阅读说明：本技术 一种针对egfr l858r基因突变的特异性t细胞受体及其应用 (Specific T cell receptor aiming at EGFR L858R gene mutation and application thereof ) 是由 杜学明 李凤娥 霍冲 邓丽刚 邹庆薇 王亚玲 于 2019-04-25 设计创作，主要内容包括：本发明提供一种针对EGFR L858R基因突变的特异性TCR及其应用,所述TCR具有结合衍生自EGFR L858R基因突变的新抗原肽KITDFGRAK-HLA-A*1101复合物的特性,所述TCR包含α链和β链的可变区和恒定区。经过该类TCR修饰的T细胞具有对EGFR L858R基因突变的HLA-A*1101肿瘤细胞的特异性杀伤作用。另外,本发明还提供了用于治疗涉及表达所述基因突变的肿瘤的药物组合,具有特异性强,个体化治疗效果好的特点。(The invention provides a specific TCR against EGFR L858R gene mutation and uses thereof, the TCR having the property of binding to a neo-antigenic peptide KITDFGRAK-HLA-A1101 complex derived from EGFR L858R gene mutation, the TCR comprising variable and constant regions of alpha and beta chains. T cells modified by the TCR have specific killing effect on HLA-A1101 tumor cells mutated by EGFR L858R gene. In addition, the invention also provides a drug combination for treating tumors related to the expression of the gene mutation, and the drug combination has the characteristics of strong specificity and good individualized treatment effect.)

1. A specific T cell receptor directed against a mutation in the EGFR L858R gene, wherein said T cell receptor is capable of binding to the KITDFGRAK-HLA-a 1101 complex.

2. The specific T cell receptor for mutation of EGFR L858R gene according to claim 1, comprising an alpha chain and a beta chain, wherein the alpha chain CDR3 region is an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the sequence shown in SEQ ID NO. 02 and having the same function; or the amino acid sequence of the alpha chain CDR3 region is the sequence shown in SEQ ID NO. 02.

3. The specific T cell receptor for EGFR L858R gene mutation according to claim 2, wherein the variable region sequence of the alpha chain comprises CDR3, and the amino acid sequence of the variable region sequence is the sequence shown in SEQ ID NO. 06.

4. The specific T cell receptor for EGFR L858R gene mutation according to claim 1, 2 or 3, wherein the beta-chain CDR3 region is an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the sequence shown in SEQ ID NO. 04 and having the same function, or the amino acid sequence of the beta-chain CDR3 region is the sequence shown in SEQ ID NO. 04.

5. The specific T cell receptor for EGFR L858R gene mutation according to claim 4, wherein the variable region sequence of the beta chain comprises CDR3, and the amino acid sequence of the variable region sequence is the sequence shown in SEQ ID NO. 08.

6. The specific T cell receptor for EGFR L858R gene mutation according to claim 1, 2, 3, or 5, being in the form of an α -chain and β -chain heterodimer structure.

7. A vector comprising a codon-optimized nucleotide sequence corresponding to a nucleotide sequence or an amino acid sequence thereof encoding said specific T cell receptor for EGFR L858R gene mutation.

8. A cell expressing said specific T cell receptor for EGFR L858R gene mutation in the cell membrane.

9. Use of a specific T cell receptor directed against mutations in the EGFR L858R gene for the manufacture of a medicament or combination of medicaments for specifically killing EGFR L858R mutated HLA-a 1101 tumor cells.

10. An anti-tumor drug or drug combination, which comprises the specific T cell receptor aiming at the EGFR L858R gene mutation or a cell expressing the specific T cell receptor aiming at the EGFR L858R gene mutation on the cell membrane.

Technical Field

The invention relates to the field of genetic engineering and tumor immunotherapy, in particular to a specific T cell receptor aiming at EGFR L858R gene mutation and application thereof.

Background

Tumor immunotherapy is a therapeutic approach to control and eliminate tumors by restarting and maintaining the tumor-immune cycle, restoring the body's normal anti-tumor immune response. Tcr (T cell receptor) therapy, a T cell receptor therapy in which endogenous T cells are isolated, engineered, and infused back into the human body. As a result, the number of T cells with the ability to target cancer cells will increase. This approach avoids the delayed effects of vaccine and immune checkpoint inhibitor therapies, in addition to the ability to rapidly kill tumors like cytotoxic chemotherapy and targeted therapies. TCR-T is one of the most promising tumor treatment technologies after tumor surgery, radiotherapy, chemotherapy and targeted therapy, and is a research hotspot of current tumor immunotherapy both internationally and domestically.

