阅读说明：本技术 一种t细胞受体及其应用 (T cell receptor and application thereof ) 是由 王智鼎 于川 李宜声 侯晨芳 于 2020-06-17 设计创作，主要内容包括：本发明公开了一种T细胞受体及其应用,表达该T细胞受体的表达载体和宿主细胞,编码该T细胞受体的核酸；以及用于治疗肿瘤或自身免疫疾病的组合物。(The invention discloses a T cell receptor and application thereof, an expression vector and a host cell for expressing the T cell receptor, and nucleic acid for encoding the T cell receptor; and compositions for treating tumors or autoimmune diseases.)

1. A T cell receptor comprising a TCR β chain variable domain having a CDR3 β amino acid sequence according to any one of sequences SEQ ID nos. 7 to 11.

2. The T cell receptor according to claim 1, wherein the TCR β chain variable domain has the amino acid sequence CDR1 β, CDR2 β according to the sequences SEQ ID No.5, SEQ ID No. 6.

3. The T cell receptor according to claim 1, wherein the TCR alpha chain variable domain has an amino acid sequence according to CDR1 a, CDR2 a, CDR3 a of the sequences SEQ ID No.1, SEQ ID No.2, SEQ ID No. 3.

4. A T cell receptor according to any one of claims 1 to 3 wherein the TCR alpha chain variable domain has an amino acid sequence according to sequence SEQ ID No. 1.

5. A T cell receptor according to any one of claims 1 to 3 wherein the TCR β chain variable domain has an amino acid sequence according to any one of sequences SEQ ID No.12 to 16.

6. A nucleic acid molecule comprising a nucleotide sequence encoding the T cell receptor of any one of claims 1-5.

7. An expression vector comprising the nucleic acid molecule of claim 6; preferably, the vector is a viral vector; preferably, the viral vector is a lentiviral vector or a gammaretrovirus vector.

8. A host cell comprising the T cell receptor of any one of claims 1-5, the nucleic acid molecule of claim 6, the expression vector of claim 7; preferably, the host cell comprises a hematopoietic progenitor cell or an immune system cell; preferably, the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD 8-double negative T cell, a gamma T cell, a natural killer cell, a dendritic cell, or any combination thereof; preferably, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

9. A composition comprising the T cell receptor of any one of claims 1-5, the expression vector of claim 7, or the host cell of claim 8.

10. Use according to any one of the following:

(1) use of a T cell receptor according to any one of claims 1 to 5 in the preparation of a TCR-T;

(2) use of a T cell receptor according to any one of claims 1 to 5 for the preparation of a chimeric antigen receptor;

(3) use of the T cell receptor of any one of claims 1-5, the nucleic acid molecule of claim 6, the expression vector of claim 7, the host cell of claim 8, or the composition of claim 9 for the preparation of a product for enhancing immunity;

(4) use of the T cell receptor of any one of claims 1-5, the nucleic acid molecule of claim 6, the expression vector of claim 7, the host cell of claim 8, or the composition of claim 9 for the preparation of a medicament for the treatment of a disease; preferably, the disease is an autoimmune disease; preferably, the disease is cancer.

(5) Use of the T cell receptor of any one of claims 1-5, the nucleic acid molecule of claim 6, the expression vector of claim 7, the host cell of claim 8, or the composition of claim 9 for the preparation of an antigen detection product.

Technical Field

The invention belongs to the fields of cellular immunology and genetic engineering, and relates to a T cell receptor and application thereof.

Background

T cell-related immunotherapy has been a major debate in recent cancer research, and the "force" is both CAR-T and TCR-T technologies. TCR-T therapy is of relatively low interest relative to CAR-T cell therapy.

The TCR-T therapy improves the affinity and the fighting power of TCR (T cell receptor) that specifically recognizes a relevant antigen by transducing a chimeric antigen receptor or TCR α/β heterodimer, enabling T lymphocytes to efficiently recognize a target cell again. TCR-T therapy confers new non-innate immune activity to the immune system by infusing genetically modified T lymphocytes that are capable of recognizing specific targets. This approach avoids the delayed effects of vaccine and T lymphocyte checkpoint therapies, in addition to killing tumors as rapidly as chemotherapy and targeted therapies.

