T-cell responses and combined immunotherapy against human carbonic anhydrase 9-expressing mouse renal cell carcinoma

Renal cell carcinoma (RCC) is known to respond to immune checkpoint blockade (ICB) therapy, whereas there has been limited analysis of T-cell responses to RCC. In this study, we utilized human carbonic anhydrase 9 (hCA9) as a model neoantigen of mouse RENCA RCC. hCA9-expressing RENCA RCC (RENCA/hCA9) cells were rejected in young mice but grew in aged mice. CD8⁺ T cells were the primary effector cells involved in rejection in young mice, whereas CD4⁺ T cells participated at the early stage. Screening of a panel of hCA9-derived peptides revealed that mouse CD8⁺ T cells responded to hCA9_288-296 peptide. Mouse CD4⁺ T cells responded to lysates of RENCA/hCA9, but not RENCA cells, and showed reactivity to hCA9 _276-290, which shares three amino acids with hCA9 _288-296 peptide. Immunohistochemistry analysis revealed that few T cells infiltrated RENCA/hCA9 tissues in aged mice. ICB therapy of anti-PD-1/anti-CTLA-4 antibodies promoted T-cell infiltration into tumor tissues, whereas no definite antitumor effect was observed. However, additional combination with cyclophosphamide or axitinib, a vascular endothelial growth factor receptor inhibitor, induced complete regression in half of the RENCA/hCA9-bearing aged mice with increased expression of PD-L1 in tumor tissues. These results indicate that hCA9 can be a useful model neoantigen to investigate antitumor T-cell responses in mice with RCC, and that RENCA/hCA9 in aged mice can serve as a non-inflamed 'cold' tumor model facilitating the development of effective combined immunotherapies for RCC.