T cell receptors (TCR for short) are characteristic markers of all T cell surfaces and have the ability to recognize human Major Histocompatibility Complex (MHC) molecule-antigenic peptide complexes on Antigen Presenting Cells (APC). The TCR is a heterodimer formed by alpha and beta peptide chains, and each peptide chain is divided into a variable region (V region), a constant region (C region), a transmembrane region, a cytoplasmic region and the like; the cytoplasmic domain is short and signaling is mainly through CD3 molecules that are non-covalently bound to it. The TCR molecule belongs to the immunoglobulin superfamily, and the antigen specificity exists in the V region; the V regions in turn each have three hypervariable regions CDR1, CDR2, CDR3, of which the largest CDR3 variation directly determines the antigen binding specificity of the TCR. In the case of TCR recognition of MHC-antigen peptide complexes, CDR3 can be directly bound to the antigen peptide.

In recognition of antigenic peptides presented by MHC molecules on APCs or target cells, TCRs recognize both antigenic peptides and polymorphic portions of self-MHC molecules, which is MHC restriction. The T cells are activated by recognizing the tumor cell surface antigen peptide-MHC complex through a specific T cell receptor, and the activated T cells can directly dissolve the tumor cells or inhibit the growth of tumors by secreting cytokines such as interferon, tumor necrosis factor and the like. CD8+ T cell-mediated specific MHC-class I molecules limit cellular immune function, and are particularly important in anti-tumor immunity.

HLA (human lymphocyte antigen), MHC-controlled gene cluster, is highly polymorphic alloantigen, is the most complex genetic polymorphism system of the human body known at present, has dozens of gene loci, each gene locus has dozens of alleles, and is expressed in codominance. HLA has A, B, C, D and DR 5 sites, which are called HLA-A, HLA-B, HLA-C, HLA-D and HLA-DR, respectively.

The egfr (epidermal growth factor receptor family) human epidermal growth factor receptor belongs to the tyrosine kinase receptor family, also known as the HER family or erbB family. The EGFR signaling pathway plays an important role in physiological processes such as growth, proliferation and differentiation of cells. The functional deficiency of protein tyrosine kinases such as EGFR and the like or the activity or the cellular localization abnormality of key factors in related signal paths of the protein tyrosine kinases can cause the occurrence of tumors, diabetes, immunodeficiency and cardiovascular diseases. EGFR induces cancer through at least 3 mechanisms: overexpression of EGFR ligand, amplification of EGFR, or mutational activation of EGFR, with mutational activation of EGFR being the primary mechanism. The EGFR gene is composed of 28 exons, wherein exons 18-21 are common gene mutation sites and are sites for combining with EGFR-TKI such as Iressa. Generally, both exon 19 deletion and L858R mutations of exon 21 of EGFR are sensitive to the iressa target. Therefore, the deletion of exon 19 and the L858R mutation of exon 21 of EGFR can be used as an important criterion for determining the suitability of EGFR-TKI for tumor patients, wherein the mutation of L858R gene, i.e., the mutation of the 858 th amino acid of EGFR protein from L (leucine) to R (arginine), is included.

Tumor cells undergo a number of genetic mutations, some of which alter the amino acid coding sequence, resulting in tumor cells expressing abnormal proteins not found in normal cells. These abnormal proteins are proteolytically cleaved into peptide fragments (epitopes) in cells (tumor cells or antigen presenting cells), are bound to MHC-class I or MHC-class II molecules with high affinity, are presented on the cell surface in the form of complexes, are bound to T cell receptors, and T cells are activated; the activated T cells expand, infiltrate the tumor microenvironment, and recognize and kill the tumor cells. This abnormal protein specific to tumor cells is called neoantigen (neoantigen).

The TCR-T cell therapy comprises key technologies and therapeutic means such as selection of tumor specific TCR, construction of TCR expression vectors, cell feedback after TCR-T modification, immune process monitoring and the like. Compared with the same type of CAR-T cell therapy technology, TCR-T has wider antigen selection space, so that the applicable tumor range can be expanded, and off-target effect can be reduced. However, since TCR-T development is still in its infancy, effective TCRs against specific mutations and correspondingly typed patient populations are still very deficient. At the same time, the effective patient population of TCR-T and the therapeutic efficacy of the new construct TCR-T remain to be verified.

Disclosure of Invention

The present invention is directed to solving, to some extent, one of the technical problems in the related art described above. Therefore, the invention aims to provide a specific TCR aiming at EGFR L858R gene mutation and application thereof.

