Combination of TOLL-like receptor stimulating agent and immune effector cell

A technology of immune effector cells and receptor agonists, applied to receptors/cell surface antigens/cell surface determinants, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc.

Pending Publication Date: 2018-11-23
CRAGE MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, due to the complexity of the tumor microenvironment, in the treatment of tumors,...

Method used

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  • Combination of TOLL-like receptor stimulating agent and immune effector cell
  • Combination of TOLL-like receptor stimulating agent and immune effector cell
  • Combination of TOLL-like receptor stimulating agent and immune effector cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0223] Example 1. Preparation of CAR-T cells

[0224] In this example, EGFRvIII is selected as the target of CAR-T cells. In order to more accurately verify the anti-tumor effect in mice, the signal peptide, transmembrane region, and intracellular region selected are of mouse origin. The preparation method is operated according to the conventional CAR-T cell preparation method in the art.

[0225] 1. Retroviral vector construction

[0226] Mouse CD8α signal peptide (SEQID NO: 1), anti-EGFRvIII monoclonal antibody (SEQID NO: 2), CD8α glue chain region and transmembrane region (SEQID NO: 3), CD28 intracellular domain (SEQID NO: 4) , CD3ζ intracellular domain (SEQID NO: 5) were sequentially connected, and the EGFRvIII-28Z gene fragment was obtained by in vitro gene synthesis, and the IRES- GFP fragment, the recombinant vector MSCV-EGFRvIII-28Z was obtained. The composition sequence of each component fragment is as follows figure 1 shown.

[0227] 2. Preparation of retrovirus...

Embodiment 2

[0238] Example 2. In vitro toxicity of EGFRvIII-28Z CAR T lymphocytes

[0239] Mouse colon cancer cell CT26 (purchased from American Type Culture Collection (ATCC)) and mouse pancreatic cancer cell E0771 (obtained from Baylor College of Medicine) were prepared by conventional techniques in the field to obtain CT26 overexpressing EGFRvIII - EGFRvIII and E0771-EGFRvIII.

[0240] CytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, USA) was used to measure the in vitro cytotoxicity of EGFRvIII-28Z CAR T lymphocytes to target cells. Adjust the concentration of target cells CT26, CT26-EGFRvIII, E0771, and E0771-EGFRvIII to 2×105 / mL, take 50 μL and inoculate them into 96-well plates; inject them into 96-well plates according to the effect-to-target ratio of 0.3:1, 1:1 and 3:1 Add CAR-T cells and control T cells (50ul), set each control well according to the kit instructions, and set 5 duplicate wells for each group; incubate for 18 hours. After incubation, the in v...

Embodiment 3

[0242] Example 3. Anti-tumor effect of EGFRvIII-28Z CAR-T combined with poly I:C in mouse breast cancer model

[0243] Inoculate 1×106E0771-EGFRvIII cells into the fourth pair of mammary gland fat pads of 6-week-old female C57 mice (weight 18g-24g, purchased from Shanghai Lingchang Biotechnology Co., Ltd.); 13 days after tumor inoculation, the tumor-bearing mice Divided into 4 groups, divided into UT cell group, UT+poly I:C group, EGFRvIII-28Z CAR-T cell group and EGFRvIII-28Z CAR-T+poly I:C group, the average tumor volume of each group was about 150mm 3 ; After grouping, infuse 5.0×10 through the tail vein 6 EGFRvIII-28Z CAR-T cells (injection day is day 0), and the U T cell group was used as the control, 50ug polyI:C was injected intratumorally on the 3rd and 6th day after the CAR-T cell reinfusion, and the UT cell group was injected intratumorally same dose of normal saline. Observe the growth of transplanted tumor, the results are as follows: Figure 4A shown.

[0244]...

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Abstract

The invention provides use of a combination of a TOLL-like receptor stimulating agent and an immune effector cell in preparation of antitumor drugs or anti-pathogen drugs or anti-immunosuppression drugs. The TOLL-like receptor stimulating agent can be used for reinforcing the effect of the immune effector cell. The invention further provides a composition of the TOLL-like receptor stimulating agent and the immune effector cell.

Description

[0001] This application claims the priority of two patents CN201710344558.5 (application date: 2017.5.16) and CN201710561825.4 (application date: 2017.7.11), which are all incorporated herein by reference. technical field [0002] The invention belongs to the field of immunotherapy and relates to the combined application of immune effector cells with targeted recognition of tumor antigens or pathogen antigens and TOLL-like receptor agonists. Background technique [0003] In recent years, genetically engineered immune cells have been increasingly used in the field of tumor treatment or infectious disease treatment, such as chimeric antigen receptor-modified T cells (CAR-T cells), chimeric antigen receptor-modified NK cells (CAR-NK cells), TCR-modified T cells (TCR-T), etc. [0004] However, due to the complexity of the tumor microenvironment, in the treatment of tumors, especially solid tumors, immune effector cells usually cannot play an anti-tumor effect well in vivo. Con...

Claims

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Application Information

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IPC IPC(8): A61K35/17A61K31/713A61K47/64A61P35/00A61P37/04
CPCA61K31/713A61K35/17A61K47/6425A61P35/00A61P37/04A61K39/0011A61K2039/5156A61K2039/5158A61K39/00A61K47/64C07K14/7051C07K2319/03C12N5/10C12N15/62
Inventor 李宗海狄升蒙
Owner CRAGE MEDICAL CO LTD
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