These data indicate that CY plus anti-CEA CAR T cells combined with ICK predominantly recruit IFN+/PD1? CD8+ T cells from TDLNs into tumors and that ICK promotes a pro-inflammatory tumor microenvironment. When the number of ICK treatments post CY plus CAR T cell therapy were increased to four times, three days apart, in the MC38/CEA tumor model (figure 6f), tumor eradication was obtained in 6/6 treated mice compared to 2/6 in the CY plus anti-CEA CAR T cell group (Figure 6g). ICK treatment 1 day after CY plus anti-CEA CAR T cell therapy in the MC38/CEA model, and two ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy in the E0771/CEA model were ineffective, while four ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy completely eradicated MC38/CEA tumor growth and induced tumor immunity when the mice were re-challenged with tumor. These studies show the therapeutic potential of anti-CEA CAR T cells combined with ICK to treat CEA-positive tumors. Abbreviations: CAR: Chimeric antigen receptor, CEA: Carcinoembryonic antigen, CEACAM5, ICK: Immunocytokine, CY: Cyclophosphamide, CEATg mouse: transgenic CEA mouse, TDLN: Tumor-draining lymph node experiments and 2.5??107 cells/mL concentration for experiments. In vitro ?.02, Supplement Figure 1c). No whole-body toxicity was observed as measured by mouse weight (Supplement Figure 1d), as well as absence of diarrhea or loss of physical mobility. These data indicate that systemic delivery of anti-CEA CAR T cells in this model resulted in modest delays in CEA+ tumor growth without severe toxicity in immune-competent CEATg mice. This is in agreement with other studies that have shown that CAR T cell therapy alone was not sufficient to eradicate solid tumors.43 Immunodepletion improves the efficacy of anti-CEA CAR T cell therapy Since the administration of exogenous T cells in immunocompetent mice leads to homeostatic reduction of T cells,44C46 T cell depletion by short-acting agents such as cyclophosphoamide (CY) can improve CAR T therapy.47C49 CY, an alkylating agent, is a commonly used chemotherapeutic agent50 that selectively depletes immunosuppressive cells, such as regulatory T cells, to increase antitumor activity.48,51 While CY treatment alone is also not sufficient to eradicate most solid tumors, it was quite effective in the treatment of MC38 tumors as shown by Myers ?.0001) and at 21?days for E0771/CEA tumors ( ?.0001) post tumor implantation. Efficacy of single anti-CEA CAR T cell therapy on the survival of s.c. MC38/CEA tumor-bearing mice ( ?.05) and orthotopic E0771/CEA tumor-bearing mice ( ?.05). B-E: Green for CY only (n?=?4C7); blue for CY+Mock (n?=?6C7); red for CY+CAR (n?=?7). ****p? ?.0001; *p? ?.05. CY i.p. injected into CEA+ tumor-bearing, immunocompetent CEATg mice 24?hours prior to CAR T cell therapy depleted both B cells and T cells, 73% and 43%, respectively (Figure 2a). CY treatment combined with anti-CEA CAR T cell therapy delayed MC38/CEA tumor growth by 30?days compared to 24?days for anti-CEA CAR T Thrombin Receptor Activator for Peptide 5 (TRAP-5) cell therapy alone (Figure 2b and Supplement Figure 1a). The median overall survival in mice treated with CY plus anti-CEA CAR T cells had a statistically significant increase to 40?days compared to 30?days for mice treated with CY and mock T Thrombin Receptor Activator for Peptide 5 (TRAP-5) cells ( ?.01) (Figure 2c). The median survival in mice treated with CY alone was also 30?days (Figure 2c). No whole-body toxicity was observed as measured by a decrease in mouse weight (Supplement Figure 3), as well as no diarrhea or loss of physical mobility. There were no morphology changes in CEA+ organs that were collected 3-days post T cell therapy and stained for human CEA (Supplement Figure 4), confirming the expression of CEA in the transgenic mice. Staining of the CEA+ tissues C collected 3?days post-T cell therapy (Supplement Figure 5a) vs. the terminal timepoint of 1500 mm3 tumor size (Supplement Figure 5b) C for murine CD3 revealed the presence of murine T cells in these tissues. In addition, there was no evidence of morphology changes in CEA+ organs at either the early or later timepoints, indicating lack of inflammatory tissue damage or presence of non-cytotoxic tissue infiltrating T cells. Open Thrombin Receptor Activator for Peptide 5 (TRAP-5) TRK in a separate window Figure 3. CEA expression on s.c. MC38/CEA tumors in CEATg mice After s.c. MC38/CEA tumor has grown to 1500 mm3 the termination tumor size in CEATg mice, tumors were collected and analyzed on immunohistochemistry (IHC) for human CEA. The.