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A t-cell receptor associated with mutations in the kras gene

A cell receptor and carrier technology, applied in gene therapy, DNA/RNA fragments, medical preparations containing active ingredients, etc.

Active Publication Date: 2021-07-16
高军 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no known T cell receptor related to KRAS gene mutation, and there is a need for a method for treating and / or preventing diseases related to KRAS gene mutation at codon 12

Method used

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  • A t-cell receptor associated with mutations in the kras gene
  • A t-cell receptor associated with mutations in the kras gene
  • A t-cell receptor associated with mutations in the kras gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 Construction of Retroviral Vector of Recombinant Human α Chain and β Chain Heteroduplex TCR Receptor Gene Combined with KRAS G12D Mutation 9-12 Epitope Peptide

[0091]An extra cysteine ​​(C) is introduced into the constant region of the above-mentioned α-chain sequence 1 and β-chain sequence 1, and its sequence is as follows: the complete amino acid sequence is shown in SEQ ID NO: 46, and the complete nucleotide sequence is shown in SEQ ID NO :47.

[0092] After the above gene sequence was synthesized, refer to figure 1 The gene arrangement was constructed into the MP71 reverse transcription vector. The MP71 vector backbone has been described in previous literature (Linnemann, C et al., High-throughput identification of antigen-specific TCRs by TCR gene capture, Nature Medicine, 2013, 19(11) 1534-1541). This vector has been used in clinical trials of gene-modified T cells to treat HIV in Germany.

Embodiment 2

[0093] Example 2 Retroviral transfection of human T cells carrying recombinant human α-chain and β-chain heterologous double-chain TCR receptor gene carrying KRAS gene G12D mutation 9-12 epitope peptide

[0094] The recombinant human α-chain and β-chain heterologous double-chain TCR receptor gene retrovirus constructed in Example 1 that can bind to the KRAS G12D mutation 9-12 epitope peptide was referred to literature (Linnemann, C, etc., High-throughput identification of antigen- specificTCRs by TCR gene capture, Nature Medicine, 2013, 19(11) 1534-1541) PBMC cells transfected with human HLA-A0301 as the experimental group (TCR1-1), and empty vector transfection as the control group (Non-Td) . Flow cytometry was used to detect the transfected CD8+T lymphocytes that could specifically bind the KRAS gene G12D mutant epitope peptide (SEQ ID NO: 49) and the epitope peptide-MHC molecule complex pMHC of HLA-A0301 molecule. The result is as figure 2 As shown, the number of cells i...

Embodiment 3

[0095] Example 3 Flow cytometric detection of target cell recognition by HLA-A0301 CD8+ T lymphocytes recombinantly expressing recombinant human α-chain and β-chain heteroduplex TCR receptor genes capable of binding KRAS G12D mutation 9-12 epitope peptide

[0096] The primary cultured HLA-A0301+ and HLA-A0301- pancreatic cancer cell lines carrying the G12D mutation of the KRAS gene were respectively selected as target cells, referring to the literature ( E et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, SCIENCE, 2016, 352 (6291): 1337-1341) and CD8+ T lymphocytes transfected with the TCR gene prepared in Example 2 (Transferred in Example 2 PBMC cells transfected with TCR gene were incubated together by CD8+ flow cytometry screening), and flow cytometry was used to detect the expression of CD8+ T lymphocyte activation molecular markers (IFN-γ and CD107a / b) after incubation. The result is as image 3 As shown, after incubation with HLA-A...

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Abstract

The invention discloses a T cell receptor, which specifically binds to the KRAS gene 12 codon mutation G12D 26aa peptide segment, the amino acid sequence of the peptide segment is: MTEYKLVVVGADGVGKSALTIQLIQN, as shown in SEQ ID NO: 1; the T cell When the receptor recognizes the HLA molecular antigen complex, the 9‑12aa antigenic epitope polypeptide with higher affinity is as follows: VVVGADGVGK, as shown in SEQ ID NO: 48; KLVVVGADGVGK, as shown in SEQ ID NO: 49; The HLA molecule is HLA‑A0301; the amino acid sequence of the α chain and β chain of its T cell receptor is disclosed when recognizing the antigen complex formed by the G12D mutant peptide of the KRAS gene and HLA‑A0301. In addition, the present invention also discloses the nucleotide sequence encoding the α chain and β chain of the above-mentioned T cell receptor, the vector containing the above-mentioned nucleotide sequence, the cell containing the above-mentioned nucleotide sequence, and the above-mentioned vector or the above-mentioned cell in the preparation Application in medicines for preventing and treating KRAS gene mutation-related diseases.

Description

technical field [0001] The invention relates to a T cell receptor (TCR), in particular to a T cell receptor related to KRAS gene mutation. The present invention also relates to the use of TCR gene transfer to generate T cells, and their pharmaceutical use for preventing and treating diseases related to KRAS gene 12 codon mutation. Background technique [0002] KRAS is a proto-oncogene, about 35kb in length, located on chromosome 12. It is a member of the RAS gene family and encodes KRAS protein. It is related to tumor formation, proliferation, migration, spread and angiogenesis. [0003] The KRAS gene (KRAS, p21) family has three genes related to human tumors—H-ras, KRAS and N-ras, which are located on chromosome 11, 12 and 1, respectively. The KRAS gene encodes a 21kD ras protein, also known as the p21 gene. Among ras genes, KRAS has the greatest impact on human cancer. It acts like a molecular switch: when normal, it controls the pathways that regulate cell growth; when...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/725C12N15/62A61K35/17A61K35/28A61K48/00A61K31/7088A61P35/00
CPCA61K35/17A61K35/28A61K48/005C07K14/7051C07K2319/00
Inventor 高军吕垚史军
Owner 高军
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