Traditional adoptive immunotherapy only increases the number of effector cells, does not improve the specificity of effector cells, and has low affinity even if the effector cells can bind to tumor cells. The TCR-T therapy directly modifies a probe (TCR) of a T cell combined with a tumor antigen, strengthens the specific recognition process of the T cell for the tumor cell, improves the affinity of the T lymphocyte for the tumor cell, and enables the original T cell without tumor recognition capability to effectively recognize and kill the tumor cell. The clinical efficacy of TCR-T therapy is relatively low, so that the search for effective TCR receptors with high affinity for tumor target antigens and the optimization of the transformation efficiency of TCR-T cells are the key points of the current research.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide a TCR receptor.

Another object of the present invention is to provide a TCR-modified cell.

It is a further object of the present invention to provide a composition comprising a TCR receptor or TCR-modified cell and its use in the treatment and diagnosis of disease.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect the invention provides a T cell receptor comprising a TCR β chain variable domain having a CDR3 β amino acid sequence according to any one of sequences SEQ ID nos. 7 to 11.

Further, the variable domain of the TCR β chain has the amino acid sequence of CDR1 β, CDR2 β according to the sequences SEQ ID No.5, SEQ ID No. 6.

Further, the TCR α chain variable domain has the CDR1 α, CDR2 α, CDR3 α amino acid sequence according to sequences SEQ ID No.1, SEQ ID No.2, SEQ ID No. 3.

Further, the variable domain of the TCR alpha chain has an amino acid sequence according to sequence SEQ ID No. 1.

Further, the variable domain of the TCR beta chain has an amino acid sequence according to any one of the sequences SEQ ID No.12 to 16.

"T cell receptor" (TCR) refers to a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region and a short cytoplasmic tail region) capable of specifically binding antigenic peptides that bind to MHC receptors, TCR being found on the cell surface or in soluble form and typically consisting of heterodimers with alpha and beta chains (also known as TCR alpha and TCR beta, respectively) or gamma and chains (also known as TCR gamma and TCR, respectively.) like immunoglobulins, the extracellular portion of a TCR chain (e.g., alpha chain, beta chain) contains two immunoglobulin domains, one variable domain at the N-terminus (e.g., alpha chain variable domain or V alpha, beta chain variable domain or V beta), and one constant domain adjacent to the cell membrane (e.g., alpha chain constant domain or C alpha, based on Kabat typically amino acids 117 to 259, beta chain domain or C beta, based on Kabat typically amino acids 117 to 295.) like immunoglobulins, the variable domains contain Complementarity Determining Regions (CDRs) separated by Framework Regions (FRs). The V α and V β of the original TCR generally have similar structures, with each variable domain comprising four conserved FRs and three CDRs. The V.alpha.domain is encoded by two separate DNA segments, a variable gene segment and a linker gene segment (V-J); the V.beta.domain is encoded by three separate DNA segments, namely, a variable gene segment, a diversity gene segment, and a connecting gene segment (V-D-J). A single va or V β domain may be sufficient to confer antigen binding specificity. In addition, the va or ν β domains from a TCR can be used to isolate TCRs that bind to a particular antigen to screen a library of complementary va or ν β domains, respectively. In certain embodiments, the TCR is found on the surface of a T cell (or T lymphocyte) and is associated with a CD3 complex. The TCR sources used in the present disclosure may be from various animal species, such as human, mouse, rat, rabbit, or other mammals.

In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a T cell receptor according to the first aspect of the invention.

The nucleic acid may be single-or double-stranded DNA, cDNA or RNA in any form, and may include the positive and negative strands of nucleic acid that are complementary to each other, including antisense DNA, cDNA and RNA.

