Specific T cell receptor targeting NY-ESO-1(157-165) epitope and anti-tumor application
阅读说明：本技术 靶向ny-eso-1（157-165）表位的特异性t细胞受体及抗肿瘤应用 (Specific T cell receptor targeting NY-ESO-1(157-165) epitope and anti-tumor application ) 是由 张卫红 谭曙光 靳文静 陈瑜 于 2020-06-28 设计创作，主要内容包括：本发明提供一种靶向NY-ESO-1(157-165)表位的特异性T细胞受体及抗肿瘤应用,该T细胞受体由α、β两条肽链组成,还提供其抗原结合片段,以及编码它的核酸,包含该核酸的载体,包含该载体的宿主细胞；还提供一种制备NY-ESO-1特异性T细胞受体或其抗原结合片段的方法。该特异性T细胞受体及其抗原结合片段能够作为免疫效应活化物刺激机体的免疫反应,从而产生抗肿瘤等疾病的作用效果。(The invention provides a specific T cell receptor targeting NY-ESO-1(157-165) epitope and anti-tumor application, wherein the T cell receptor consists of alpha and beta peptide chains, and also provides an antigen binding fragment, a nucleic acid for encoding the T cell receptor, a vector containing the nucleic acid, and a host cell containing the vector; also provided is a method of making a NY-ESO-1 specific T cell receptor or antigen-binding fragment thereof. The specific T cell receptor and the antigen binding fragment thereof can be used as an immune effect activator to stimulate the immune response of an organism, thereby having the effect of resisting diseases such as tumor and the like.)
1. A T Cell Receptor (TCR), or an antigen-binding fragment thereof, which TCR, or antigen-binding fragment thereof, is capable of binding to the NY-ESO-1(157-165) epitope and HLA-a2 complex, and which TCR comprises an alpha chain variable region and a beta chain variable region, wherein the TCR, or antigen-binding fragment thereof, comprises the following alpha chain Complementarity Determining Regions (CDRs) and beta chain Complementarity Determining Regions (CDRs):
an alpha chain Complementarity Determining Region (CDR)1 comprising the amino acid sequence shown in SEQ ID NO. 3 or a modification thereof;
an alpha chain Complementarity Determining Region (CDR)2 comprising the amino acid sequence shown in SEQ ID NO. 4 or a modification thereof;
an alpha chain Complementarity Determining Region (CDR)3 comprising the amino acid sequence shown in SEQ ID NO. 5 or a modification thereof;
beta-strand Complementarity Determining Region (CDR)1 comprising the amino acid sequence shown in SEQ ID NO. 6 or a modification thereof;
beta-strand Complementarity Determining Region (CDR)2 comprising the amino acid sequence shown in SEQ ID NO. 7 or a modification thereof; and
beta-strand Complementarity Determining Region (CDR)3 comprising the amino acid sequence shown in SEQ ID NO. 8 or a modification thereof.
2. A T Cell Receptor (TCR), or an antigen-binding fragment thereof, which TCR, or antigen-binding fragment thereof, is capable of binding to the NY-ESO-1(157-165) epitope and HLA-a2 complex, and which TCR comprises an alpha chain variable region and a beta chain variable region, wherein the TCR, or antigen-binding fragment thereof, comprises:
an alpha chain variable region having 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% or 100% identity to the sequence of SEQ ID NO. 1, and
a beta-chain variable region having 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% or 100% identity to the sequence of SEQ ID NO. 2.
3. The TCR, or antigen-binding fragment thereof, of claim 1 or 2, wherein the TCR is a murine TCR, a human murine chimeric TCR, or a humanized TCR.
4. A polynucleotide encoding a TCR, or antigen-binding fragment thereof, according to any one of claims 1-3.
5. An expression vector comprising the polynucleotide of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. A method of making a TCR, or antigen-binding fragment thereof, according to any one of claims 1-3, comprising:
1) culturing the host cell of claim 6;
2) recovering the TCR, or antigen-binding fragment thereof, of any one of claims 1-3 from the host cell or culture medium thereof.
8. A pharmaceutical composition comprising a TCR, or antigen-binding fragment thereof, according to any one of claims 1-3, and a pharmaceutically acceptable carrier.
9. Use of a TCR, or antigen-binding fragment thereof, according to any one of claims 1-3 in the manufacture of a medicament for increasing the level of IL-2 secretion by a T cell.
10. Use of a TCR, or an antigen-binding fragment thereof, according to any one of claims 1 to 3 in the manufacture of an anti-tumour medicament for the treatment of multiple myeloma and soft tissue sarcoma, melanoma, lung cancer, ovarian cancer and the like, preferably the soft tissue sarcoma is synovial sarcoma.
