CN111748029B - Specific T cell receptor aiming at EGFR L858R gene mutation and application thereof - Google Patents

Abstract

The invention providesbase:Sub>A specific TCR against EGFR L858R gene mutation, said TCR comprising the variable and constant regions of the alpha and beta chains, and the use thereof, said TCR having the property of binding to the neo-antigenic peptide kitfgrak-HLA-base:Sub>A 1101 complex derived from EGFR L858R gene mutation. T cells modified by the TCR have specific killing effect on HLA-A x 1101 tumor cells mutated by EGFR L858R genes. 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.

Description

Specific T cell receptor aiming at EGFR L858R gene mutation and application thereof

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) molecules-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 signal transmission occurs primarily through the CD3 molecule to which it is non-covalently bound. The TCR molecule belongs to the immunoglobulin superfamily, the antigen specificity of which resides in the V region; the V region has three hypervariable regions CDR1, CDR2 and CDR3, wherein the CDR3 variation is the largest, which directly determines the antigen binding specificity of the TCR. In the case where the TCR recognizes an MHC-antigen peptide complex, 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. T cells are activated by recognizing tumor cell surface antigen peptide-MHC complexes through specific T cell receptors, and the activated T cells can directly dissolve tumor cells or inhibit tumor growth 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 human body at present, and has dozens of gene loci, each gene locus has dozens of alleles, and the alleles are co-dominant expression. HLA has 5 sites of A, B, C, D and DR, which are respectively called HLA-A, HLA-B, HLA-C, HLA-D and HLA-DR.

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 EGFR and other protein tyrosine kinases or the activity or cellular localization of key factors in related signal pathways 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 EGFR-TKI such as Iressa. Generally, the exon 19 deletion of EGFR and the L858R mutation of exon 21 are sensitive to the iressa target. Therefore, the deletion of exon 19 and the mutation of L858R in 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 in 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.

Disclosure of Invention

The invention aims to provide a specific T cell receptor aiming at EGFR L858R gene mutation and application thereof.

The invention providesbase:Sub>A specific T cell receptor against EGFR L858R gene mutation, said T cell receptor being capable of binding to the kitfgrak-HLA-base:Sub>A 1101 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.

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

Furthermore, 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 mutated HLA-A1101 tumor cells.

An application of the T cell in the field of preparing antitumor drugs or drug combinations.

The T cell receptor and the TCR expression cell are applied to the preparation of products capable of specifically killing EGFR L858R mutant HLA-A1101 tumor cells.

An anti-neoplastic drug or drug combination comprising the TCR or a T cell expressing the TCR.

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

The anti-tumor medicament and the medicinal composition are used for treating malignant tumors related to EGFR L858R mutation expression.

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 pharmaceutical combination comprising administering to a subject at least one immune checkpoint inhibitor.

The methods may be administered simultaneously or sequentially in any order.

The beneficial effects of the invention are: 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 T cell receptor 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 embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 flow cytometry analysis of TCR-T cell TCR expression;

figure 2 flow cytometry analysis of target cells for HLA-base:Sub>A 1101 expression;

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

FIG. 4ELISA 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:

example 1 TCR Lentiviral preparation

Connecting coding sequences of TCR alpha chain and TCR beta chain with a F2A sequence through furin cutting sites, SGSG joints and the like, totally synthesizing alpha chain and beta chain genes, cloning the TCR genes into a lentivirus expression vector pCDH (purchased from SBI) by using restriction endonucleases EcoRI and BamHI, and obtaining recombinant plasmids which can express amino acid sequences of TCR alpha chain and beta chain variable regions shown in sequences 6 and 8.

The recombinant plasmid is transformed into XL-10 competent cells, evenly coated on an LB solid culture medium plate containing ampicillin, cultured for 12h at 37 ℃, then a single colony is picked up to an LB liquid culture medium containing ampicillin, shake-cultured for 14-16h at 37 ℃ and 220rpm/min, and the plasmid is extracted.

