Abstract:

Objective

To explore the efficacy and mechanism of OX40 antibody agonist （ES102） in humanized OX40 mouse NSCLC model.

Methods

Mouse Lewis lung cancer cells were detected using flow cytometry （Lewis lung cancer cells，CD44 expression in LLC）； OX40 humanized mouse NSCLC tumor-bearing model （vehicle control group，ES102 treatment group at different doses） and colon cancer tumor-bearing model（positive vehicle control group and positive control group） were constructed respectively，To evaluate the tumor suppressive level of ES102 in different groups； Serum interferon- γ was measured by ELISA （interferon- γ，IFN- γ） and interleukin-17 （interleukin-17，IL-17） secretory level； Expression of PI3K，NFAT，CD44，NapsinA and SYN by western blot （Western blot）； The expression of the immune-infiltrating cells was determined by RNA transcriptome sequencing.

Results

The CD44 positive rate in LLC cells showed 99.58%and low expression of NapsinA protein，suggesting that LLC murine-derived lung cancer cell lines had NSCLC properties.In the humanized OX40 mouse NSCLC model，ES102 inhibited NSCLC，but on Day 11，Day 14，Day 18，and Day 21 in the positive vehicle control （P=0.0004， t=5.712； P＜0.0001， t=9.368； P＜0.0001，t=10.364； P＜0.0001， t=13.37）.The content of plasma IL-17 in humanized mice was significantly differently expressed between different samples （F=7.703， P=0.0004）； The secretion content of IL-17 in the positive control group （58.24±18.11）pg/ml than the ES102 （2 mg/ml） group （21.87±11.74）pg/ml，The ES102（5 mg/ml） group （18.49±10.17）pg/ml and the ES102 （10 mg/ml） group （20.47±10.52）pg/ml（P＜0.05）；Higher IFN- γ in NSCLC ES102 （10 mg/ml） than the control，ES102 （2 mg/ml） and ES102 （5 mg/ml）（P＜0.05）.PI3K expression was not seen in the positive control group and was highly expressed in the untreated group and the ES102 experimental group （2 μg/kg to 10 μg/kg）（P＜0.05）.As a nuclear factor in activated T cells，NFAT was expressed in a trend consistent with PI3K，without a statistically significant difference between the groups（P＞0.05）.After transcriptome sequencing of NSCLC and positive control mice，further transcriptome sequencing and deconvolution analysis showed that the proportion of Tregs cells infiltrated was （1.95±0.02）%，which was lower than that of Tregs cells infiltrated in the positive control group （7.2±0.03）%（P＜0.05）.

Conclusion

The efficacy of ES102 in humanized NSCLC mice is lower than colon cancer，and the difference may be related to the decreased secretion of IL-17 and the infiltration of Tregs cells in the microenvironment.

Key words: Non-small cell lung cancer, Humanized mouse, Tumor necrosis factor receptor superfamily member 4, ES102, Transcriptome sequencing