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Screening and anti-tumor application of KRAS mutation specific T cell receptor

A cell receptor and specific technology, applied in the field of medicine, can solve the problems of lack of detection and treatment of tumor drugs

Active Publication Date: 2021-02-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no relevant therapeutic drug for other KRAS mutations, and there is also a lack of a tumor drug that uses the body's immune mechanism for detection and treatment

Method used

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  • Screening and anti-tumor application of KRAS mutation specific T cell receptor
  • Screening and anti-tumor application of KRAS mutation specific T cell receptor
  • Screening and anti-tumor application of KRAS mutation specific T cell receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. KRAS-G12 mutant polypeptide-specific T cell sorting and TCR gene cloning

[0073] In this example, HLA-A11-restricted epitope polypeptides predicted to have KRAS-G12 mutations were first synthesized, and these polypeptides were used to immunize mice, and T cell response screening was performed by ELISPOT experiments, and the immunogenic mutant polypeptides were selected. At the same time, tetramers of these KRAS mutant polypeptides and HLA-A11 were prepared, and CD3 was selected from splenocytes of immunized mice by staining with CD3 and CD8 antibodies. + CD8 + The T cells were sorted to obtain the KRAS-G12 mutant polypeptide-specific T cells.

[0074] 1. Prediction of HLA-restricted epitopes of KRAS-G12 mutant polypeptides

[0075] Through the NetMHC-4.0 online prediction system, the HLA-A11 restricted T cell epitope polypeptide was predicted for the polypeptide with KRAS-G12 mutation. It was found that the KRAS-G12V epitope has a strong affinity with HLA...

Embodiment 2

[0120] Example 2. KRAS-G12V 8-16 (VVGAVGVGK) / HLA-A11 tetramer binding to cells expressing 1-2C and 3-2E TCR

[0121] In this example, the inventors further confirmed that the screened 1-2C and 3-2E TCRs have the ability to target KRAS-G12V 8-16 (VVGA V GVGK) / HLA-A11 specific recognition. In addition, this example also found that 1-2C and 3-2E can be combined with KRAS-G12V presented by HLA-A11 8-16 (VVGA V In addition to GVGK), it can also bind to KRAS-G12V presented by HLA-A03 8-16 (VVGA V GVGK) combined.

[0122] 1. 1-2C and 3-2E TCR binding specificity verification

[0123]First, the α chain and β chain variable region (V region) of 1-2C and 3-2E TCR (1-2C: nucleic acid sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 6 , and 3-2E: the nucleic acid sequence is shown in SEQ ID NO: 7, the amino acid sequence is shown in SEQ ID NO: 11) gene and human TCR α chain and β chain constant region (C region) gene (synthesized by Hongxun Biotechnolo...

Embodiment 3

[0145] Example 3. Preparation of 1-2C and 3-2E TCR-T cells and their responses to different mutant polypeptides of KRAS

[0146] In this example, the 1-2C or 3-2E TCR gene was introduced into peripheral blood mononuclear cells (PBMC) or CD8 T cells isolated from healthy volunteers with HLA-A11 genetic background as TCR-T effector cells. KRAS-G12 wild-type polypeptide 8-16 (VVGA G GVGK), G12V 8-16 (VVGA V GVGK), G12D 8-16 (VVGA D GVGK), G12C 8-16 (VVGAC GVGK) mutant polypeptides were added to the above-mentioned TCR-T effector cell system for co-cultivation, and the level of IFN-γ secreted by the effector cells and target cells presenting KRAS wild-type and mutant polypeptides was detected, and the effect on 1-2C or The effect of 3-2E TCR on target cells expressing KRAS wild-type and mutant polypeptides / HLA-A11 was evaluated. The specific operation is as follows.

[0147] 1. Preparation of lentivirus expressing 1-2C and 3-2E TCR

[0148] The 1-2C and 3-2E TCR lentivira...

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Abstract

The invention provides two specific T cell receptors targeting G12V or G12C mutation epitopes of a KRAS gene and an anti-tumor application. The two T cell receptors respectively consist of an alpha peptide chain and a beta peptide chain. The invention also provides an antigen binding fragment of the T cell receptors, nucleic acid encoding the antigen binding fragment, a vector containing the nucleic acid, and a host cell containing the vector. The invention further provides a method for preparing a G12V mutation-specific T cell receptor of KRAS or an antigen-binding fragment thereof. The specific T cell receptor and the antigen binding fragment thereof can be used as immune effect activators to stimulate the immune response of the body, thereby generating the action effect of resisting tumors and other diseases.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to a T cell receptor (TCR) or an antigen-binding fragment thereof capable of specifically recognizing antigenic polypeptides of tumor KRAS gene G12V and G12C mutations. Background technique [0002] In 2011, cancer surpassed heart disease as the leading cause of death worldwide. The WHO announced in December 2013 that the number of new cancer patients worldwide has exceeded 14 million each year, which is a substantial increase compared with the 2008 statistics of 12.7 million. As of 2020, cancer will cause 9.6 million deaths every year, and about US$1 trillion is invested in cancer diagnosis and treatment worldwide every year. [0003] In the early 1980s, Allison and others determined the gene structure of the αβ T-cell receptor (TCR), which is responsible for recognizing antigens on the surface of T cells. In the late 1980s, Boone, Rosenberg, Old and others found that there ar...

Claims

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Application Information

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IPC IPC(8): C07K14/725C12N15/12C12N15/867C12N5/10G01N33/68G01N33/574A61K39/00A61P35/00
CPCC07K14/7051C12N15/86C12N5/0686G01N33/68G01N33/574G01N33/57484A61K39/001111A61P35/00C12N2740/15043C12N2800/107C12N2510/02G01N2333/7051C12N2740/16043A61K39/4611A61K39/4632A61K39/464464
Inventor 高福谭曙光卢丹
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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