Packaging of the recombinant plasmid: 293T cells (purchased from the basic medicine cell center of the institute of basic medicine of Chinese academy of medical sciences) in the logarithmic growth phase were used as packaging cells, and inoculated into a T25 cell culture flask containing a medium (DMEM medium containing 10% FBS) and transfected when the cell confluence reached 80-90%. The recombinant plasmid and the mixed packaging vector plasmid pPACKH1 (purchased from SBI) are mixed uniformly, 500 microliter of 1 microgram/microliter liposome transfection reagent Lip2000 (Invitrogen, 11668-027) is added after the mixture is blown up and down by a pipette and is blown up and down by the pipette and is mixed uniformly, and the mixture is kept stand for 10-15 minutes at room temperature. The DNA/liposome compound is added into a culture dish drop by drop and mixed evenly. Placing the culture dish at 37 ℃ and 5% CO ₂ And (4) an incubator for 6-8 hours, removing the culture medium containing the transfection reagent, and replacing the culture medium with a fresh complete culture medium. After 48 hours, the culture broth was collected into a sterile centrifuge tube, centrifuged at 2000g for 10 minutes at 4 ℃, the supernatant was filtered through a 0.45 μm PES membrane, a fresh sterile ultracentrifuge tube was added to the virus-containing culture supernatant (i.e., filtrate), and centrifuged at 20,000g for 3 hours at 4 ℃. Carefully sucking the liquid in the centrifugal tube, obtaining the precipitate as the target lentivirus, adding 1mL of PBS buffer solution to resuspend the precipitate to obtain the lentivirus suspension, and storing at-80 ℃.

EXAMPLE 2 preparation of TCR-T cells

Peripheral blood from healthy volunteers was collected and isolated using lymphocyte separator (Stemcell, 07861, usa) to obtain human Peripheral Blood Mononuclear Cells (PBMCs). Dynabeads (Gibco, 11141D) were mixed with PBMC and incubated at room temperature for 20min to isolate activated T cells. 3mL of X-Vivo 15 medium (Lonza, DL-201) was added to the T cells, the mixture of cells and Dynabeads (Thermo, 11141D) was resuspended with a pipette, and the cell density was adjusted to 0.5 to 1X10 ⁶ Individual cells/mL, cell suspension was obtained. Subjecting the cell suspension to 37 ℃ and 5% CO ₂ After continuous culture in an incubator for 48h, the cell density was adjusted to 1x10 ⁶ one/mL. Removing lentivirus from-80 deg.C ultra-low temperature refrigerator, rapidly thawing in 37 deg.C water bath, adding into prepared T cells,polybrene (Santa Cruz, sc-134220) was added to a final concentration of 6. Mu.g/mL, the above lentivirus was added, gently blown and mixed well, and centrifuged at 800g for 1h at room temperature. Then placed at 37 ℃ and 5% CO ₂ The culture was continued for 24 hours, the virus-containing culture supernatant was removed, the cell pellet was resuspended in fresh medium, the cells were transferred to a new culture vessel and continued to be cultured, and the positive rate of TCR-T cells was indicated by FACS detection of the expression of TCR of the cells on day 4, see fig. 1. In FIG. 1A, T cells with untransfected TCR are shown, and in FIG. 1B, T cells with transfected TCR, the TCR transfection efficiency obtained by comparison is 17.3%.

EXAMPLE 3 preparation of target cells

Construction of lentiviral expression vector pCDH-A1101 overexpressing HLA-A1101 molecules, transient transfection of 293T cells, preparation of recombinant lentiviruses, as in example 1. The virus transduced T2 cell line, andbase:Sub>A T2 cell line expressing HLA-base:Sub>A 1101 was constructed according to the method of example 2. After labeling with antibodies to HLA molecules, expression of HLA-base:Sub>A 1101 on the surface of T2 cells was detected by FACS, see fig. 2. In FIG. 2A, T2 cells not transfected with HLA-A1101 and in FIG. 2B, T2 cells transfected with HLA-A1101, the transfection efficiency of HLA-A1101 was 96.6% compared.

Example 4 cytokine assay to detect killing of target cells by specific TCR-T cells

T2 cell lines lack the transporter associated with antigen processing (TAP) and can therefore be efficiently loaded with foreign peptides to serve as antigen presenting cells that present the loaded antigenic peptides for T cell recognition. The artificially synthesized mutant antigen peptide KITDGRAK and wild-type peptide (KITDGLAK) were subjected to the concentration reduction at 37 ℃ and 5% by CO in T2 cells obtained in example 3 ₂ Incubation under conditions for 24h (polypeptide concentration 50. Mu.g/ml, T2 cell concentration 1X10 ⁶ one/mL), washing to remove the unbound antigen peptide, and collecting the cells, namely the T2 cells loaded with the antigen peptide.

