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Immunologic effector cell targeting CPC3 and application thereof

A technology of immune response cells and cells, applied in blood/immune system cells, genetically modified cells, applications, etc., can solve problems such as death, weight loss, and side effects

Pending Publication Date: 2019-03-15
CRAGE MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in clinical trials, it was found that when IL12 was administered systemically, serious side effects occurred in multiple organs, such as abnormal liver function, high fever, severe hemodynamic instability, etc., and severe cases led to death, which limited the application of IL12
[0004] In addition, due to the particularity of tumors, especially solid tumors, the microenvironment of each tumor is completely different. Whether the immune effector cells co-expressing IL12 can enhance the anti-tumor effect, especially significantly enhance the anti-tumor effect is unknown, and will Cause certain side effects, such as weight loss in experimental mice
In addition, the combination of CAR T cells and other cytokines also has defects such as large side effects or poor in vivo effects.
Therefore, it is not reasonably predictable what effect the combination of specific chimeric antigen receptor immune response cells and specific cytokines will have

Method used

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  • Immunologic effector cell targeting CPC3 and application thereof
  • Immunologic effector cell targeting CPC3 and application thereof
  • Immunologic effector cell targeting CPC3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] Example 1. Preparation of GPC3-CAR-T cells / IL12-GPC3-CAR T cells

[0182] 1. Plasmid construction

[0183] The chimeric antigen receptor used in this example is a second-generation chimeric antigen receptor, and the nucleotide coding sequence of the scFv targeting the extracellular domain of the GPC3 receptor is shown in SEQ ID NO: 1, Also has the transmembrane domain of CD28, the intracellular domain of CD28, and CD3ζ. refer to Figure 1A As indicated, construct the plasmids of GPC3-CAR-T cells and IL12-GPC3-CAR T cells, respectively, as follows:

[0184] The GPC3-CAR-T sequence consists of CD8α signal peptide (SEQ ID NO: 2), scFv targeting GPC3 (SEQ ID NO: 1), CD8 hinge region (SEQ ID NO: 3), CD28 transmembrane region (SEQ ID NO: 6) and the intracellular signaling domain (SEQ ID NO: 4) and the intracellular segment of CD3 CD3ζ (SEQ ID NO: 5).

[0185] The IL12-GPC3-CAR plasmid is based on the GPC3-CAR-T plasmid with the NFAT6-IL12 sequence inserted, and a second-ge...

Embodiment 2

[0205] Example 2, Cytotoxicity assay targeting GPC3CAR-T / IL12-GPC3-CAR T cells

[0206] The CytoTox 96 non-radioactive cytotoxicity detection kit (Promega Company) was used to detect cytotoxicity, specifically referring to the instructions of the CytoTox 96 non-radioactive cytotoxicity detection kit.

[0207] 1) Target cells: select Huh-7, PLC / PRF / 5, and SK-HEP-1 cells as target cells, wherein Huh-7 cells, PLC / PRF / 5 cells are GPC3 positive, and SK-HEP-1 cells are GPC3 negative;

[0208] 2) Effector cells: add UTD, GPC3-CAR-T and IL12-GPC3-CAR T cells according to the effect-to-target ratio of 3:1, 1:1 or 1:3;

[0209] Experimental results such as figure 2 As shown, compared with the UTD group, GPC3-CAR-T / IL12-GPC3-CAR T showed strong cytotoxicity in the in vitro toxicity test, and there was no significant difference between the two. For GPC3-negative liver cancer cells, GPC3-CAR-T / IL12-GPC3-CAR T did not enhance the killing effect.

Embodiment 3

[0210] Example 3, GPC3-CAR-T / IL12-GPC3-CAR T cytokine secretion

[0211] UTD / GPC3-CAR-T / IL12-GPC3-CAR T was co-incubated with liver cancer cell lines Huh-7, PLC / PRF / 5, SK-HEP-1 cells at a ratio of 1:1 for 24 hours, then the supernatant was collected, and the supernatant was detected by ELISA Cytokine secretion levels.

[0212] The samples for detecting TNF-α and IL12 do not need to be diluted, and the samples for detecting IL2 and IFN-γ are diluted 20 times and 25 times respectively. The ELISA kit adopts double-antibody sandwich enzyme-linked immunosorbent detection technology. The results are shown in Figures 3A and 3B. Figure 3A It shows that IL12-GPC3-CAR T has a higher level of IL12 secretion when co-incubating with GPC3+ tumor cells Huh-7 and PLC / PRF / 5, and almost IL12 could not be detected, indicating that the CAR-T cells could secrete IL12 only when GPC3 was recognized. Figure 3B In order to detect the release of cytokines IL-2, TNF-α, and IFN-γ when UTD / GPC3-CAR-...

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Abstract

The invention relates to an immunologic effector cell which expresses a chimeric antigen receptor targeting GPC3 and exogenous IL12. The invention also provides a medicinal composition containing theimmunologic effector and a method for treating tumor, particularly GPC3 positive tumor through the immunologic effector cell or the medicinal composition. The immunologic effector cell is effective toentity tumor cell in vitro, and is outstanding in effect of extinguishing the entity tumor cell in vivo.

Description

technical field [0001] The invention belongs to the field of cellular immunotherapy, in particular to a gene-modified immune effector cell targeting GPC3. Background technique [0002] Chimeric antigen receptor engineered T lymphocyte (CAR-T) technology is a new strategy for tumor biological therapy that has emerged in recent years. Chimeric antigen receptor (Chimeric antigen receptor, CAR) is a combination of single-chain antibodies that recognize tumor-associated antigens and immune cell activation motifs, and T lymphocytes are genetically engineered to express chimeric antigen receptors in vitro. Endows T cells with the ability to specifically recognize and kill tumor cells. At present, CAR-T has achieved good results in the treatment of hematological malignancies, for example, it has achieved great success in patients with CD19-positive B lymphocytic leukemia. However, the application of CAR-T in the treatment of solid tumors still faces great challenges. [0003] Int...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/861A61K35/17A61P35/00
CPCA61P35/00A61K35/17C12N5/0636C12N5/0637C12N5/0646C12N15/86C12N2740/10043C12N2510/02C12N2710/10043C12N2740/15043
Inventor 李宗海蒋华
Owner CRAGE MEDICAL CO LTD
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