A preparation method for enhancing the lethality of tumor-infiltrating lymphocytes

A technology of lymphocyte and tumor infiltration, applied in the field of preparation of anti-tumor cells, can solve the problems of limiting the clinical application and toxicity of TIL therapy

Active Publication Date: 2022-06-17
BEIJING AEONVITAL BIOMEDICINE RES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high doses of IL-2 usually cause systemic toxicity, requiring intensive monitoring and care; in addition, IL-2 may also promote the activity of suppressor T cells, thereby inhibiting the anti-tumor response of TIL; the above drawbacks greatly limit the application of TIL therapy. Clinical application

Method used

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  • A preparation method for enhancing the lethality of tumor-infiltrating lymphocytes
  • A preparation method for enhancing the lethality of tumor-infiltrating lymphocytes
  • A preparation method for enhancing the lethality of tumor-infiltrating lymphocytes

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preparation example Construction

[0045] The invention provides a preparation method of infiltrating lymphocytes, which is specifically as follows:

[0046] 1) Preparation of recombinant lentivirus containing the gene of interest;

[0047] Specifically: obtaining and optimizing the synthesis of IL-7 sequence, IL-15 sequence and IL-7-IL-15 fusion sequence, inserting the sequence into a lentiviral vector, and then transfecting HEK-293T cells for virus preparation and amplification, and after culturing Obtain recombinant lentivirus;

[0048] Wherein, the IL-7 sequence is shown in SEQ ID No: 1; the IL-15 sequence is shown in SEQ ID No: 2; the IL-7-IL-15 fusion sequence is shown in SEQ ID No: 3 Show;

[0049] 2) After the tumor tissue was digested, a single cell suspension was obtained, and the single cell suspension was cultured with IL-2 to separate and purify the infiltrating lymphocytes; then in a culture environment coated with recombinant human fibrin and CD3 antibody, purified IL-2 was added to the infilt...

Embodiment 1

[0052] Example 1 Construction of lentiviral expression vectors of IL-7, IL-15 and IL-7-IL-15 genes

[0053] 1. Construction of plasmid containing IL-7 or IL-15 or IL-7-IL-15 gene

[0054] Download the CDS sequences of IL-7 and IL-15 from NCBI, and then perform codon optimization to enhance their expression. These optimization methods include but are not limited to: human codon usage preference, moderate GC content, stable mRNA secondary structure, etc., eliminate repetitive sequences and cryptic splice sites and unwanted restriction sites while preventing depletion of the tRNA pool in the cell. The optimized sequence was directly used for gene synthesis, and the ECoRI and BamHI restriction sites were used to connect the Tet-on expression plasmid pLVX-TetOne-Puro with the same double restriction. The plasmids verified by restriction digestion and sequencing were named pLVX-IL-7, pLVX-IL-15 and pLVX-IL-7-IL15, respectively. IRES is used to connect IL-7 and IL-15. IRES is an i...

Embodiment 2

[0060] Example 2 Preparation of non-small cell lung cancer TIL cells

[0061] 1. Tissue pretreatment: Tumor tissue sample collection tubes, reagents, operating instruments, etc. are wiped with 75% ethanol and placed in a biological safety cabinet. Open the tumor tissue sample collection bottle in the biological safety cabinet, wash the tumor tissue, and remove blood vessels. , capsule and other necrotic tissue.

[0062] 2. Tissue digestion:

[0063] 1) Mince the tumor tissue in the tissue enzymolysis solution to a near-meat-like shape, add the tissue enzymolysis solution to suspend, transfer the suspension to a centrifuge tube, put it into an electrothermal constant temperature shaker, set the temperature to 37°C, and set the shaking frequency to 150rpm / min, time 30-60min, constant temperature digestion.

[0064] 2) Take 40mL of stop protection solution and put it in a 50mL centrifuge tube for use, take a 70µm filter and place it at the mouth of the 50mL centrifuge tube, an...

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Abstract

The invention belongs to the field of anti-tumor cell preparation, and specifically relates to a method for preparing tumor-infiltrating lymphocytes. The method includes the following steps: preparing a recombinant lentivirus containing a target gene, and the target gene includes an IL-7 sequence and an IL-15 sequence and IL-7-IL-15 fusion sequence; isolate infiltrating lymphocytes from tumor tissue, add IL-2, and culture the cells to the logarithmic growth phase; add the recombinant lentivirus to the infiltrating lymphocytes cultured to the logarithmic growth phase In the cells, after 2-4 days of culture, add screening agents for screening culture, and culture until all cells are transgene-positive cells; use Tet-on system, add inducers to induce the expression of IL-7 or / and IL-15, and maintain the expression of cells Expansion culture. The expansion efficiency of TIL cells obtained by the preparation method is significantly improved, which is about 5-10 times higher than that of common TIL expansion methods, and the expanded lymphocytes have higher tumor killing effect.

Description

technical field [0001] The invention relates to a preparation method of anti-tumor cells, in particular to a preparation method for enhancing the killing power of tumor-infiltrating lymphocytes. Background technique [0002] Tumor infiltrating lymphocytes (TIL), as a personalized adoptive cell transfer therapy (ACT), have been developed for more than 30 years and show great clinical application prospects. TIL has TCR clonal diversity and superior tumor homing ability. It has significant efficacy and good safety in the treatment of solid tumors, and has advantages unmatched by other ACT methods. So far, TIL therapy is currently undergoing clinical trials primarily as a second-line treatment. TIL therapy has shown impressive clinical responses in melanoma, non-small cell lung cancer, and advanced cervical cancer. [0003] Usually, TIL is prepared by mechanical treatment and enzymatic digestion of tumor tissue to obtain a single cell suspension. T lymphocytes are selected and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/24C12N15/62C12N5/10A61K35/17A61K38/20A61K48/00A61P35/00
CPCC12N15/86C07K14/5418C07K14/5443C12N5/0636A61K35/17A61K38/2046A61K38/2086A61K48/005A61K48/0008A61P35/00C12N2740/15043C12N2800/107C07K2319/00C12N2501/2302C12N2501/515C12N2533/56C12N2510/00
Inventor 张秀军刘磊王星星
Owner BEIJING AEONVITAL BIOMEDICINE RES CO LTD
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