Technical Field
The invention belongs to the field of medicines, and particularly relates to a method capable of specifically identifying NY-ESO-1(157-165) epitope (SLLMWITQC, HLA-A)*0201) Or an antigen binding fragment thereof.
Background
In 2011, cancer surpasses heart disease, and becomes the first leading cause of death worldwide. WHO published 12 months in 2013, the number of newly added cancer patients worldwide has exceeded 1400 million every year, which is greatly increased compared to 1270 million patients as a statistical result in 2008. At the same time, the number of deaths among cancer patients has increased, from 760 to 820 million in the past.
Early in the 80's of the 20 th century, Allison and other researchers determined the genetic structure of the α β T Cell Receptor (TCR) responsible for antigen recognition on the surface of T cells. In the later 80 s, Boone, Rosenberg, Old and other people respectively found that some tumor specific antigens exist in different tumor patients, can be recognized by T cells and specifically kill the tumor cells, so that the hope of tumor immunotherapy is reignited, and a great deal of research is devoted to the research and development of tumor therapeutic vaccines. The immune anticancer therapy in 2013 is judged as the first breakthrough of 10 years of Science journal.
In recent years, with the rapid development of stem cell biology, immunology, molecular techniques, tissue engineering techniques, and the like, cellular immunotherapy has been increasingly used as a safe and effective therapeutic means for the treatment of tumors and the like. Currently, research and development of novel cell therapy techniques have become an important research field for solving related diseases such as tumor.
Adoptive Cell Therapy (ACT) is a highly personalized cancer treatment that can achieve anti-tumor effects by reconstituting missing or weakened immune systems in cancer patients. ACT therapy refers to the isolation of immunocompetent cells from a tumor patient, amplification and functional identification in vitro, and reinfusion to the patient to achieve the goal of directly killing the tumor or stimulating the immune response of the body to kill the tumor cells. The search for antigens that are expressed only on cancer tissues, but not on normally essential tissues, is a limiting factor in ACT therapy. At present, ACT therapy can be achieved by including T cell receptor engineered cell (TCR-T) therapy techniques and chimeric antigen receptor engineered T cell (CAR-T) therapy techniques. By these methods, ACT is highly effective against a variety of cancers, such as melanoma, cervical cancer, lymphoma, leukemia, common bile duct cancer, and neuroblastoma.
TCR is a characteristic marker of the surface of all T cells, and binds non-covalently to CD3, forming a TCR-CD3 complex. The TCR is composed of alpha and beta peptide chains, belongs to an immunoglobulin superfamily, and has antigen specificity in V regions (CDR1, CDR2 and CDR3), and the CDR3 directly determines the antigen binding specificity of the TCR. In peripheral blood, 90% to 95% of T cells express TCR. The T cells of the TCR are genetically modified to specifically recognize antigen molecules on the surface of tumor cells, and then generate immune response aiming at the tumor cells.
TCR-T cell immunotherapy is a new cell therapy technology developed in recent years, and is a typical "precision medicine" therapy technology. At present, the technology has shown a positive treatment prospect in the treatment of myeloma, melanoma, esophageal cancer, liver cancer and the like. In the application of TCR-T cell immunotherapy to HIV treatment at the end of the 20 th century, researches in recent years find that the autoimmune cells based on the specific TCR engineering transformation of tumor antigens such as MART-1, MAGE-A4, NY-ESO-1, WT-1 and the like show good development prospects in treating melanoma, esophageal cancer, multiple myeloma, synovial cell sarcoma and the like. In particular, clinical phase I/II of 20 cases of multiple myeloma reported in 2015 by NYESO-1 specific TCR engineered cell immunotherapy, 80% of cases showed positive clinical therapeutic effect after receiving TCR-T therapy. Currently, TCR-T cell immunotherapy technology has become a hot spot for international research on tumor and infectious disease treatment, and some technologies and products have entered preclinical or clinical research.
Currently, CAR-T also realizes a major breakthrough in the treatment of acute/chronic myelocytic leukemia, lymphoma and other diseases, and greatly improves the survival rate and the quality of life of patients. However, CAR-T cell therapy has not shown promise in solid tumor therapy research due to limited specific targets. Research shows that T cell surface receptor (TCR) is the main molecule for body to recognize endogenous tumor antigen, and TCR based cell immunotherapy has wide prospect in breaking through solid tumor and clinical treatment of serious infectious disease.
