸序列如SEQ ID NO:10所示；优选地，所述胞内信号结构域还包括共刺激信号结构域；更优选地，所述共刺激信号结构域包括选自下列蛋白质的功能性信号转导结构域：4-1BB(CD137)、HVEM、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、 CARD11、2B4；最优选地，所述共刺激信号结构域为2B4的功能性信号转导结构域；最优选地，所述2B4的功能性信号转导结构域的氨基酸序列如SEQ ID NO:11所示；最优选地，所述肿瘤抗原结合肽由信号肽、抗原识别区、铰链区、跨膜结构域、共刺激信号结构域、信号转导结构域依次连接而成；最优选地，所述肿瘤抗原结合肽包括以下任一项所述的氨基酸序列：(1)如SEQ ID NO:2所示的氨基酸序列；(2)与如SEQ ID NO:2所示的氨基酸序列相比，具有一个或几个氨基酸的置换、缺失或添加的氨基酸序列；(3)与如SEQ ID NO:2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的相同性的氨基酸序列；最优选地，所述肿瘤抗原结合肽的氨基酸序列如SEQ ID NO:2所示。
- 一种缀合物，其特征在于，所述缀合物包括权利要求1所述的多肽；优选地，所述缀合物还包括连接所述多肽的修饰部分；更优选地，所述修饰部分包括可检测的标记或治疗剂；最优选地，所述可检测的标记包括酶、成像剂、造影剂、荧光染料、发光物质或生物素；最优选地，所述治疗剂包括具有抗肿瘤活性的药物或细胞毒剂。
- 一种分离的核酸分子，其特征在于，所述核酸分子包括编码权利要求1所述的多肽的核苷酸序列、权利要求2所述的肿瘤抗原结合肽的核苷酸序列；优选地，所述核酸分子包括选自下列组中的核酸分子：(1)编码如SEQ ID NO:1所示的氨基酸序列的多肽、或其活性片段、类似物的核酸分子；(2)编码权利要求2所述的肿瘤抗原结合肽的核酸分子；(3)与(1)或(2)互补的核酸分子；(4)与(1)或(2)或(3)有至少70％相同性的核酸分子；更优选地，所述核酸分子为编码如SEQ ID NO:1所示氨基酸序列的核酸分子、如SEQ ID NO:3所示核苷酸序列的核酸分子；最优选地，编码如SEQ ID NO:1所示氨基酸序列的核酸分子的核苷酸序列如SEQ ID NO:12所示。
- 一种重组载体，其特征在于，所述载体包含权利要求4所述的核酸分子；优选地，所述载体为权利要求4所述的核酸分子与DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体、病毒载体表达载体构建而成的重组载体；更优选地，所述病毒载体包括慢病毒载体、腺病毒载体、逆转录病毒载体。
- 一种经改造的宿主细胞，其特征在于，所述经改造的宿主细胞包含权利要求1所述的多肽、权利要求2所述的肿瘤抗原结合肽、权利要求3所述的缀合物、权利要求4所述的核酸分子、权利要求5所述的载体；优选地，所述宿主细胞包括大肠杆菌、酵母、昆虫细胞、哺乳动物细胞；优选地，所述宿主细胞包括免疫细胞；更优选地，所述免疫细胞包括T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突状细胞及 其任意组合；最优选地，所述免疫细胞包括T淋巴细胞、NK细胞；最优选地，所述免疫细胞为NK细胞。
- 一种经改造的宿主细胞群体，其特征在于，所述经改造的宿主细胞群体包含权利要求6所述的经改造的宿主细胞；优选地，所述经改造的宿主细胞群体还包含不包含权利要求1所述的多肽、权利要求2所述的肿瘤抗原结合肽、权利要求3所述的缀合物、权利要求4所述的核酸分子、权利要求5所述的载体的宿主细胞；更优选地，所述宿主细胞包括免疫细胞；最优选地，所述免疫细胞包括T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突状细胞及其任意组合。
- 一种组合物或试剂盒，其特征在于，所述组合物或试剂盒包括权利要求1所述的多肽、权利要求2所述的肿瘤抗原结合肽、权利要求3所述的缀合物、权利要求4所述的核酸分子、权利要求5所述的载体、权利要求6所述的经改造的宿主细胞、权利要求7所述的经改造的宿主细胞群体。