The invention provides a specific T cell receptor aiming at EGFR L858R gene mutation, and the T cell receptor can be combined with KITDFGRAK-HLA-A1101 complex.

Wherein the T cell receptor comprises an alpha chain and a beta chain, and the alpha chain CDR3 region is an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the sequence of SEQ ID NO. 02 and having the same function.

Further, the amino acid sequence of the alpha chain CDR3 region is the sequence shown in SEQ ID NO. 02.

Further, the nucleotide sequence of the alpha chain CDR3 region is the sequence shown in SEQ ID NO. 01.

Wherein the variable region sequence of the alpha chain comprises CDR3, and the amino acid sequence of the variable region sequence of the alpha chain is the sequence shown in SEQ ID NO. 06.

Further, the nucleotide sequence of the variable region sequence of the alpha chain is the sequence shown in SEQ ID NO. 05.

Wherein the T cell receptor beta chain CDR3 region is an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the sequence of SEQ ID NO. 04 and having the same function.

Further, the amino acid sequence of the beta chain CDR3 region is the sequence shown in SEQ ID NO. 04.

Further, the nucleotide sequence of the beta chain CDR3 region is the sequence shown in SEQ ID NO. 03.

The variable region sequence of the beta chain comprises CDR1, CDR2 and CDR3, wherein in the nucleotide of the CDR3 region of the beta chain, the variable region sequence of the beta chain comprises CDR3, and the amino acid sequence of the variable region sequence is the sequence shown in SEQ ID NO. 08.

Further, the nucleotide sequence of the variable region sequence of the beta chain is a sequence shown as SEQ ID NO: 07.

Wherein, the T cell receptor is a heterodimer structure of an alpha chain and a beta chain, and the constant region sequences of the alpha chain and the beta chain can be human-derived or murine-derived constant region sequences.

The invention provides a vector which contains the nucleic acid molecule of the TCR sequence or the nucleic acid molecule with optimized codons corresponding to the amino acid sequence of the TCR sequence.

Preferably, the vector is a viral vector.

More preferably, the vector is a lentiviral vector.

The invention also provides a cell expressing the T cell receptor on the cell membrane.

Preferably, the cell is a T cell.

More preferably, the cells are CD8 positive T cells.

Wherein the T cells have specific killing effect on EGFR L858R mutant HLA-A1101 tumor cells.

An application of T cells in the field of preparing anti-tumor drugs or drug combinations.

The T cells can be used for preparing T cell receptors.

The T cell receptor and the TCR-expressing cells are used for preparing a medicament or a medicament combination capable of specifically killing EGFR L858R mutant HLA-A1101 tumor cells.

The anti-tumor drug or drug combination comprises the TCR or the T cell expressing the TCR.

The antineoplastic agent or combination of agents is used for treating HLA-A1101 patients.

The anti-tumor drug and the drug combination are used for treating malignant tumors which are involved in expressing EGFR L858R mutation.

The tumor includes lung cancer, central nervous system tumor, colorectal cancer, gastric cancer, endometrial cancer, etc.

A method for treating cancer, administering a therapeutically effective amount of the prepared anti-tumor drug or drug combination to a mammal.

The method for treating cancer comprises the step of administering the cells expressing the TCR, one or more of chemotherapeutic drugs, targeted therapeutic drugs and immune checkpoint inhibitor drugs to a subject.

The method for treating cancer can be used for simultaneously or sequentially administering one or more of TCR-expressing cells, chemotherapeutic drugs, targeted therapeutic drugs and immune checkpoint inhibitor drugs in any order.

The invention has the beneficial effects that:

the infusion of genetically modified T cells capable of recognizing specific targets confers new non-innate immune activity to the immune system.

The antitumor drug and the drug combination prepared by applying the TCR or the T cell provided by the invention have the characteristics of strong specificity and good individualized treatment effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1A flow cytometry analysis of T cells without TCR transfection;

FIG. 1B flow cytometry analysis of TCR transfected T cells;

figure 2A flow cytometry analysis of T2 cells that were not transfected with HLA-a 1101;

FIG. 2B flow cytometry analysis of HLA-A1101 transfected T2 cells;

FIG. 3 ELISA detection of IFN- γ expression after TCR-T cell incubation with target cells;

FIG. 4 ELISA detects IL-2 expression after TCR-T cells are incubated with target cells;

FIG. 5 luciferase assay detects the rate of killing of specific TCR-T cells against target cells.

The specific implementation mode is as follows: