Construction Of A B Cell-deficient Mouse Model Based On Igh Gene Modification And Its Efficacy Verification

Objective:In this study,a B cell-deficient hPD-1/hPD-L1,Igh-J KO mouse model was generated by gene editing technology and validated by in vivo and in vitro experiments,aiming to establish an animal model without immunogenicity of large molecule biological drugs,and to provide an efficient and rational model for the screening of antibody drug candidates and preclinical efficacy evaluation studies.Methods:1.B cell-deficient hPD-1/hPD-L1,Igh-J KO mice were generated using gene editing technology.2.PCR was used to detect the genotype of the offspring mice to determine whether the offspring mice were the target mice.Mouse Ig M & Ig D protein expression in the spleen and bone marrow cells of the mice were analyzed by flow cytometry to determine whether the B cells were missing in hPD-1/hPD-L1,Igh-J KO mice.3.Flow cytometry was used to analyze changes in leukocyte subsets and T cell subsets in the spleen,peripheral blood and lymph nodes so as to assess the effect of B cell-deficient on the distribution of various lymphocyte subpopulations in hPD-1/hPD-L1,Igh-J KO mice.4.Levels of granzyme B,gamma interferon and proliferation marker Ki-67 in the spleen and peripheral blood of hPD-1/hPD-L1,Igh-J KO mice were analyzed by flow cytometry to assess whether hPD-1/hPD-L1,Igh-J KO mice affect the proliferation and activation of NK cells and T cells in the mice.5.A mouse model of MC38 colon cancer was established and M7824 analogue was administered intraperitoneally to hPD-1/hPD-L1,Igh-J KO tumor-bearing mice and normal B cell-competent hPD-1/hPD-L1 tumor-bearing mice to observe the changes of tumor volume and body weight in both types of mice.6.Enzyme-linked immunosorbent assay was used to determine M7824 analogue and anti-M7824 analogue antibody levels in mouse serum to assess whether hPD-1/hPDL1,Igh-J KO mice could avoid the immunogenic effects of M7824 and also showed anti-tumor effects of M7824.Results:1.PCR results showed that only human PD-1,human PD-L1 and Igh-J were detected in hPD-1/hPD-L1,Igh-J KO mice compared to wild-type C57BL/6 mice,while murine PD-1 and murine PD-L1 were not detected.2.Flow cytometry results showed that:(1)Mouse Ig M and Ig D could be detected in wild-type C57BL/6 mice but not in hPD-1/hPD-L1,Igh-J KO mice.(2)Only murine PD-1 was detectable in wild-type C57BL/6 mice,and only human PD-1 was detectable in hPD-1/hPD-L1,Igh-J KO mice.(3)Only murine PD-L1 was detectable in wild-type C57BL/6 mice,and only human PD-L1 was detectable in hPD-1/hPD-L1,Igh-J KO mice.3.Flow cytometry results showed that:(1)Analysis of splenic leukocyte subpopulations revealed a failure of B cell development in hPD-1/hPD-L1,Igh-J KO mice,resulting in the absence of B cells and the proportion of T cells,granulocytes and monocytes increased significantly(P(27)0.001)in this mouse,while other cells such as dendritic cells and macrophages were not detected due to the low cell content.(2)Analysis of splenic T cell subsets revealed that the percentage of CD8+ T cells,the percentage of CD4+ T cells,and the percentage of regulatory T cells in hPD-1/hPDL1,Igh-J KO mice were similar to those in C57BL/6 mice without significant changes.(3)The same results were obtained in lymph nodes and blood.4.Flow cytometry results showed that:(1)The levels of granzyme B and gamma interferon secreted by NK cells in peripheral blood and splenocytes were significantly higher in hPD-1/hPD-L1,Igh-J KO mice and hPD-1/hPD-L1 mice after polyinosinic acid cytidylic acid stimulation compared to mice that did not receive polyinosinic acid cytidylic acid(P(27)0.01).(2)The levels of granzyme B,gamma interferon and Ki-67 secreted by CD4+ T cells and CD8+ T cells in splenocytes were significantly higher in hPD-1/hPD-L1,Igh-J KO mice and hPD-1/hPD-L1 mice after anti-CD3ε antibody stimulation compared to mice that did not receive anti-CD3ε antibody(P<0.01).5.The results of in vivo experiments showed that:(1)Under the same administration conditions,M7824 showed significant anti-tumor effects in hPD-1/hPD-L1,Igh-J KO tumor-bearing mice(TGI=69.2%)compared to hPD-1/hPD-L1 tumor-bearing mice with normal B cell function,while M7824 in hPD-1/hPD-L1 tumor-bearing mice showed no anti-tumor effect.(2)Compared to the control group,body weight changes before and after administration in hPD-1/hPD-L1,Igh-J KO tumor-bearing mice remained the same as those in B cell-normal hPD-1/hPD-L1 tumor-bearing mice,with no significant differences.6.In vitro results showed that:(1)Serum M7824 analogues could be detected in hPD-1/hPD-L1,Igh-J KO tumor-bearing mice,and the blood concentration was increased with the number of doses,while anti-M7824 analogues were undetectable.(2)Serum M7824 analogues were not detectable in hPD-1/hPD-L1 tumor-bearing mice after day10 of administration,and anti-M7824 analogues were detectable in all mice on day 6.Conclusion:1.The B cell-deficient hPD-1/hPD-L1,Igh-J KO mouse can be used as an effective and reliable screening animal model for tumor immunological drugs,retaining important immune cell types for immunotherapeutic evaluation while not producing anti-drug antibodies.2.The B cell-deficient hPD-1/hPD-L1,Igh-J KO mouse has only B-cell deficiency,while the proliferation and activation of NK cells and T cells in the mice were not affected,indicating that this mouse model we established can be used for NK cell and T cell-related immunotherapy studies.3.The B cell-deficient hPD-1/hPD-L1,Igh-J KO mouse models can effectively reduce or even avoid immunogenicity and contribute to the screening and efficacy assessment of antibody drugs.