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

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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 experimenta

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
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Example Embodiment

[0039] Example 1

[0040] This embodiment provides a method of constructing a CTLA4 gene and a PD1 gene for humanized BALB / C mouse model comprising the following model construct:

[0041] The PD1 gene-modified humanized mouse model was constructed according to the method of patent CN 109266656 A, and then the expression of CTLA4 gene was used in accordance with Patent CN 109022443 A, and then constructed to carry CTLA4 human sources according to the obtained SGRNA. The carrier of the sequence is then injected with the carrier of the BALB / C mouse CTLA4 gene with the carrier carrying a CTLA4 human sequence, and CAS9 mRNA or Cas9 protein to the above-mentioned humanized mouse-modified humanized rats. The quality or nucleus is transplanted into the mouse to produce the Human mouse model in the simultaneous modification of the Mothermouse and CTLA4 gene. The resulting mouse model was named BALB / C-HPD1 / HCTLA4 mice (i.e., BALB / C mice described in this application). BALB / C-HPD...

Example Embodiment

[0044] Example 2

[0045] Toxicological evaluation tests were carried out using BALB / C-HPD1 / HCTLA4 adult mice constructed in Example 1. 120, 5-6 week old, female. All data is made via GraphPad Prism. Two groups of data are differential analysis by Unpaired TWO-TAILED STUDENT'S TEST. YERVOY ANALOG Supplier BioIntron, KEYTRUDA supplier MSD Ireland, Yervoy supplier BRISTOL-MYERS SQUIBB.

[0046] 5-6 week old BALB / C-HPD1 / HCTLA4 mice were given a KEYTRUDA antibody, Yervoy antibody, Yervoy Analog, or Keytruda antibody and Yervoy antibody, KEYTRUDA antibody, and YervoyanAlog combined with administration. Through the method of intraperitoneal injection (I.p.), the number of administrations is shown in Table 1 every 3 days. On the 9th day after administration or day 21, mice were erect, serum, organized organ collection, and testing toxicological correlation indicators.

[0047] IgG 1, IgG 4 is an independent antibody for blank control; "Q3D X 3" means once every 3 days; Q3D × 7 me...

Example Embodiment

[0050] Example 3

[0051] This example gives the expression of inflammatory cytokines in plasma of mice after administration. The first group (G1) to 6 (G6) perform eyelid blood collection at D0 and D9; G7-G12 groups perform eyelid blood collection in D0, D21, blood separation plasma, using CBA (Cytometric Bead Array) kits (BD, 560485) Cell factor detection.

[0052] Cell factor test results figure 1 As shown in the control group, day 9 and 21, Yevoy + keytruda combined with a group treated BALB / C-HPD1 / HCTLA4 mouse plasma hyperficial factor IL-4, IL-6, IFN-R The expression of IL-17A indicates that after the joint treatment of PD1 / CTLA4 antibody, mice showed immunization-related inflammatory response, similar to the immune-related side effects caused by the patient after treatment with clinical HPD1 / HCTLA4 antibody .

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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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