Specific TCR-T cells and antigen peptide KITDGRAK-loaded T2 cells at 37 ℃ with 5% CO ₂ The cells were incubated under the conditions for 24h, and the concentration of T2 cells was 2X 10 ⁴ piece/mL, effective target ratio (1, 2.5. The control cells were T2 cells and negative cells not loaded with antigen peptideT2 cells loaded with negative control antigen peptide (kitfglak). ELISA was performed to detect the expression of the cytokines IFN-. Gamma.and IL-2 in the co-culture system (Human IFN-. Gamma.ELISA Kit, human IL-2 ELISA Kit from David). The killing effect of specific TCR-T cells on target cells was evaluated, see figure 3, figure 4. FIG. 3 shows that after co-culturing TCR-T with T2 target cells, T2 cells incubated with mutant antigen peptides can specifically stimulate TCR-T to secrete IFN- γ, and T2 cells incubated with wild-type polypeptide and T2 cells alone cannot cause TCR-T to secrete IFN- γ. The TCR can specifically recognize the antigen peptide KITDGRAK, the T cell transfected with the TCR can secrete IFN-gamma after recognizing the target cell, and the secretion can reach 419pg/mL when the effective target ratio is 10. FIG. 4 shows that T2 cells incubated with mutant antigen peptide can specifically stimulate TCR-T to secrete IL-2 after TCR-T is co-cultured with T2 target cells, and T2 cells incubated with wild-type polypeptide and T2 cells alone cannot cause TCR-T to secrete IL-2. The TCR can specifically recognize the antigen peptide KITDGRAK, the T cell transfected with the TCR can secrete IL-2 after recognizing the target cell, and the secretion amount can reach 306pg/mL when the effective target ratio is 10.

Example 5 luciferase assay to detect killing of target cells by specific TCR-T cells

Based on the construction of the target cell line in example 3, target cells were transfected with a lentivirus carrying luciferase, and target cells stably expressing luciferase were selected for in vitro co-culture experiments. The luciferase catalyzes the oxidation reaction of a luciferin substrate to emit light, only living cells can emit light, and the intensity of the emitted light is linearly related to the number of the cells. The number of target cells is detected through fluorescence intensity, and the killing effect of the TCR-T on the target cells is reflected.

Using the TCR-T cells prepared above and target cells expressing luciferase, a co-culture system was established, and co-culture was continued for 24 hours according to different effective target ratios (1, 2.5; untransfected control T cells were used as controls. Using luciferase assay kit (ONE-Glo) ^TM Luciferase Assay System Promega E6110) detection of surviving tumor cells in the Co-culture System, aspiration of cell culture fluid, PBS gentlyThe cells were washed and 100. Mu.L of lysis solution was added. At room temperature, the cells were shaken gently for 15min to completely dissolve the cells, and the fluorescence intensity was measured at full wavelength for staining 3. The percent killing of the target cells by TCR-T was calculated. T cells that were not transfected with TCR were used as controls.

As shown in fig. 5, the results indicate that the obtained specific TCR-T cells can specifically kill T2 target cells loaded with the antigenic peptide kitfgrak, and the killing efficiency can reach 78% at an effective target ratio of 10.

The specific TCR provided by the invention for EGFR L858R gene mutation and the application thereof are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the principle of the invention, and these changes and modifications also fall into the protection scope of the claims of the present invention.

Sequence listing

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<120> specific T cell receptor aiming at EGFR L858R gene mutation and application thereof

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Claims (6)

1. base:Sub>A specific T cell receptor directed against EGFR L858R gene mutation, wherein said T cell receptor is capable of binding to the kitfgrak-HLA-base:Sub>A 1101 complex; the T cell receptor comprises an alpha chain and a beta chain, 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; 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.

2. A vector comprising a nucleic acid encoding the T cell receptor of claim 1.

3. The vector of claim 2, wherein the vector comprises a codon-optimized nucleic acid corresponding to the amino acid sequence encoding the T-cell receptor of claim 1.

4. A cell that expresses the T cell receptor of claim 1 on a cell membrane.

5. Use of the T cell receptor of claim 1 or the cell of claim 4 in the preparation ofbase:Sub>A product capable of specifically killing HLA-base:Sub>A 1101 tumor cells mutated at EGFR L858R.

6. An anti-neoplastic drug or drug combination comprising the T-cell receptor of claim 1 or the cell of claim 4.

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