| Objective:Since C5AR1 targets play a role in tumour development,blocking C5AR1 has become a new strategy for the treatment of tumours,leading to the continuous development of C5AR1-targeted drugs.In order to reduce the development cost of C5AR1-targeted drugs and accelerate the development process of C5AR1-targeted drugs,it is urgent to develop a humanized mouse model for preclinical validation of human C5AR1-targeted drugs.In this study,we first constructed and validated the C5AR1 humanized(hC5AR1)mice.Then we validated C5/C5AR1 humanized(hC5/hC5AR1)mice and mouse colon cancer MC38 cell lines with high expression of human C5 protein(hC5 MC38)in order to construct an environment suitable for tumour growth with highC5a expression.Finally,since immune checkpoint inhibitors and C5AR1-targeted drugs can simultaneously enhance T-cell effects and inhibit tumour growth,PD-1/PD-L1/C5AR1 humanized(hPD-1/hPD-L1/hC5AR1)mice and MC38 cell lines with high expression of human PD-L1 protein(hPD-L1 MC38)were analyzed.The cell lines were also sequentially inoculated into humanized mice to construct tumour models,administrated C5AR1antibody drugs and analyzed for efficacy to determine whether they could be used to validate the efficacy and safety of C5AR1-targeted drugs for use in humans.Methods:1.Analysis of the expression of C5a/C5AR1:cultured mouse colon cancer cell lines(MC38 cell lines)and tumor tissues growing to approximately 100 mm~3after inoculation of MC38 cell lines on C57BL/6 wild-type mice were taken,and expression of murine-derived C5AR1 protein in tumour tissues and MC38 cell lines was detected using flow cytometry;supernatants of MC38 cell cultures and tumor tissue homogenate supernatant were collected,and the expression of complement C5a protein was detected using ELISA.2.Analysis of hC5AR1 mice.(1)Gene editing strategy in hC5AR1 mice:the mouse C5ar1 gene was replaced with the human C5AR1 gene in wild-type C57BL/6mice.(2)Expression of C5AR1 m RNA:Bone marrow from hC5AR1 mice was taken,total RNA was extracted,reverse transcription was performed and appropriate primers were selected for amplification.(3)Expression of C5AR1 protein:Bone marrow from hC5AR1 mice was taken,and the expression of human-derived C5AR1 protein was analyzed using anti-human C5AR1 antibody by flow cytometry.(4)Immune cell typing assay:spleen,peripheral blood and lymph nodes of hC5AR1 mice were taken and flow cytometry was used to analyze the difference of the proportions of T/B cells,NK cells,dendritic cells(DCs),monocytes/macrophages and T cell subpopulations CD4+T cells,CD8+T cells,Treg cells between hC5AR1 mice and wild-type mice using species-specific antibodies with fluorescent markers.(5)Tumor model construction and efficacy analysis:MC38 cell lines were inoculated into hC5AR1mice to construct an animal model of colon cancer,grouped and administrated the day before inoculation,weighed twice a week and tumor volume was measured to observe tumor growth trends and analyze efficacy.3.Analysis of hC5/hC5AR1 mice.(1)Gene editing strategy for hC5/hC5AR1mice and hC5 MC38 cell lines:In wild-type C57BL/6 mice,the C5 humanized(hC5)mice were constructed by replacing the murine C5 gene with the human C5 gene,and then mated with hC5AR1 mice to obtain hC5/hC5AR1 mice;the murine C5 gene was replaced with the human C5 gene in MC38 cell lines to construct the hC5 MC38 cell lines.(2)Expression of C5 and C5AR1 m RNA:Lung tissues from hC5/hC5AR1 mice were taken,total RNA was extracted,reverse transcription was performed and appropriate primers were selected for amplification,respectively.(3)Expression of C5,C5AR1 and C5a proteins:Bone marrow from hC5/hC5AR1 mice was taken,and the expression of human-derived C5AR1 protein was analyzed by flow cytometry using anti-human C5AR1 antibody;serum from hC5/hC5AR1 mice was taken,and the expression of C5 and C5a protein was analyzed using ELISA kits;culture supernatant from hC5 MC38 cells was taken,and the expression of human-derived C5protein was analyzed using ELISA kits.(4)Immune cell typing assay:The spleen,peripheral blood and lymph nodes of hC5/hC5AR1 mice were collected and analyzed in the same way as hC5AR1 mice.(5)Tumour model construction and efficacy analysis:hC5 MC38 cell line was inoculated into hC5/hC5AR1 mice to construct an animal model of colon cancer.hC5/hC5AR1 mice were grouped and administrated when the tumour volume reached 100-150 mm~3,the tumour volume was weighed and measured twice a week.4.Analysis of hPD-1/hPD-L1/hC5AR1 mice(1)Gene editing strategy for hPD-1/hPD-L1/hC5AR1 mice and hPD-L1 MC38 cell lines:In wild-type C57BL/6mice,PD-1 humanized(hPD-1)mice were constructed by replacing the murine-derived PD-1 gene with the human-derived PD-1 gene,and replacing the murine-derived PD-L1 gene with the human-derived PD-L1 gene to construct PD-L1humanized(hPD-L1)mice,mating the two mice with each other to obtain PD-1/PD-L1 humanized(hPD-1/hPD-L1)mice,and finally mating hC5AR1 mice with hPD-1/hPD-L1 mice to obtain hPD-1/hPD-L1/hC5AR1 mice;the murine-derived PD-L1 gene was replaced with the human-derived PD-L1 gene in MC38 cell lines to construct the hPD-L1 MC38 cell lines.