CN111848781A

CN111848781A - Specific T cell receptor aiming at EGFR L858R gene mutation and application thereof

Info

Publication number: CN111848781A
Application number: CN201910338977.7A
Authority: CN
Inventors: 杜学明; 李凤娥; 霍冲; 邓丽刚; 邹庆薇; 王亚玲
Original assignee: Tianjin Hengjia Biotechnology Development Co ltd
Current assignee: Tianjin Hengjia Biotechnology Development Co ltd
Priority date: 2019-04-25
Filing date: 2019-04-25
Publication date: 2020-10-30

Abstract

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.

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) 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:

example 1 TCR Lentiviral preparation

The coding sequences of the TCR alpha chain and the TCR beta chain are connected with the F2A sequence through furin cutting sites, SGSG joints and the like, alpha chain and beta chain genes are completely synthesized, the TCR genes are cloned into a lentivirus expression vector pCDH (purchased from SBI) by using restriction endonucleases EcoRI and BamHI, and recombinant plasmids are obtained, and the recombinant plasmids can express the amino acid sequences of the TCR alpha chain and the beta chain variable region shown in the 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 12 h at 37 ℃, then a single colony is picked up to an LB liquid culture medium containing ampicillin, shake-cultured for 14-16 h at 37 ℃ and 220rpm/min, and the plasmid is extracted.

Packaging of the recombinant plasmid: 293T cells in a logarithmic growth phase (purchased from the basic medicine cell center of the institute of basic medicine of Chinese academy of medical science) are taken as packaging cells, inoculated into a T25 cell culture bottle containing a culture medium (DMEM culture medium containing 10% FBS), and transfected when the cell confluence reaches 80-90%. The recombinant plasmid and the mixed packaging vector plasmid pPACKH1 (purchased from SBI) are mixed uniformly, 500 muL of 1 mug/muL liposome transfection reagent Lip2000 (Invitrogen, 11668) is added after the mixture is blown up and down by a pipette and is fully mixed uniformly, the mixture is immediately blown up and down by a pipette and is mixed uniformly, and the mixture is kept stand for 10 to 15 minutes at room temperature. The DNA/liposome complex is added into a culture dish drop by drop and mixed evenly. After 6-8 hours of incubation in a 37 ℃ 5% CO2 incubator, the medium containing the transfection reagent was removed and replaced with fresh complete medium. After 48 hours, the culture medium was collected into a sterile centrifuge tube, centrifuged at 4 ℃ and 2000g for 10 minutes, the supernatant was filtered through a 0.45 μm PES membrane, a new sterile ultracentrifuge tube was added to the virus-containing culture medium supernatant (i.e., filtrate), and centrifuged at 4 ℃ and 20,000g for 3 hours. 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 and flow cytometry analysis of TCR expression 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 1X10⁶Individual cells/mL, cell suspension was obtained. Placing the cell suspension at 37 deg.C and 5% CO₂After continuous culture in an incubator for 48 h, the cell density was adjusted to 1X10⁶one/mL. The lentivirus was removed from an ultra-low temperature refrigerator at-80 ℃ and rapidly thawed in a water bath at 37 ℃, polybrene (Santa Cruz, sc-134220) was added to the prepared T cells to a final concentration of 6. mu.g/mL, the lentivirus was added thereto, blown gently and thoroughly mixed, and centrifuged at 800 g at room temperature for 1 hour. Culturing in an incubator at 37 deg.C and 5% CO2 for 24 hr, removing virus-containing culture medium supernatant, re-suspending the cell pellet with fresh culture medium, transferring the cells to a new culture vessel, culturing, and detecting TCR expression by FACS on day 4 Indicating the positive rate of TCR-T cells, see figure 1. In FIG. 1A, T cells with untransfected TCR and FIG. 1B, T cells with transfected TCR resulted in a 15.1% TCR transfection efficiency comparison.

EXAMPLE 3 preparation of target cells

Construction of lentiviral expression vector pCDH-A1101 overexpressing HLA-A1101 molecule, transient transfection of 293T cells, preparation of recombinant lentivirus, the procedure was the same as in example 1. The virus transduced T2 cell line and constructed T2 cell line expressing HLA-a 1101 as per example 2. After labeling with antibodies to HLA molecules, expression of HLA-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 gave a transfection efficiency of 96.6% for HLA-A1101 in comparison.

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

The T2 cell line lacks a 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 KITDFGRAK and wild-type peptide (KITDFGLAK) were incubated with the T2 cells obtained in example 3 at 37 ℃ and 5% CO2 for 24 h (the concentration of the polypeptide was 50. mu.g/ml and the concentration of the T2 cells was 1X 10) ⁶Pieces/ml), washing to remove unbound antigen peptide, and collecting the cells, namely the T2 cells loaded with the antigen peptide.

The specific TCR-T cells and T2 cells loaded with the antigenic peptide KITDFGRAK are incubated for 24 hours at 37 ℃ and 5% CO2, and the concentration of the target cells is 2 multiplied by 10⁴Number/ml, effective target ratio (1: 1, 2.5:1, 5:1, 10: 1). The control cells were T2 cells not loaded with the antigen peptide and T2 cells loaded with the negative control antigen peptide (KITDFGLAK). 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 T2 cells incubated with mutant antigenic peptides specifically stimulate TCR-T to secrete IFN-. gamma.after co-culture of TCR-T with T2 target cells, incubated with wild-type polypeptideT2 cells and T2 cells alone were unable to cause IFN-. gamma.secretion by TCR-T. The TCR can specifically recognize the antigen peptide KITDFGRAK, the T cell transfected with the TCR can secrete IFN-gamma after recognizing the target cell, and the secretion amount can reach 685pg/mL when the effective target ratio is 10:1, so that the TCR can kill the target cell. FIG. 4 shows that T2 cells incubated with the 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 and T2 cells alone, which are incubated with the wild-type polypeptide, cannot cause TCR-T to secrete IL-2. The TCR can specifically recognize the antigen peptide KITDFGRAK, the T cell transfected with the TCR can secrete IL-2 after recognizing the target cell, and the secretion amount can reach 422pg/mL when the effective target ratio is 10:1, so that the TCR has the effect of killing the target cell.

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 TCR-T on the target cells is reflected.

Establishing a co-culture system by using the prepared TCR-T cells and target cells expressing luciferase, and continuously co-culturing for 24 hours according to different effective target ratios (1: 1, 2.5:1, 5:1 and 10: 1); untransfected control T cells were used as controls. Luciferase activity of tumor cells surviving in the co-culture System was detected using a Luciferase Assay kit (ONE-Glo ™ Assay System Promega E6110), cell culture fluid was aspirated, cells were gently washed with PBS, and 100. mu.l of lysate 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 of catalysis 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 show that the obtained specific TCR-T cells can specifically kill T2 target cells loaded with the antigenic peptide KITDFGRAK, and the killing efficiency can reach 82% when the effective target ratio is 10: 1.

The specific TCR aiming at EGFR L858R gene mutation and the application thereof provided by the invention 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 present invention, and these changes and modifications also fall into the protection scope of the appended claims.

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Claims

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.