Preparation method for enhancing lethality of tumor infiltrating lymphocytes

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

Active Publication Date: 2022-04-29
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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  • Preparation method for enhancing lethality of tumor infiltrating lymphocytes
  • Preparation method for enhancing lethality of tumor infiltrating lymphocytes
  • Preparation method for enhancing lethality of tumor infiltrating lymphocytes

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preparation example Construction

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

[0046] 1) Prepare a recombinant lentivirus containing a gene of interest;

[0047] Specifically: obtain and optimize the synthesis of IL-7 sequence, IL-15 sequence and IL-7-IL-15 fusion sequence, insert the sequence into a lentiviral vector, and then transfect HEK-293T cells for virus preparation and amplification. 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 digesting the tumor tissue, obtain a single cell suspension, culture the single cell suspension with IL-2, separate and purify infiltrating lymphocytes; Continue to add IL-2 to the infiltrating lymphocytes, and culture until the logarithmic growth phase;

[0050] 3) The recombinant lentivirus was added to the infiltrating lymph...

Embodiment 1

[0052] The construction of the lentiviral expression vector of embodiment 1 IL-7, IL-15 and IL-7-IL-15 gene

[0053] 1. Plasmid construction 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 bias, moderate GC content, and stable secondary mRNA structures, etc., eliminating repetitive sequences and cryptic splice sites as well as unwanted restriction sites, while preventing depletion of the tRNA pool in the cell. The optimized sequence was directly synthesized, and the ECoRI and BamHI restriction sites were used to link into the Tet-on expression plasmid pLVX-TetOne-Puro, which was also double-digested. The plasmids verified by digestion and sequencing were named pLVX-IL-7, pLVX-IL-15 and pLVX-IL-7-IL15, respectively. IL-7 and IL-15 are linked by IRES. IRES is an internal ribos...

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 on the outer surface and placed in a biosafety cabinet. Open the tumor tissue sample collection bottle in the biosafety cabinet, clean and process the tumor tissue, and remove blood vessels. , capsule and other necrotic tissue.

[0062] 2. Tissue digestion:

[0063] 1) Shred the tumor tissue in the tissue enzymatic solution until it is almost like meat powder, add the tissue enzymatic solution to suspend, transfer the suspension to a centrifuge tube, put it into an electric constant temperature oscillator, set the temperature at 37°C, and the oscillation frequency at 150rpm / min, time 30-60min, constant temperature digestion.

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

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Abstract

The invention belongs to the field of preparation of anti-tumor cells, and particularly relates to a preparation method of tumor infiltrating lymphocytes, the method comprises the following steps: preparing a recombinant lentivirus containing target genes, the target genes comprising an IL-7 sequence, an IL-15 sequence and an IL-7-IL-15 fusion sequence; separating infiltrating lymphocytes from tumor tissues, adding IL-2, and culturing the cells to a logarithmic phase; adding the recombinant lentivirus into infiltrated lymphocytes cultured to a logarithmic phase, culturing for 2-4 days, and then adding a screening agent for screening culture until all cells are transgenic positive cells; a Tet-on system is used, an inducer is added to induce expression of IL-7 or/and IL-15, and multiplication culture of cells is maintained. The amplification efficiency of the TIL obtained by the preparation method is obviously improved and is about 5-10 times higher than that of a common TIL amplification method, and the amplified lymphocyte has a 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 lethality of tumor infiltrating lymphocytes. Background technique [0002] Tumor infiltrating lymphocytes (Tumor infiltrating lymphocytes, TIL), as a personalized adoptive cell therapy (Adoptive cell transfer therapy, ACT), has been developed for more than 30 years, showing great prospects for clinical application. TIL has TCR clonal diversity, superior tumor homing ability, significant curative effect and good safety in the treatment of solid tumors, and has advantages that other ACT methods cannot match. So far, TIL therapy is currently undergoing clinical trials mainly 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, the preparation method of TIL is to obtain a single cell suspension after mechanical treatmen...

Claims

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Application Information

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Patent Type & Authority Applications(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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