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Preparation method and kit of cytokine-induced killing cell for inducing antibody-dependent cellular cytotoxicity

An antibody-dependent, cytokine-based technology, applied in cytokines/lymphokines/interferons, genetically modified cells, botanical equipment and methods, etc., can solve the problems of limited efficacy of CIK cells and inability to meet clinical treatment of malignant tumors.

Inactive Publication Date: 2016-08-31
ZICHENG RUISHENGHUI BEIJING BIOTECH DEV CO LTD
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Problems solved by technology

[0003] However, the clinical efficacy of CIK cells in the treatment of malignant tumors is still very limited. According to the statistics of clinical treatment data, the effective rate including complete remission with complete tumor disappearance, partial remission with partial tumor shrinkage and stable disease without tumor enlargement is within 10%. -20%, far from meeting the needs of clinical treatment of malignant tumors

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  • Preparation method and kit of cytokine-induced killing cell for inducing antibody-dependent cellular cytotoxicity
  • Preparation method and kit of cytokine-induced killing cell for inducing antibody-dependent cellular cytotoxicity
  • Preparation method and kit of cytokine-induced killing cell for inducing antibody-dependent cellular cytotoxicity

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preparation example Construction

[0035] The present invention relates to a method for preparing cytokine-induced killer cells that mediate antibody-dependent cytotoxicity, which is characterized in that the interferon gamma signal peptide and tumor cell binding polypeptide are constructed and linked to the second heavy chain of the crystallizable fragment domain. and 3 constant region gene fragments, recombined into viral vectors and transfected into human cytokine-induced killer cells, high expression of new polypeptide-the fusion protein of the 2nd and 3rd constant regions of the heavy chain of the crystallizable fragment domain, which can trigger antibody-dependent Cytotoxic activity: Activation of cytokines induces the proliferation of killer cells to reach more than 1000 times, and its expression of CD3+ / CD56+ is more than 40%, and CD16+ is more than 30%. It has strong anti-tumor toxicity in vivo and in vitro tests.

[0036] The new polypeptide-crystallizable fragment domain recombination gene of the pres...

Embodiment 1

[0065] Recombinant SP-(TCBP) 2 -L-Fc gene construction and lentivirus preparation

[0066] The new polypeptide-crystallizable fragment domain recombinant gene is composed of interferon gamma signal peptide, two tumor cell binding polypeptides and the second and third constant region gene fragments of the crystallizable fragment domain heavy chain, and the second and third constant region gene fragments of the crystallizable fragment domain heavy chain 2 and 3 constant region fragments, the 24th serine is mutated to aspartic acid and the 117th isoleucine is mutated to glutamic acid, namely Fc-D / E, see figure 1 , the schematic diagram of polypeptide-crystallizable fragment domain fusion protein.

[0067] Human IFNγ-SP, two TCBP and Fc-D / E gene sequences were obtained by chemical synthesis, and a linking polypeptide was introduced between the two TCBPs and between TCBP and Fc-D / E, and its gene sequence was GGCGGCGGCGGCAGT, forming Complete human IFNγ-SP, (TCBP) 2 and the cDNA ...

Embodiment 2

[0073] Preparation of cytokine-induced killer cells that mediate antibody-dependent cytotoxicity

[0074] Collect 30ml of autologous peripheral blood from patients with advanced prostate cancer and breast cancer (with informed consent), add an equal amount of 1×PBS to dilute, and gently superimpose on the lymphocyte separation medium along the wall of the centrifuge tube to form a clear interface, 2000rpm at room temperature After centrifugation for 20 min, the PBMCs in the interface layer were aspirated and washed twice with 1×PBS (pH 7.4). After counting orchids, resuspend with RPMI 1640 medium and dilute to 3×10 6 cells / ml, and supplemented with 1000 activity units (IU) / ml IFN-γ and 10% autologous serum, every 20ml was added to 75cm 2 culture flask at 37°C with 5% CO 2 Cultivate in the incubator for 24 hours; add 50ng / ml IL-15, 50ng / ml IL-18, 20ng / ml mouse anti-human anti-CD3 monoclonal antibody (OKT3) and 500IU / ml IL-2 after 24 hours. 5%CO 2 Cultured in the incubator f...

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Abstract

The invention provides a preparation method of a cytokine-induced killing cell for inducing antibody-dependent cellular cytotoxicity. The preparation method comprises constructing an interferon gamma signal peptide-tumor cell combined peptide, bonding 2th and 3th constant region gene segments of a crystallizable fragment domain heavy chain to the combined peptide, recombining the combined chain to a viral vector, transfecting a human cytokine-induced killing cell and carrying out high expression of a 2th and 3th constant region fusion protein of a novel polypeptide-crystallizable fragment domain heavy chain. The preparation method can cause antibody-dependent cellular cytotoxicity, improve a cytokine-induced killing cell proliferation multiple to 1000 times or more, a CD3+ / CD56+ expression rate to 40% or more and a CD16+ expression rate to 30% or more. In-vivo and in-vitro tests prove that the cytokine-induced killing cell has strong tumor killing toxicity. The invention also provides a kit for autologous cytokine-induced killing cell proliferation culture and has a high-efficiency anti-tumor function.

Description

technical field [0001] The present invention relates to a method for preparing cytokine-induced killer cells that mediate antibody-dependent cytotoxicity, which includes the following features: constructing interferon gamma signal peptide (Interferon gamma signal peptide, IFN gamma-SP) and tumor cell binding polypeptide ( Tumor cells binding peptide (TCBP) and link the second and third constant domain (Ch2 and Ch3) gene fragments of the heavy chain of Fragment crystallizable domain (Fc) heavy chain, recombine into viral vector and transfect Human cytokine-induced killer cells (Cytokine-induced killer cells, CIK), high expression of the new peptide-crystallizable fragment domain heavy chain 2 and 3 constant region fusion protein, can trigger antibody-dependent cytotoxic activity, in vivo and in vitro The test anti-tumor has strong killing toxicity; the present invention also provides a kit containing the autologous cytokines obtained by the above-mentioned method to induce the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/62C12N5/10
CPCC07K14/57C07K2319/02C07K2319/30C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 陈建勋黄启明
Owner ZICHENG RUISHENGHUI BEIJING BIOTECH DEV CO LTD
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