Abstract To determine therapeutic mechanism of action (MOA), it is standard to analyze the tumor immune response at a single timepoint. Therefore, to examine the impact of checkpoint inhibition on early and late phases of the immune response, we dosed CT26 tumor-bearing mice with anti-mCTLA-4, or isotype control, and analyzed tumors at day 12 and day 18 post-implant. Tumor measurements at day 17 revealed a 77% growth inhibition in response to anti-mCTLA-4 treatment. To explore MOA, 11 tumor-infiltrating myeloid and lymphoid subsets were quantified and analyzed for various phenotypic and functional markers. We first examined immune cell recruitment in the control group tumors from day 12 to day 18. As expected, dynamic changes were observed in various subsets. Absolute numbers of CD8+ T cells, M1 and M2 tumor-associated macrophages (TAM) increased by 203%, 208%, and 100% respectively. In contrast, a 31% reduction in monocytic myeloid-derived suppressor cells (M-MDSC) was observed. The effects of anti-mCTLA-4 on tumor infiltration was then examined. Enhanced CD4+ helper, CD8+, and regulatory T cell recruitment was observed at both timepoints compared to the isotype control group. While NK and NKT cell recruitment was also enhanced, the increase in NK cells was only transient (130% at day 12). A similar transient increase was observed for M1 TAMs. In contrast, a 33% reduction in M2 TAMs occurred following treatment from day 12 to day 18, compared to controls. Finally, an increase in M-MDSC was observed in the anti-mCTLA-4 group at both timepoints (33% and 58% respectively). To evaluate effects of anti-mCTLA-4 on proliferation, we measured Ki-67 expression as a surrogate marker in CD8+ T cells. A 43% increase in Ki-67 expression was induced by anti-mCTLA-4 compared to controls at day 12. No difference was observed on day 18 suggesting anti-mCTLA-4 increased CD8+ T cell infiltration by triggering an early and transient enhancement in proliferation. Analysis of CD62L and CD44 in the control group demonstrated the pool of memory CD8+ T cells increased in the tumors over time. Anti-mCTLA-4 treatment delayed memory formation resulting in a larger pool of CD8+ T cells with an effector phenotype (CD62L-CD44+). To examine anti-tumor activity in CD8+ T cells, IFNγ production was measured after ex vivo stimulation. Anti-mCTLA-4 enhanced IFNγ responses on day 18 in CD8+ T cells and NKT cells (25% and 17% respectively), but similar responses were observed between groups on day 12. Taken together, these results suggest that checkpoint blockade inhibited tumor growth in part by enhancing early T cell proliferation and recruitment of NK and M1 TAMs. This was followed by a late enhancement of anti-tumor T cell and NKT responses, as well as reduced M2 TAM infiltration. These findings demonstrate that kinetic analysis can help gain insight into therapeutic MOA by revealing dynamic responses that can be missed by analysis at a single point. Citation Format: David W. Draper, Alden Wong, Stacey Roys, Scott Wise, Maryland Franklin. Anti-mCTLA-4 treatment results in early and late immune response effects in a murine model of colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4982.