(2)Expression of PD-1,PD-L1 and C5AR1 proteins:The bone marrow of hPD-1/hPD-L1/hC5AR1 mice was taken and the expression of human-derived C5AR1 protein was analyzed by flow cytometry using anti-human C5AR1 antibody;the spleen of hPD-1/hPD-L1/hC5AR1mice after 24 h of anti-mouse CD3 antibody stimulation was taken and the expression of human-derived PD-1,PD-L1 proteins were analyzed by flow cytometry using anti-human PD-1 and PD-L1 antibodies;hPD-L1 MC38 cells were taken to analyze the expression of human-derived PD-L1 proteins using anti-human PD-L1 antibodies by flow cytometry.(3)Immune cell typing assay:The spleen,peripheral blood and lymph nodes of hPD-1/hPD-L1/hC5AR1 mice were obtained in the same way as hC5/hC5AR1 mice.(4)Antibody binding assay,tumour model construction and efficacy analysis:The binding ability of the internally synthesized positive drug Avdoralimab to hPD-1/hPD-L1/hC5AR1 mice was analyzed by flow cytometry;hPD-L1 MC38 cell line was inoculated into hPD-1/hPD-L1/hC5AR1 mice to construct an animal model of colon cancer.Other methods were the same as for hC5/hC5AR1 mice.Results:1.Analysis of the expression of C5a/C5AR1:C5a was expressed in tumour tissue homogenate supernatant and C5AR1 was expressed in CD45+cells in tumour tissue,indicating that C5AR1 is expressed in immune cells in the tumour tissue microenvironment and not in tumour cells.2.Analysis of hC5AR1 mice.(1)hC5AR1 mice were genotypically identified to confirm the acquisition of successful gene editing positive mice.(2)hC5AR1 mice express only human C5AR1 m RNA,not mouse C5AR1 m RNA.(3)hC5AR1 mice express only human C5AR1 protein,not mouse C5AR1 protein.(4)Using wild-type mice as controls,the proportions of T/B cells,NK cells,dendritic cells(DCs),monocytes/macrophages,and T cell subpopulations CD4+T cells,CD8+T cells,and Treg cells in the spleen,lymph nodes,and peripheral blood of hC5AR1 mice were not significantly different from those of wild-type mice.(5)Tumour models in hC5AR1mice were successfully constructed,but without significant pharmacological effects.3.Analysis of hC5/hC5AR1 mice.(1)hC5/hC5AR1 mice and hC5 MC38 cell lines were genotyped to confirm the acquisition of successful gene editing positive mice and positive clones.(2)hC5/hC5AR1 mice express only human-derived C5,C5AR1 m RNA and not murine C5,C5AR1 m RNA.(3)hC5/hC5AR1 mice express only human-derived C5,C5a,C5AR1 protein and not murine C5,C5a,C5AR1protein;human-derived C5 protein was detected in the culture supernatant of the hC5MC38 cell lines.(4)The proportions of each immune cell in the spleen,lymph nodes and peripheral blood of hC5/hC5AR1 mice were not significantly different from those of wild mice when wild mice were used as controls.(5)The tumour model in hC5/hC5AR1 mice was successfully constructed but without pharmacological effect.4.Analysis of hPD-1/hPD-L1/hC5AR1 mice.(1)hPD-1/hPD-L1/hC5AR1 mice and hPD-L1 MC38 cell lines were genotyped to confirm the acquisition of successful gene editing positive mice and positive clones.(2)The hPD-1/hPD-L1/hC5AR1 mice expressed only human-derived PD-1,PD-L1 and C5AR1 proteins,but not murine PD-1,PD-L1 and C5AR1 proteins;human-derived PD-L1 protein was also detected in the hPD-L1 MC38 cell lines.(3)The proportions of each immune cell in the spleen,lymph nodes and peripheral blood of hPD-1/hPD-L1/hC5AR1 mice were not significantly different from those of wild mice when wild mice were used as controls.(4)The anti-human C5AR1 antibody drug Avdoralimab specifically bound only to hPD-1/hPD-L1/hC5AR1 mice and not to wild-type mice.hPD-1/hPD-L1/hC5AR1mice tumour models were successfully constructed and the combination of anti-human PD-1 antibody and anti-human C5AR1 antibody was more effective in inhibiting tumour growth than single inhibitor.Conclusions:The hC5AR1 mice,hC5/hC5AR1 mice,hPD-1/hPD-L1/hC5AR1mice,hC5 MC38 cell lines and hPD-L1 MC38 cell lines,which have been successfully genotyped were analyzed and all three humanized mice and both cell lines were successfully constructed.Tumour models were constructed based on these three humanized mice and humanized cell lines,and the results showed that:(1)no tumor inhibition was observed for the anti-human C5AR1 antibody alone in either the hC5AR1 mouse tumour model or in the hC5/hC5AR1 mouse tumour model;(2)however,by combining the anti-human PD-1 antibody and the anti-human C5AR1antibody in the hPD-1/hPD-L1/hC5AR1 mouse tumor model,we demonstrated that the drug combination can effectively inhibit tumor growth.In conclusion,hPD-1/hPD-L1/hC5AR1 mice can be used as a preclinical efficacy validation model for C5AR1-targeted drugs,which has implications for accelerating the development process of C5AR1-targeted drugs. |
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