Abstract P003: In vitro and in vivo characterization of CCR8 humanized mouse model (HuGEMM™)

Abstract

Abstract Background: CCR8 is a C-C chemokine receptor with 7-transmembrane regions which belongs to the G-protein coupled receptor family. Recently, tumor-infiltrating CCR8+ CD4+ Foxp3+ regulatory T (Treg) cells have been identified as a major immune-suppressive subset in the tumor microenvironment (TME), with CCR8 upregulated in tumor-infiltrating Tregs in patients with breast, lung and colorectal cancer. Among the tumor-infiltrating Treg cells, only 8%-10% of Tregs are CCR8 positive, which are regarded as the master driver populations for Treg-mediated immunosuppression. Thus, CCR8 is increasingly believed to be a potential therapeutic target with monoclonal antibodies specific to human CCR8 (hCCR8) inducing depletion of tumor-resident CCR8+ Treg cells, reverse immunosuppressive signals to enhance the anti-tumor role of effector T cells, particularly in combination with other immune checkpoint inhibitors. Given the lack of cross-binding to mouse targets of human-specific antibodies, we developed a hCCR8 knock-in mouse model (CCR8 HuGEMM) to evaluate the in vivo therapeutic efficacy of hCCR8 antibodies. Methods: Since there is only one coding exon (exon 2) in both hCCR8 and mouse CCR8 genes, CCR8 HuGEMM was developed by CRISPR/Cas9 engineering to substitute the mouse CCR8 exon 2 for hCCR8 exon 2 in C57BL/6 mice. The hCCR8 expression in CCR8 HuGEMM was confirmed by flow cytometry analysis. The non-tumor-bearing CCR8 HuGEMM mice were then treated with anti-hCCR8 antibodies to investigate the immune cell phenotyping in blood and spleen. Finally, the CCR8 HuGEMM mice were inoculated with either MC38-OVA or Hepa1-6 syngeneic tumors in parallel to evaluate the anti-tumor responses of anti-hCCR8 antibodies in vivo. Results: The surface expression of hCCR8 on CD4+ and CD8+ T cells, derived from thymus as well as on FoxP3+ Treg cells from CCR8 HuGEMM mice, was characterized by flow cytometry analysis. Monocytic myeloid-derived suppressor cells (MDSCs) (CD3-CD11b+Ly6Chi) populations decreased significantly in non-tumor-bearing CCR8 HuGEMM mice upon treatment of anti-hCCR8 antibody compared to the control mice, which indicates that anti-hCCR8 may play a critical role in abrogating the immunosuppressive role of MDSC and further retard the MDSC-mediated Treg induction. Furthermore, MC38-OVA and Hepa1-6 syngeneic tumors were inoculated into the homozygous CCR8 HuGEMM mice and the mice were treated with 10mg/kg anti-hCCR8,4 days post tumor engraftment. The anti-hCCR8 alone led to ~30% tumor growth inhibition (TGI) in MC38-OVA tumor model, and ~50% tumor growth inhibition (TGI) in Hepa1-6 tumor model. Similarly, we found the tumor-resident CCR8+ CD4+ FoxP3+ populations decreased significantly post-treatment which suggests that CCR8+ Tregs potentially play an important immunosuppressive role in the TME. Conclusions: Our CCR8 HuGEMM model provides an important predictive preclinical model to evaluate the efficacy of hCCR8 antibodies alone or in combination regimens with other immune modulators. Citation Format: Daniel He, Henry Li. In vitro and in vivo characterization of CCR8 humanized mouse model (HuGEMM™) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P003.