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Application of CTLA4 gene and PD1 gene humanized mouse model

A mouse model, humanized technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of complex operation, unstable results, and high experimental costs.

Pending Publication Date: 2020-11-17
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of this evaluation method has the problems of unstable results and complicated operation: 1) young mice need to be operated in breeding cages, and the experimental cost is high; 2) the toxicological reaction of young mice to antibodies is different from that of clinical drugs. inter-measured differences in toxicological reactions in adults

Method used

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  • Application of CTLA4 gene and PD1 gene humanized mouse model
  • Application of CTLA4 gene and PD1 gene humanized mouse model
  • Application of CTLA4 gene and PD1 gene humanized mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This embodiment provides a method for constructing a CTLA4 gene and PD1 gene humanized BALB / c mouse model, which includes the following model construction method:

[0041] According to the method of patent CN 109266656 A, a humanized mouse model of PD1 gene modification was constructed, and then according to the method of patent CN 109022443 A, an sgRNA expressing the CTLA4 gene of BALB / c mice was constructed, and then according to the obtained sgRNA, a human source of CTLA4 was constructed. sequence carrier, and then inject the sgRNA against the BALB / c mouse CTLA4 gene and the carrier carrying the CTLA4 human sequence, as well as Cas9 mRNA or Cas9 protein, into the fertilized egg cells of the above-mentioned (patent CN 109266656 A) PD1 gene-modified humanized mouse Humanized mouse model in which the PD1 gene and CTLA4 gene are simultaneously modified by transplantation into the recipient mother mouse in the cytoplasm or nucleus. The obtained mouse model is named BALB / c...

Embodiment 2

[0045] BALB / c-hPD1 / hCTLA4 adult mice constructed in Example 1 were used for toxicological evaluation test, 120 of them were 5-6 weeks old, female. All data were plotted by GraphPad Prism. The data between the two groups were analyzed by unpaired two-tailed Student's t-test. The supplier of Yervoy analog is BIOINTRON, the supplier of Keytruda is MSD Ireland, and the supplier of Yervoy is Bristol-Myers Squibb.

[0046] BALB / c-hPD1 / hCTLA4 mice aged 5-6 weeks were treated with Keytruda antibody, Yervoy antibody, Yervoy analog alone, or in combination with Keytruda antibody and Yervoy antibody, or in combination with Keytruda antibody and Yervoyanalog. By intraperitoneal injection (i.p.), once every 3 days, the administration frequency is shown in Table 1. On the 9th or 21st day after administration, the mice were euthanized, serum, tissues and organs were collected, and toxicology-related indicators were tested.

[0047] IgG 1 and IgG 4 are irrelevant antibodies, used for blank...

Embodiment 3

[0051] This example shows the expression of inflammatory cytokines in plasma of model mice after administration. Group 1 (G1) to Group 6 (G6) received orbital blood collection at D0 and D9; groups G7-G12 received orbital blood collection at D0 and D21, and separated blood plasma using CBA (Cytometric Bead Array) kit (BD, 560485) ) for cytokine detection.

[0052] Cytokine test results refer to figure 1 As shown, compared with the control group, on the 9th and 21st day, the inflammatory factors IL-4, IL-6, IFN-r , the expression of IL-17A increased, indicating that after the combined treatment of PD1 / CTLA4 antibody, the mice showed an immune-related inflammatory response, which was similar to the immune-related side effect phenotype caused by clinical hPD1 / hCTLA4 antibody drug treatment in patients .

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Abstract

The invention discloses application of a CTLA4 gene and PD1 gene humanized mouse model, and relates to the field of animal gene engineering and gene genetic modification. The CTLA4 and PD1 immune checkpoint humanized model with the BALB / c background is constructed and obtained, the model can reproduce toxic reactions of CTLA4 antibody drugs, PD1 antibody drugs and combined drugs, the problem thatother rodent mice cannot be subjected to toxicity evaluation is solved, meanwhile, the model is not as high as primate in experimental cost, and the model fills the market blank of PD1 and CTLA4 antibody safety evaluation of the rodents. The model constructed by adopting the model construction method can be used for evaluating the effectiveness of the targeted drug, screening and developing the targeted drug, evaluating the anti-tumor efficacy of the targeted drug in combination with other drugs, or researching the toxicology of the targeted drug.

Description

technical field [0001] The present invention relates to the field of animal genetic engineering and genetic modification, in particular to the application of a CTLA4 gene and PD1 gene humanized mouse model. Background technique [0002] As early as 2013, Science magazine rated tumor immunotherapy as the first of the top 10 scientific breakthroughs, among which the immune checkpoint therapy (Immuno-checkpoint therapy) headed by PD1 / PDL1 has shown significant effects in the clinical treatment of various cancers. Antitumor efficacy. At present, there are successful drugs on the market and used in clinical practice. Merck’s PD-1 humanized antibody Keytruda (Pembrolizumab, MK-3475) and Bristol-Myers Squibb’s PD-1 antibody Opdivo (Nivolumab) were approved by the FDA in 2014 The marketed first-line tumor treatment drug has a robust anti-tumor effect. Anti-CTLA4 monoclonal antibodies have also achieved excellent anti-tumor therapeutic effects in mouse models and clinical patients....

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/90C12N15/85A01K67/027
CPCC07K14/70521C12N15/907C12N15/8509A01K67/0278A01K2217/072A01K2227/105A01K2267/03
Inventor 赵静琚存祥梁娟
Owner GEMPHARMATECH CO LTD
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