Development Of A Novel Humanized Mouse Model For Development Antitumor Drug For Preclinical Evaluation Of CD36 Targeted Drugs

ObjectivesTo construct novel humanized CD36 mice by gene editing technology（Unless otherwise stated,hCD36 mice below refer to homozygous hCD36 mice）and characterize the phenotype of the mice.Evaluation of the effect of humanized editing on immune function,blood cell composition,morphology,and vital organ function in hCD36 mice by gene editing technique.To validate that hCD36 mice can be used to evaluate the efficacy of tumor immunotherapeutic antibody drugs targeting CD36.MethodsExperiment 1:Based on the C57BL/6 mouse background,heterozygous humanized CD36 mice were obtained by replacing exons 4 to 15 of the extracellular region of CD36 with human exons 3 to 14 by gene editing techniques.The heterozygous mice were mated for expansion to obtain hCD36 mice.The mRNA expression of CD36in the lung tissue was measured by RT-PCR in 7-week-old female C57BL/6 mice and hCD36 mice.Experiment 2:Protein expression of CD36 on peritoneal exudative macrophages from 7-week-old female C57BL/6 mice and hCD36 mice was measured by flow cytometry.CD36 expression on immune cells such as macrophages,granulocytes,B cells,and T cells in the peripheral blood of 7-week-old female C57BL/6 mice and hCD36 mice was then further examined.Experiment 3:Immunophenotyping of leukocyte subsets and T-cell subsets in the spleen,blood,and mesenteric lymph nodes of 8-week-old female C57BL/6 mice and hCD36 mice by flow cytometry.Blood biochemistry and routine blood tests were performed in 8-week-old female C57BL/6 mice and hCD36 mice using a mouse fully automatic biochemical analyzer and a mouse fully automated modular blood fluid analyzer.Experiment 4:The PDX model was established by subcutaneously inoculating mouse colon cancer MC38 cells at a volume of 0.1 ml/each into the right dorsum of thirty female hCD36 mice at 7 weeks of age.The tumor volume was measured once a day from day 0 after inoculation,and the tumor volume was calculated by the formula:0.5×long diameter×short diameter².The mice were randomly divided into three groups of six mice each after the tumors had grown to a volume of about 100 mm³.The three groups of mice were injected with PBS in the tail vein（G1）,1G04 at 3 mg/kg（G2）and anti-CD36 chimeric human-mouse monoclonal antibody（subsequently abbreviated as 1G04 which was made in house.）at 10 mg/kg（G3）,and the dosing frequency was twice a week for a total of six doses.During the experiment,the health and behavior of the animals were monitored once a day.We measured the tumor volume and weight of the animals twice a week.At the end of the experiment,the animals were euthanized and the relative tumor volume growth inhibition rate(TGI_TV)was measured.TGI_TV was used to evaluate the anti-tumor efficacy.The TGI_TVin percent was calculated as below:TGI_TV（%）=[1-（T_i-T₀）/（C_i-C₀）]×100;Where T_i=mean tumor volume of the drug-treated group on the final day of the study,T₀=mean tumor volume of the drug-treated group on first dosing day,C_i=mean tumor volume of the control group on the final day of the study,C₀=mean tumor volume of the control group on the first dosing day.ResultsExperiment 1:Only mouse CD36 mRNA expression was detected in C57BL/6mice,whereas only human CD36 mRNA expression was detected in hCD36 mice.Experiment 2:Only mouse CD36 protein was identified on the surface of peritoneal exudative macrophages in C57BL/6 mice,and only human CD36 protein expression was identified in hCD36 mice.hCD36 mice also had only human CD36protein expression identified on immune cells such as macrophages,granulocytes,B cells,and T cells in peripheral blood,which is consistent withCD36 protein expression on immune cells in human peripheral blood.Experiment 3:The development,differentiation,and distribution of leukocyte subpopulations and T cell subpopulations in the spleen,blood,and mesenteric lymph nodes of hCD36 mice were not statistically different from those of C57BL/6 mice（P>0.05）.The routine blood and blood biochemistry of hCD36 mice were also not statistically different from those of C57BL/6 mice（P>0.05）.Experiment 4:The TGI_TV（%）of the G2 group was 31.6%and the TGI_TV（%）of the G3 group was 47.3%compared to the control group（P<0.05）.In addition,there was no significant change in the body weight of the experimental animals in both the G2and G3 groups compared to the control group（P>0.05）.ConclusionThe hCD36 mice were constructed by gene editing technology to express only human CD36 mRNA and CD36 protein.hCD36 mice were successfully generated without affecting the immune system of the mice,without altering the composition and morphology of blood cells,and without affecting the health of organs such as the heart,liver,and kidney.In vivo,efficacy successfully validated the inhibitory effect of 1G04on colon cancer tumor growth in hCD36 mice.hCD36 mice serve as a validated preclinical mouse model for immunotherapeutic studies targeting CD36.