As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" refers to any deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments produced, for example, by the Polymerase Chain Reaction (PCR), or by in vitro translation, and fragments produced by any ligation, fragmentation, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., the α -enantiomeric form of a naturally occurring nucleotide), or a combination of both. The modified nucleotides may have modifications or substitutions of sugar moieties or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphordiselenoate, phosphoroanilino phosphorothioate, phosphoroanilino, phosphoroamidate, and the like. The nucleic acid molecule may be single-stranded or double-stranded.

In a third aspect the present invention provides an expression vector comprising a nucleic acid molecule according to the second aspect of the invention. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule. The vector may be, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule, which may include chromosomal, nonchromosomal, semisynthetic, or synthetic nucleic acid molecules. Exemplary vectors are vectors (expression vectors) capable of autonomous replication (episomal vectors) or expression of the nucleic acid molecules to which they are linked.

Further, the vector is a viral vector. Viral vectors include, but are not limited to, retroviruses; an adenovirus; parvoviruses (e.g., adeno-associated viruses); a coronavirus; negative strand RNA viruses, such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and sendai); positive strand RNA viruses, such as picornaviruses and alphaviruses; and double-stranded DNA viruses including adenoviruses, herpes viruses (e.g., herpes simplex virus types 1 and 2, epstein-barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus (Norwalk virus), togavirus, flavivirus, reovirus, papova virus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian type C, type B viruses, type D viruses, HTLV-BLV group, lentiviruses, foamy viruses.

Further, the viral vector is a lentiviral vector or a gamma retroviral vector.

In a fourth aspect, the present invention provides a host cell comprising a T cell receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, and an expression vector according to the third aspect of the invention.

Further, the host cell includes hematopoietic progenitor cells or cells of the immune system. A "hematopoietic progenitor cell" is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into a mature cell type (e.g., an immune system cell). By "immune system cell" is meant any cell of the immune system that is derived from a hematopoietic stem cell in the bone marrow, giving rise to two major lineages, namely myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells such as T cells, B cells, and natural killer cells (NK).

Further, the immune system cells are CD4+ T cells, CD8+ T cells, CD4-CD 8-double negative T cells, gamma T cells, natural killer cells, dendritic cells, or any combination thereof.

Further, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

A "T cell" is an immune system cell that matures in the thymus and produces a T Cell Receptor (TCR). T cells can be naive (no exposure to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA, and decreased expression of CD45RO compared to TCM), memory T cells (TM) (undergoing antigen and long life) and effector cells (undergoing antigen, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 compared to naive T cells or TCM). Effector T cells (TEs) refer to CD8+ cytotoxic T lymphocytes that undergo antigen, have reduced expression of CD62L, CCR7, CD28 compared to TCM, and are positive for granzyme and perforin. Other exemplary T cells include regulatory T cells such as CD4+ CD25+ (Foxp3+) regulatory T cells and Treg17 cells, as well as Tr1, Th3, CD8+ CD 28-and Qa-1 restricted T cells.

A fifth aspect of the invention provides a composition comprising a T cell receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, an expression vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention.

A sixth aspect of the invention provides the use of any one of:

(1) use of a T cell receptor according to the first aspect of the invention in the preparation of a TCR-T;

(2) use of a T cell receptor according to the first aspect of the invention in the preparation of a chimeric antigen receptor;

(3) use of the T cell receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the expression vector of the third aspect of the invention, the host cell of the fourth aspect of the invention or the composition of the fifth aspect of the invention in the manufacture of a product for enhancing immunity;

(4) use of the T cell receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the expression vector of the third aspect of the invention, the host cell of the fourth aspect of the invention or the composition of the fifth aspect of the invention in the manufacture of a medicament for the treatment of a disease;

(5) use of a T cell receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, an expression vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a composition according to the fifth aspect of the invention for the preparation of an antigen detection product.

Further, the disease is an autoimmune disease.

Further, the disease is cancer.