NY-ESO-1(New York Eyophagal Squamous Cell
Disclosure of Invention
In a first aspect, the present invention provides a T Cell Receptor (TCR) or an antigen-binding fragment thereof which is capable of binding to the NY-ESO-1(157-165) epitope and the HLA-A2 complex and which comprises an alpha chain variable region and a beta chain variable region, wherein the TCR or antigen-binding fragment thereof comprises the following alpha chain Complementarity Determining Regions (CDRs) and beta chain Complementarity Determining Regions (CDRs):
an alpha chain Complementarity Determining Region (CDR)1 comprising the amino acid sequence shown in SEQ ID NO. 3 or a modification thereof;
an alpha chain Complementarity Determining Region (CDR)2 comprising the amino acid sequence shown in SEQ ID NO. 4 or a modification thereof;
an alpha chain Complementarity Determining Region (CDR)3 comprising the amino acid sequence shown in SEQ ID NO. 5 or a modification thereof;
beta-strand Complementarity Determining Region (CDR)1 comprising the amino acid sequence shown in SEQ ID NO. 6 or a modification thereof;
beta-strand Complementarity Determining Region (CDR)2 comprising the amino acid sequence shown in SEQ ID NO. 7 or a modification thereof; and
beta-strand Complementarity Determining Region (CDR)3 comprising the amino acid sequence shown in SEQ ID NO. 8 or a modification thereof.
The NY-ESO-1 specific TCR or antigen binding fragment thereof is capable of specifically binding SLLMWITQC-HLA-A0201 molecule.
In a second aspect, the invention provides a T Cell Receptor (TCR), or an antigen-binding fragment thereof, which TCR, or antigen-binding fragment thereof, is capable of binding to the NY-ESO-1(157-165) epitope and the HLA-A2 complex, and which TCR comprises an alpha chain variable region and a beta chain variable region, wherein the TCR, or antigen-binding fragment thereof, comprises:
an alpha chain variable region having 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% or 100% identity thereto, and
a beta-chain variable region having 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% or 100% identity to the sequence of SEQ ID NO. 2.
In a specific embodiment, the inventive TCR described above comprises an α chain variable region having the sequence SEQ ID No. 1 and a β chain variable region having the sequence SEQ ID No. 2.
In a preferred embodiment, in the TCR or antigen-binding fragment thereof of the invention described above, the TCR is a murine TCR, a human murine chimeric TCR, or a humanized TCR.
A third aspect of the invention provides a polynucleotide encoding a TCR of the invention, or an antigen-binding fragment thereof, as described above.
In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide of the present invention as described above.
A fifth aspect of the present invention provides a host cell comprising the above-described expression vector of the present invention.
A sixth aspect of the invention provides a method of preparing a TCR, or an antigen-binding fragment thereof, of the invention as described above, which method comprises:
1) culturing the host cell of the present invention described above;
2) recovering the inventive TCR, or antigen-binding fragment thereof, described above from the host cell or culture medium thereof.
A seventh aspect of the invention provides a pharmaceutical composition comprising a TCR of the invention, or an antigen-binding fragment thereof, as described above, and a pharmaceutically acceptable carrier.
An eighth aspect of the invention provides the use of a TCR, or an antigen-binding fragment thereof, of the invention as described above, in the manufacture of a medicament for increasing the level of IL-2 secretion from a T cell.
A ninth aspect of the invention provides the use of a TCR, or an antigen-binding fragment thereof, of the invention as defined above, in the preparation of an anti-tumour medicament for the treatment of multiple myeloma and soft tissue sarcoma, melanoma, lung cancer, ovarian cancer and the like, preferably the soft tissue sarcoma is synovial sarcoma.
The NY-ESO-1 specific TCR or the fragment thereof provided by the invention can specifically identify SLLMWITQC epitope polypeptide which is presented by human HLA-A2 and is derived from tumor-associated antigen NY-ESO-1, and further combine with NY-ESO-1 molecules, can be used as an immune effect activator to stimulate the immune reaction of organisms, thereby generating the effect of resisting diseases such as tumors and the like and a series of biological effects. These biological effects include, for example: can improve the level of IL-2 and IFN-gamma secretion of tumor specific T cells in tumor cases, and particularly can inhibit the growth of tumors in mice.
Drawings
FIG. 1 NY-ESO-1(157-165)/HLA-A2 protein purification and SDS-PAGE identification.
FIG. 2 biotinylation of NY-ESO-1(157-165)/HLA-A2 protein purification and biotinylation efficiency identification.
FIG. 3 Single cell sorting of NY-ESO-1(157-165)/HLA-A2 tetramer-specific T cells.
FIG. 4 verification that YW-TCR-1 TCR specifically binds NY-ESO-1 (157-165)/HLA-A2.
