Single chimeric converter for T-cell signal and application thereof

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

Active Publication Date: 2014-08-06
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 cel

Method used

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  • Single chimeric converter for T-cell signal and application thereof
  • Single chimeric converter for T-cell signal and application thereof
  • Single chimeric converter for T-cell signal and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[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 whole fusion amino acid sequence and coding DNA expression cassette are spliced ​​together, as follows:

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

[0077] GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGA (SEQ IDNO:1)

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

[0079] GGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCGCTTCCGTGTCACACACAACTGCCCAACGGGGGGCGTTGAACGGCGGGTTGAACGGCGGGCGTGCCACACACAACTGCCCAACGGGCGGGACTTCCACATCCAGGTCGGT:

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

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

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

[008...

Example Embodiment

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

[0134] Collect newly resected liver cancer specimens and immediately process them under aseptic 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 1-2 mm3 from different areas of the specimen, and place one piece in each hole of the 24-well plate. Add 2mL complete medium (GT-T551 medium containing 10% FBS) and 3000IU / mL IL-2 to each well. Place the 24-well plate in a 37°C, 5% CO2 incubator for culture. On the 5-6 days after the initiation of the culture, half of the medium was exchanged for all wells. After that, according to the growth of TIL, a half-volume exchange is performed every 1-2 days. Once the TIL in the well is overgrown and all adherent cells have been removed, collect the TIL in each overgrown well.

[0135] Subsequently, 1×10 6 TIL is resuspended in T175 culture containi...

Example Embodiment

[0136] Example 3: Genetic modification of TIL

[0137] At 175-cm 2 Culturing Amphotropic packaging cells in flasks (purchased from CellBioLabs, the number of cells is about 1–2×10 7 ), using Lipofectamine2000 (Invitrogen) to convert the purified high-quality pMXs-pSCC1-IRES-GFP, pMXs-pSCC2-IRES-GFP, pMXs-pSCC3-IRES-GFP, pMXs-epSCC-IRES-GFP, pMXs-peSCC-IRES -GFP plasmid is transfected into cells. After 3 days, collect the cell culture medium containing virus particles, centrifuge at 4000g for 10 minutes, collect the supernatant, and filter with a 0.45μm filter. The filtered liquid is placed in a 40mL ultracentrifuge tube and centrifuged at 4°C at 25000r / min for 20 minutes. Use 500ul ice PBS to resuspend the virus pellet to obtain recombinant retroviruses carrying expression cassettes of pSCC1, pSCC2, pSCC3, epSCC, and peSCC, respectively. Subsequently, divide the virus suspension with 2×10 6 TIL cells are co-cultured, and the infected TIL cells are collected separately. pSCC1 ,...

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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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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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