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Chimeric protein, immune effector cell expressing chimeric protein and application thereof

A technology of immune effector cells and chimeric proteins, applied in the field of chimeric proteins and genetically engineered immune effector cells, can solve the problems of lack of immune cell proliferation and surviving immune effector cells, and insignificant efficacy enhancement

Pending Publication Date: 2019-06-14
CRAGE MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wang et al. (“Augmented anti-tumor activity of NK-92 cells expressing chimeric receptors of TGF-βR II and NKG2D”, Cancer Immunol Immunother, 2017) reported chimerism of the extracellular domain of TGF-β receptor II with the intracellular domain of NKG2D , the chimeric receptor can enhance the secretion of IFNγ by NK-92 cells and the killing of target cells, but its therapeutic effect is not significantly enhanced
[0004] In the prior art, there is a lack of immune effector cells that can effectively promote the proliferation and survival of immune cells, which has been a research topic in this field for a long time

Method used

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  • Chimeric protein, immune effector cell expressing chimeric protein and application thereof
  • Chimeric protein, immune effector cell expressing chimeric protein and application thereof
  • Chimeric protein, immune effector cell expressing chimeric protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1. Construction of vectors expressing chimeric proteins

[0133] Follow the steps below to build a build that includes figure 1 Plasmids expressing chimeric proteins of the indicated constructs.

[0134] (1) Human TGF-β receptor I signal peptide (SEQ ID NO: 2), extracellular domain of TGF-β receptor I (SEQ ID NO: 4) and human IL-2RG transmembrane region (SEQ ID NO: 6), the coding nucleotide sequence of the IL-2RG intracellular domain (SEQ ID NO:8) is connected, and the nucleotide sequence (SEQ ID NO:37) of the first chimeric protein chIL2RG is constructed, and its coded amino acid sequence is as shown in SEQ IDNO:27).

[0135] (2) Human TGF-β receptor II signal peptide (SEQ ID NO:10), TGF-β receptor II extracellular domain (SEQ ID NO:12) and human IL-2Rβ transmembrane region (SEQ ID NO:14 ), the coding nucleotide sequence of IL-2Rβ intracellular region (SEQ ID NO: 16) was connected to construct the nucleotide sequence (SEQ ID NO: 38) of the second chimeric pr...

Embodiment 2

[0139] Example 2. Determination of T cell phosphorylation level

[0140] The constructed lentivirus plasmid 1, lentivirus plasmid 2, and lentivirus plasmid 3 were respectively co-transfected with lentivirus packaging plasmids into HEK-293T cells to prepare corresponding lentiviruses, which are respectively denoted as lentivirus 1, lentivirus 2, lentivirus 2, Lentivirus3.

[0141] Human PBMC were cultured in AIM-V medium, added with 2% human AB serum, 500 U / mL recombinant human IL-2, and activated with CD3 / CD28 antibody combined with magnetic beads for 48 hours to obtain activated T cells. After T cells were activated, they were infected with lentivirus 1, lentivirus 2, and lentivirus 3, respectively, to obtain T cells chTR2, chTR7, and chTR21 expressing chimeric proteins.

[0142] Remove serum and rest for 24 hours, take blank T cell UTD (untransfected T cell, Untransfected) as blank control, use UTD, chTR2, chTR7, chTR21 cells, divide into TGF-β1 positive group (+) and negat...

Embodiment 3

[0144] Example 3. Effects of T cells expressing chimeric proteins on the differentiation level of Treg under the stimulation of recombinant human TGF-β1

[0145] Human PBMC were cultured in AIM-V medium, and 2% human AB serum and 500 U / mL recombinant human IL-2 were added to obtain the corresponding cell culture medium. The T cells chTR7 and chTR21 obtained in Example 2 and the empty control T cell UTD were cultured in the above cell culture medium, treated with recombinant human TGF-β1 (5 ng / mL) and cultured for 4 days, and the cells were collected to label Treg markers with antibodies CD4, CD25, and Foxp3 were detected by flow cytometry, and the test group not treated with recombinant human TGF-β1 was used as a control (CTRL). The result is as Figure 4A and 4B As shown, UTD cells stimulated by TGF-β1 Foxp3 + CD4 + CD25 + The proportion of the population increased, indicating that TGF-β1 induced Treg differentiation, while the Foxp3 of the cells in the chimeric receptor...

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Abstract

The present invention relates to an immune effector cell expressing a chimeric protein and an application thereof, in particular to the immune effector cell of the chimeric protein expressing an extracellular domain containing a TGF-beta receptor and an intracellular signal domain of IL-2 family protein, a pharmaceutical composition comprising the same, and the application of the immunological effector cell or a pharmaceutical composition as described above for preparing a drug for preventing or treating a tumor or pathogenic microorganism infection. The immune effector cell expressing the chimeric protein is capable of transforming the stimulation of TGF-beta into a positive signal of IL-2, IL-7 or IL-21, inhibiting TGF-beta-induced Treg differentiation, and promoting the proliferation and survival of immune cells; also reduces the effect of TGF-beta on cell killing ability, and shows a better anti-tumor ability.

Description

technical field [0001] The invention belongs to the field of adoptive cell therapy; specifically, the invention relates to a chimeric protein and genetically engineered immune effector cells, which can significantly enhance the proliferation and survival of immune cells, and can enhance their cell killing ability . Background technique [0002] In recent years, adoptive cell therapy such as CAR-T therapy, TCR-T therapy, etc. have shown good results in the field of hematological tumors, but the treatment of solid tumors is lacklustre, because cancer cells in solid tumors can The tumor microenvironment is formed to support the growth and metastasis of cancer cells. Immunosuppressive cytokines such as IL-4, IL-10, and TGF-β, which are abundantly expressed in the tumor microenvironment, inhibit the anti-tumor activity of CAR-T cells. Moreover, the presence of TGF-β can also induce T cells to differentiate into regulatory T cells (Treg), and the regulatory T cells can further i...

Claims

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Application Information

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IPC IPC(8): C12N5/10C07K19/00A61K35/17A61P35/00A61P31/00
CPCA61P31/00A61P35/00C07K19/00C12N5/10C07K2319/03C07K14/7051A61K2239/28A61K39/464474A61K39/4631A61K39/4611A61K2239/53A61K39/4621
Inventor 李宗海王益
Owner CRAGE MEDICAL CO LTD
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