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Method for screening tumor specific T cell from TIL (tumor infiltrating lymphocyte)

A tumor-specific, cell-based technology, applied in the field of screening tumor-specific T cells from TIL, can solve the problems of limited quantity, unsatisfactory effect, and low ratio, and achieve the effect of not being easy to separate and inhibiting the growth of tumors

Pending Publication Date: 2019-07-09
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These tumor-specific antigens can be recognized by the immune system, but the immune cells are restricted by many factors in the tumor microenvironment and cannot be amplified in large quantities. The ratio is too low and the number is limited, so they cannot play a good role in killing tumors
The traditional TIL culture method also screens, but only divides the tumor tissue into different culture wells. The cells in some culture wells contain tumor-specific T cells, but the proportion is not high; The proportion of tumor-specific T cells in TIL reinfused to patients is still very low, which may be an important reason for the unsatisfactory effect of TIL therapy

Method used

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  • Method for screening tumor specific T cell from TIL (tumor infiltrating lymphocyte)
  • Method for screening tumor specific T cell from TIL (tumor infiltrating lymphocyte)
  • Method for screening tumor specific T cell from TIL (tumor infiltrating lymphocyte)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Isolation and cultivation of TIL in human tumor tissue

[0069] TIL medium: add IL2 to X-VIVO to obtain TIL medium; in TIL medium, the concentration of IL2 is 1000U / mL.

[0070] 1. Take fresh human tumor tissue, cut it into pieces, and get about 1mm 3 human tumor tissue blocks.

[0071] 2. After completing step 1, place the human tumor tissue block in a 24-well plate, then add TIL medium, and store at 37°C, 5% CO 2 Culture for 5 days.

[0072] After completing step 2, the human tumor tissue block was observed under a microscope.

[0073] See the experimental results figure 1 : Both single cells and clustered cells in the field of view are TIL cells. The results showed that there was a large distribution of TILs in the medium.

[0074] 3. After completing step 2, take the 24-well plate, pour off half of the culture solution, and then add an equal amount of fresh TIL medium (i.e., change the medium in half), at 37°C, 5% CO 2 Cultured for 12 days (during ...

Embodiment 2

[0077] Embodiment 2, screening experiment

[0078] 1. Screening of human tumor-specific antigen peptides

[0079] The human tissue to be tested is human tumor tissue or human normal tissue.

[0080] 1. Exome sequencing

[0081] Genomic DNA of the human tissue to be tested was extracted, and then exome sequencing (WES, >200x) was performed using a BGIseq500 sequencer to obtain sequencing sequences.

[0082] 2. RNA seq

[0083] The total RNA of the human tissue to be tested was extracted, then reverse-transcribed into cDNA, and sequenced with a BGIseq500 sequencer to obtain the sequencing sequence.

[0084] 3. HLA typing detection

[0085] The target sequencing region includes HLA class I and class II exon regions, including HLA-A gene, HLA-B gene, HLA-C gene, HLA-DQB1 gene and HLA-DRB1 gene. The HLA typing detection method can not only accurately detect subtypes, but also detect mutation sites in the target sequencing region. Specific steps are as follows:

[0086] (1) D...

Embodiment 3

[0191] Embodiment 3, mouse experiment

[0192] Mouse experiments were used to verify the feasibility of the above TIL screening method.

[0193] 1. Modeling

[0194] 1. Take the mouse colon cancer cell line CT26, place it in DMEM medium containing 10% (v / v) fetal bovine serum, 100U / mL penicillin and 100U / mL streptomycin, at 37°C, 5% CO 2 Cultivate for 48h.

[0195] 2. After completing step 1, centrifuge at 3000 rpm for 5 minutes to collect tumor cells.

[0196] 3. After completing step 2, take tumor cells and wash them with sterile saline for 3 times.

[0197] 4. After completing step 3, take tumor cells and add pH 7.2, 0.01mol / L PBS buffer solution to dilute to obtain a concentration of 1×10 8 cells / mL of tumor cell suspension.

[0198] 5. Take 4-week-old BALB / c mice, inoculate each mouse with 100 μL of tumor cell suspension subcutaneously, and then observe whether there is infection at the inoculation site every day, and whether the tumor has naturally subsided after gr...

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Abstract

The invention discloses a method for screening a tumor specific T cell from a TIL (tumor infiltrating lymphocyte). The method includes the steps of screening a tumor specific antigen peptide of a to-be-tested tumor patient; constructing a composite of an MHC (major histocompatibility complex) and the tumor specific antigen peptide, wherein the MHC and antigen peptide composite can be combined witha TCR (T cell receptor) of a T cell of a tumor specific antigen; adding an IFN-r positive TIL of the to-be-tested tumor patient to the MHC and antigen peptide composite, performing incubation, and screening the tumor specific T cell. The invention also provides a tumor specific polypeptide screened by the method, and application of the screened tumor specific T cell and the polypeptide in products related to cancer therapy. By the method, the tumor specific T cell and the tumor specific polypeptide can be efficiently screened out, and accordingly, an effective choice for tumor immunotherapy is provided.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for screening tumor-specific T cells from TILs. Background technique [0002] Tumor infiltrating lymphocytes (TIL) are lymphocytes distributed in and around tumor tissues. In the 1980s, scientists discovered that most of the surgically resected tumor tissues were tumor cells and a small number of lymphocytes. Further studies have found that among these cells, some lymphocytes can kill tumor cells, that is, some T cells in tumor-infiltrating lymphocytes can recognize tumor antigens presented on the surface of tumor cells, including tumor-specific antigens, which are new tumor cells. However, due to some reasons, the function is inhibited (for example, PD-1 on the surface of TIL binds to PD-L1 on the surface of tumor cells, and the tumor killing function is shielded), so it cannot effectively kill tumor cells in tumor tissue. If these lymphocytes are enriched and returned to t...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C07K7/06A61K39/00A61P35/00
CPCC12N5/0636C07K7/06A61K35/17C12N2509/00A61K39/00
Inventor 唐云霞李波张秀清
Owner SHENZHEN HUADA GENE INST
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