- 如下任一种方法，其特征在于，所述方法包括：(1)一种替代单链抗体用于制备肿瘤抗原结合肽的多肽的筛选方法，其特征在于，所述方法包括如下步骤：以癌细胞为阳性靶标，非癌细胞为阴性靶标，采用噬菌体-多肽文库筛选出所述多肽；优选地，所述肿瘤细胞包括实体瘤肿瘤细胞、非实体瘤肿瘤细胞；更优选地，所述实体瘤包括前列腺癌、膀胱癌、肾癌、肝癌、乳腺癌、卵巢癌、肺癌、肺腺癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、直肠癌、血管内皮瘤、睾丸癌、皮肤癌；更优选地，所述非实体瘤包括急性淋巴细胞白血病、慢性淋巴细胞白血病、造血系肿瘤、多发性骨髓瘤、非霍奇金氏淋巴瘤、急性髓性白血病、B细胞淋巴瘤、T细胞淋巴瘤；最优选地，所述肿瘤细胞为前列腺癌细胞；优选地，所述多肽为权利要求1所述的多肽；(2)一种制备权利要求6所述的经改造的宿主细胞的方法，其特征在于，所述方法包括如下步骤：将权利要求1所述的多肽、权利要求2所述的肿瘤抗原结合肽、权利要求3所述的缀合物、权利要求4所述的核酸分子、权利要求5所述的载体引入到宿主细胞中，得到权利要求6所述的经改造的宿主细胞；优选地，所述核酸分子或载体通过病毒、脂质体、电转染或转座子系统转染至细胞中；(3)一种生产新型多肽的方法，其特征在于，所述方法包括如下步骤：培养权利要求6所述的经改造的宿主细胞或权利要求7所述的经改造的宿主细胞群体，从细胞培养物中分离出包含如SEQ ID NO:1所示的氨基酸序列的多肽。
- 如下任一方面的应用，其特征在于，所述应用包括：(1)权利要求1所述的多肽在制备肿瘤抗原结合肽、缀合物、核酸分子、载体中的应用；(2)权利要求2所述的肿瘤抗原结合肽在制备缀合物、核酸分子、载体中的应用；(3)权利要求3所述的缀合物在制备核酸分子、载体中的应用；(4)权利要求4所述的核酸分子在制备载体中的应用；(5)权利要求1所述的多肽、权利要求2所述的肿瘤抗原结合肽、权利要求3所述的缀合物、权利要求4所述的核酸分子、权利要求5所述的载体在制备用于肿瘤免疫治疗的免疫细胞中的应用；(6)权利要求1所述的多肽、权利要求2所述的肿瘤抗原结合肽、权利要求3所述的缀合物、权利要求4所述的核酸分子、权利要求5所述的载体、权利要求6所述的经改造的宿主细胞在制备用于肿瘤免疫治疗的免疫细胞群体中的应用；(7)权利要求1所述的多肽、权利要求2所述的肿瘤抗原结合肽、权利要求3所述的缀合物、权利要求4所述的核酸分子、权利要求5所述的载体、权利要求6所述的经改造的宿主细胞、权利要求7所述的经改造的宿主细胞群体在制备用于预防和/或治疗肿瘤的药物中的应用；(8)权利要求1所述的多肽、权利要求2所述的肿瘤抗原结合肽、权利要求3所述的缀合物、权利要求4所述的核酸分子、权利要求5所述的载体、权利要求6所述的经改造的宿主细胞、权利要求7所述的经改造的宿主细胞群体在制备用于诊断受试者是否患有肿瘤的试剂盒中的应用；(9)多肽替代单链抗体在制备肿瘤抗原结合肽、肿瘤抗原结合肽修饰的免疫细胞、预防和/或治疗肿瘤的药物中的应用；(10)权利要求6所述的经改造的宿主细胞在预防和/或治疗肿瘤中的应用；(11)权利要求7所述的经改造的宿主细胞群体在预防和/或治疗肿瘤中的应用；(12)权利要求8所述的组合物或试剂盒分别在预防和/或治疗肿瘤中的应用、在诊断受试者是否患有肿瘤中的应用；优选地，所述肿瘤抗原结合肽为权利要求2所述的肿瘤抗原结合肽；优选地，所述缀合物为权利要求3所述的缀合物；优选地，所述核酸分子为权利要求4所述的核酸分子；优选地，所述载体为权利要求5所述的载体；优选地，所述肿瘤包括实体瘤、非实体瘤；更优选地，所述实体瘤包括前列腺癌、膀胱癌、肾癌、肝癌、乳腺癌、卵巢癌、肺癌、肺腺癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、直肠癌、血管内皮瘤、睾丸癌、皮肤癌；更优选地，所述非实体瘤包括急性淋巴细胞白血病、慢性淋巴细胞白血病、造血系肿瘤、多发性骨髓瘤、非霍奇金氏淋巴瘤、急性髓性白血病、B细胞淋巴瘤、T细胞淋巴瘤；最优选地，所述肿瘤为前列腺癌。
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