antigen-specific helper t cell receptor gene

An auxiliary and specific technology, applied in the field of polynucleotides, can solve the problem of not knowing the specificity of the identification of auxiliary peptide antigens, and achieve the effect of treating or preventing cancer

Inactive Publication Date: 2018-09-21
INT INST OF CANCER IMMUNOLOGY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the antigen-specific CD4 that recognizes the helper peptide is completely unknown + Sequence of the T cell receptor (TCR) gene of helper T cells

Method used

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  • antigen-specific helper t cell receptor gene
  • antigen-specific helper t cell receptor gene
  • antigen-specific helper t cell receptor gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0167] Example 1. WT1 332 specific CD4 + Establishment of T cell clones and isolation and sequencing of T cell receptor (TCR) genes

[0168] The experimental steps are as follows.

[0169] (1) Create WT1 332 specific CD4 + Methods of T cell cloning

[0170] (i) Harvest peripheral blood mononuclear cells (PBMCs) from healthy subjects at 3x10 6 cells / well were seeded into a 24-well plate. X-VIVO 15 medium supplemented with 10% AB serum and 40 IU / ml IL-2 was used as the medium.

[0171] (ii) Add WT1 at a final concentration of 20 µg / ml 332 Peptides were added to i above and cells were cultured for 7 days.

[0172] (iii) After 7 days, the cells were collected and prepared with X-VIVO 15 medium supplemented with 10% AB serum to make the cell density 1x10 7 cells / ml, and 100 μL of each was seeded into a 96-well round-bottom plate.

[0173] (iv) At final concentrations of 40 µg / ml, 4 µg / ml and 4 µg / ml, WT1 332 Peptides, BD GolgiStopTM (BD Bioscience) and CD28 / CD49d Costi...

Embodiment 2

[0225] Example 2. Will be derived from WT1 332 specific CD4 + T cell receptor (TCR) gene of T cell into human CD4 + in T cells

[0226] confirmed the use of WT1-derived 332 specific CD4 + Human CD4 transduced with T cell receptor (TCR) gene for T cells + WT1 332 Specific and HLA class II-restricted manner displays proliferative responses and produces cytokines.

[0227] The TCR genes shown in Table 3 were isolated from clone 9, which specifically recognized WT1 in a HLA-DPB1*05:01 restricted manner 332 CD4 + T cell cloning. By using lentiviral vectors, these TCR genes were transduced into CD4 cells derived from the peripheral blood of healthy subjects. + T cells, and by using cytokine (interferon-γ and IL-2) production as indicators, the response to WT1 332 Reaction( Figure 2A and B). In addition, CD4 transduced with a lentiviral vector that does not carry the TCR gene +T cells (labeled mock) were used as controls. use WT1 332 CD4-specific TCR gene transd...

Embodiment 3

[0235] Example 3. By using the source from WT1 332 specific CD4 + Human CD4 transduced with TCR gene of T cells + T cells enhance the induction of WT1-specific CTL

[0236] In general, it is known that CD4 + T cells serve as helper T cells and respond to CD8 as primary effector cells attacking cancer cells + Introduction and maintenance of T cells (CTL) are important. Therefore, WT1 was analyzed 332 -TCR-transduced CD4 + Whether T cells enhance the induction of WT1-specific CTLs.

[0237] PBMCs from HLA-A*24:02 and HLA-DPB1*05:01-positive healthy subjects were compared with WT1 prepared from the same healthy subjects 332 -TCR-transduced CD4 + T cells were mixed at a ratio of 10:1 and 5:1 (labeled 1:0.1 and 1:0.2 in Figure 3) and incubated in modified WT1 235 Incubate for 1 week in the presence of peptide (wherein M, the second amino acid of natural WT1 peptide combined with HLA-A*24:02 molecule, is modified into Y (CYTWNQMNL) (SEQ ID No: 125)), the modified WT1...

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Abstract

The present invention relates to a polynucleotide encoding CDR3 in the TCR-α chain and TCR-β chain genes of CD4+ helper T cells specific for a WT1 helper peptide having the amino acid sequence of SEQ ID NO: 123 . The invention also relates to the peptides encoded by said polynucleotides. The present invention also relates to CD4+T cells into which the TCR gene containing the polynucleotide has been introduced, the use of CD4+T cells to induce WT1-specific cytotoxic T-lymphocytes (CTL), cancer treatment, and the like.

Description

technical field [0001] The present invention relates to polynucleotides contained in T cell receptor (TCR) genes of cancer antigen-specific helper T cells. In particular, the present invention relates to encoding a CD4 specific for the WT1 helper peptide having the amino acid sequence shown in SEQ ID NO: 123 + A polynucleotide of complementarity determining region 3 (CDR3) of each α chain and β chain of the TCR of a helper T cell. The present invention also relates to polypeptides encoded by these polynucleotides. In addition, the present invention relates to the CD4 gene introduced by the TCR gene containing these polynucleotides + T cells, use of the cells to induce and enhance WT1-specific cytotoxic T cells (WT1-specific CTLs), and use of the cells to treat cancer and the like. Background technique [0002] The WT1 gene (Wilms tumor 1 gene) is a gene group identified as a gene responsible for Wilms tumor, which is childhood renal cancer (Non-Patent Documents 1 and 2)....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N5/10C12N15/85C12N5/0783A61K35/17A61P35/00C12Q1/6876C07K14/725C07K16/28G01N33/569
CPCA61K38/00A61K35/17C12Q1/6886G01N2333/70514C12N5/0636C07K14/7051C12Q1/6881A61P35/00C07K16/28G01N33/56972G01N2333/4703C12N2510/00C12Q2600/158
Inventor 杉山治夫藤木文博
Owner INT INST OF CANCER IMMUNOLOGY INC
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