In the present invention, in addition to a vector, certain embodiments relate to a host cell comprising a vector disclosed herein. Those skilled in the art will readily appreciate that many suitable host cells are available in the art. Host cells can include any acceptable carrier or nucleic acid and/or protein incorporation of individual cells or cell culture, as well as any progeny cells. The term also encompasses progeny of the host cell, whether genetically or phenotypically identical or different. Suitable host cells depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells can be induced to incorporate the vector or other material by using viral vectors, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods.

Cells (e.g., T cells) can be transfected or transduced using any suitable method, or nucleic acids or compositions of the methods of the invention can be administered. Known methods for delivering nucleic acids to host cells include, for example, the use of cationic polymers, lipid-like molecules, and certain commercial products, such as IN-VIVO-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated transfer, and the like. Still other methods of transfecting or transducing host cells employ vectors, which are described in further detail herein.

As an alternative embodiment, the composition of the invention further comprises a pharmaceutically acceptable carrier, diluent or excipient. Suitable excipients include water, saline, dextrose, glycerol, and the like, and combinations thereof. In embodiments, the composition comprising a T cell receptor or host cell as disclosed herein further comprises a suitable infusion medium. Suitable infusion media may be any isotonic medium formulation, physiological saline, Normosol R (Abbott) or Plasma-Lite A (Baxter), 5% dextrose in water, Ringer's lactate may generally be used. The infusion medium may be supplemented with human serum albumin or other human serum components. The compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to maintain stability of the formulation until infused into a patient.

An "effective amount" of a composition is that amount which is sufficient to achieve the desired therapeutic result or beneficial treatment at the necessary dosage and for the necessary time, as described herein. An effective amount may be delivered in one or more administrations. The term "therapeutic amount" may be used for treatment if administered to a subject known or confirmed to have a disease or disease state, while a "prophylactically effective amount" may be used to describe the administration of an effective amount to a subject susceptible to or at risk of having a disease or disease state (e.g., relapse) as a prophylactic process.

The composition may be administered in a manner appropriate to the disease or condition to be treated (or prevented), as determined by one of skill in the medical arts. The appropriate dosage of the composition, as well as the appropriate duration and frequency, will be determined by factors such as: the health of the patient, the size (i.e., weight, mass, or body weight) of the patient, the type and severity of the condition of the patient, the particular form of the active ingredient, and the method of administration. Generally, the appropriate dosage and treatment regimen provide the composition in an amount sufficient to provide a therapeutic and/or prophylactic benefit (e.g., as described herein, including improved clinical outcomes, such as more frequent complete or partial remission, or longer periods of no disease and/or overall survival, or reduction in severity of symptoms). For prophylactic use, the dosage should be sufficient to prevent, delay the onset of, or lessen the severity of the disease or condition-related disorder. The prophylactic benefit of a composition administered according to the methods described herein can be determined by conducting preclinical (including in vitro and in vivo studies) and clinical studies and analyzing the data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques.

Suitable dosing and treatment regimens are developed for using the particular compositions described herein in a variety of treatment regimens, including, for example, parenteral or intravenous administration or formulation. If the compositions of the present invention are administered parenterally, the compositions may also include sterile aqueous or oily solutions or suspensions. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline solution, 1, 3-butanediol, ethanol, propylene glycol or a mixture of polyethylene glycol and water. The aqueous solution or suspension may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained release formulations and formulations. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of modified cells or active compound calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier.

As used herein, administration of a composition refers to its delivery to a subject regardless of the route or mode of delivery. Administration may be continuous or intermittent, and may be parenteral. Administration may be for treating a subject who has been identified as having a recognized condition, disease, or disease state, or for treating a subject who is susceptible to or suffering from such a condition, disease, or disease state. Co-administration with adjuvant therapy may include the simultaneous and/or sequential delivery of multiple agents (e.g., modified cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose mycophenolic acid prodrugs, HDAC inhibitors, DNA hypomethylation agents, or any combination thereof) in any order and according to any dosing schedule.

Drawings

FIG. 1 is a TCR-T killing function assay.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.