FIG. 5 molecular sieve chromatographic purification and SDS-PAGE identification of YW-TCR-1 TCR protein. In the figure, DTT is dithiothreitol, a strong reducing agent, which, when added, opens the TCR alpha and beta inter-chain disulfide bonds, showing two bands in SDS-PAGE, indicating that the TCR protein is a heterodimer formed by alpha and beta chains.
FIG. 6 shows molecular sieve chromatography and SDS-PAGE identification of complexes formed by incubation of YW-TCR-1 TCR with NY-ESO-1 (157-165)/HLA-A2. In the figure, DTT is dithiothreitol, a strong reducing agent, which, when added, opens the TCR alpha and beta inter-chain disulfide bonds, showing two bands in SDS-PAGE, indicating that the TCR protein is a heterodimer formed by alpha and beta chains.
FIG. 7 BIAcore assay to determine the affinity of YW-TCR-1 TCR for NY-ESO-1 (157-165)/HLA-A2.
FIG. 8 measurement of cytokine secretion levels after incubation of YW-TCR-1 TCR with target cells.
Detailed Description
NY-ESO-1 is an important tumor testis (CT) antigen with strong spontaneous immunogenicity in human body, including complex reactions in cellular and humoral immune systems. NY-ESO-1 is a member of a multigene family on the X chromosome (Xq 28). It is expressed in high amounts (
Based on the principle, the NY-ESO-1 specific TCR or the antigen binding fragment thereof is specifically bound with a compound molecule of NY-ESO-1-derived SLLMWITQC polypeptide and HLA-A2, so that T cells are stimulated to be activated, the T cells are induced to secrete cytokines such as IFN-gamma and IL-2, and tumor cells expressing NY-ESO-1 and HLA-A2 are further killed.
In the present invention, the expression "NY-ESO-1 specific TCR" or "murine NY-ESO-1 specific TCR" is a murine TCR, in a particular embodiment YW-TCR-1 TCR, directed against a HLA-A2 restricted CTL epitope polypeptide (SLLMWITQC in NY-ESO-1).
The present application includes TCRs or derivatives which specifically bind to a complex molecule of the NY-ESO-1 derived SLLMWITQC polypeptide at position 157 and 165 and HLA-A2, and also includes TCR fragments which exhibit substantially the same antigenic specificity as the original TCR. "fragments of a TCR" or "antigen-binding fragments" refer to antigen-binding fragments and TCR analogs of TCRs, which typically include at least a portion of the antigen-binding or variable region, e.g., one or more CDRs, of the parent TCR. Fragments of the TCR retain at least some of the binding specificity of the parent TCR.
"specific" binding, when referring to a ligand/receptor, antibody/antigen or other binding pair, refers to determining the presence or absence of a binding reaction of a protein, e.g., SLLMWITQC polypeptide, to an HLA-A2 complex molecule in a heterogeneous population of proteins and/or other biological agents. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.
The invention also provides pharmaceutical compositions comprising a NY-ESO-1 specific TCR of the invention or antigen-binding fragment thereof. For the preparation of a pharmaceutical composition, various desired dosage forms can be prepared by mixing the NY-ESO-1 specific TCR or antigen-binding fragment thereof with a pharmaceutically acceptable carrier or excipient. Examples of the dosage form of the pharmaceutical composition of the present invention include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated preparations, pellets, sublingual tablets, and ointments, which are oral preparations, and examples of non-oral preparations include injections, suppositories, transdermal preparations, ointments, plasters, and external liquid preparations, and those skilled in the art can select an appropriate dosage form according to the administration route, the administration target, and the like.
The dose of the active ingredient of the pharmaceutical composition of the present invention varies depending on the subject, the target organ, the symptom, the administration method, and the like, and can be determined by the judgment of the doctor in consideration of the type of the formulation, the administration method, the age and weight of the patient, the symptom of the patient, and the like.
The pharmaceutical compositions of the invention may also contain other agents, including but not limited to cytotoxic, cytostatic, antiangiogenic or antimetabolic agents, tumor-targeting agents, immunostimulants or immunomodulators or TCRs in combination with cytotoxic, cytostatic or other toxic agents.
The technical scheme of the invention is further illustrated by the detailed description and the attached drawings, but the technical scheme can be understood by those skilled in the art: the following detailed description and examples are intended to illustrate the invention and should not be construed as limiting the invention in any way. It will be apparent to those skilled in the art that many modifications can be made to the present invention without departing from the spirit thereof, and such modifications are intended to be within the scope of the invention.
The following experimental methods are all conventional experimental methods in the art unless otherwise specified, and the experimental materials used are all experimental materials that can be easily obtained from commercial companies unless otherwise specified.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：具有人源化FC-γ受体的非人动物