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A kind of chimeric molecular converter of T cell signal and its use

A molecular and signal peptide technology, applied in the fields of molecular biology and immunology, which can solve the problems of malignant proliferation, invasion and metastasis of tumor cells

Active Publication Date: 2018-10-30
SHANGHAI CELL THERAPY GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the tumor microenvironment, there are many factors that can negatively regulate the immune response of T cells, making tumor cells escape the monitoring and elimination of the body's immune system, making tumor cells continue to malignantly proliferate, invade and metastasize

Method used

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  • A kind of chimeric molecular converter of T cell signal and its use
  • A kind of chimeric molecular converter of T cell signal and its use
  • A kind of chimeric molecular converter of T cell signal and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Synthesis of Fusion Protein Expression Cassette and Construction of Expression Vector

[0075] According to the amino acid sequence and coding sequence of each component of the fusion protein, the entire fusion amino acid sequence and coding DNA expression frame are spliced, as follows:

[0076] The amino acid residue sequence of PD1-IGV is:

[0077] GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGA (SEQ ID NO: 1)

[0078] The coding sequence of PD1-IGV is:

[0079] GGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCCAGCCCGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCNOGT2GCAATGACAGCGGCACG

[0080] The amino acid residue sequence of CD28 transmembrane region (CD28TM) is:

[0081] PFWVLVVVGGVLACYSLLVTVAFIIFWVRS (SEQ ID NO: 3).

[0082] The coding sequence of the CD28 transmembrane region (CD28TM) is:

[0083] CCCTTTTGGGTGCTGGT...

Embodiment 2

[0133] Example 2: Isolation and culture of liver cancer tissue-derived TIL cells

[0134] Collect freshly resected HCC specimens and process them immediately under sterile conditions. The specific method is as follows: remove the normal tissue and necrotic area around the liver cancer specimen, remove small tissue pieces with a size of 1-2mm3 from different areas of the specimen, and place one piece in each well of a 24-well plate. Add 2 mL complete medium (GT-T551 medium containing 10% FBS) and 3000 IU / mL IL-2 to each well. Place the 24-well plate in a 37°C, 5% CO2 incubator. On the 5th to 6th day after the initiation of culture, a half-volume medium change was performed for all wells. Afterwards, according to the growth of TILs, a half-volume medium change was performed every 1-2 days. Once the wells were overgrown with TILs and all adherent cells had been removed, the TILs in each overgrown well were collected.

[0135] Subsequently, 1×10 6 TILs were resuspended in T...

Embodiment 3

[0136] Example 3: Genetic modification of TILs

[0137] at 175-cm 2 Amphotropic packaging cells (purchased from CellBioLabs) were cultured in flasks, the number of cells was about 1–2×10 7 ), purified high-quality pMXs-pSCC1-IRES-GFP, pMXs-pSCC2-IRES-GFP, pMXs-pSCC3-IRES-GFP, pMXs-epSCC-IRES-GFP, pMXs-peSCC-IRES -GFP plasmid was transfected into cells. After 3 days, collect the cell culture medium containing virus particles, centrifuge at 4000g for 10min, collect the supernatant, and filter it with a 0.45μm filter. Use 500ul ice-cold PBS solution to resuspend the virus pellet, and obtain recombinant retroviruses carrying the expression cassettes of pSCC1, pSCC2, pSCC3, epSCC, and peSCC, respectively. Subsequently, the virus suspension was mixed twice (100 μL each time) with 2 × 10 6 TIL cells were co-cultured, and the TIL cells after infection were collected separately. pSCC1 、TIL pSCC2 、TIL pSCC3 、TIL epSCC 、TIL peSCC .

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Abstract

The invention relates to a single chimeric converter for a T-cell signal and application thereof, which belongs to the fields of molecular biology and immunology. Specifically speaking, the single chimeric converter is formed by connection of a polypeptide highly efficiently bonding with immunosuppression molecules on the surfaces of tumor cells and / or tumor matrix cells to a transmembrane domain originated from a high-affinity receptor and an intracellular peptide fragment of a costimulatory signal molecule through a hinge structure. Out-membrane polypeptide receives a signal of the immunosuppression molecules on the surfaces of tumor cells and / or tumor matrix cells and transmits the signal into a cell, a second signal of immune cells is activated through the intracellular peptide fragment of the costimulatory signal molecule, so multiplication capacity of the immune cells and the secretion function of cell factors are enhanced, and the survival time of the activated immune cells is prolonged; thus, side-effects of a tumor immunosuppression microenvironment on adoptive cell therapy effector cells are overcome.

Description

technical field [0001] The invention belongs to the field of molecular biology and immunology, and relates to a T cell signal chimeric molecular converter (Signal Chimeric Convertor, SCC) and its application. Background technique [0002] Adoptive cell therapy (ACT) is a method to reinfuse processed autologous or allogeneic immune cells (mainly autologous cells) to tumor patients to enhance the patient's immune function and achieve therapeutic purposes. Currently, tumor ACT is developing rapidly, and has achieved very good results in the clinical treatment of various types of malignant tumors (Nature.2011;480:480-9; J Clin Oncol.2011;29:4828-36). [0003] The stability of the immune system is crucial, underreaction can cause serious infection, overreaction can lead to allergic reaction. Therefore, the human immune system has evolved a set of sophisticated and complex two-way immune regulation mechanism to regulate the positive and negative two-way immune response. When the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/79C12N15/867C12N5/10A61K48/00A61K38/16A61P35/00
Inventor 钱其军金华君丁娜俞德超李林芳吴孟超
Owner SHANGHAI CELL THERAPY GRP CO LTD
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