Abstract B12: Immune checkpoint blockade to improve T cell infiltration and function in colorectal cancer
Authors: Kodumudi Krithika N. , Charles James , Luu Carrie , Mullinax John , Li Julie , Hall MacLean , Weber Amy , Sarnaik Amod A. , Pilon-Thomas Shari 
Published: March 2017

Abstract
Adoptive transfer of tumor infiltrating lymphocytes (TIL) has emerged as a promising approach to induce effective anti-tumor immunity and tumor regression in various types of cancers. Current studies suggest that the presence of TIL play a role in patient outcomes and can predict tumor stage, node positivity, and in some reports, disease free and overall survival in patients with colorectal cancer. TIL may be inactivated in patients due to factors in the local tumor microenvironment, such as regulatory T cells, myeloid derived suppressor cells and the expression of a number of co-inhibitory immune checkpoints including PD-L1, BTLA, PD-1, CTLA-4, Lag-3 and Tim-3. We first examined the feasibility of expanding TIL from the tumors of three colorectal patients. Tumors were minced into fragments and cultured in media supplemented with 6000 IU IL2 / mL. TIL growth was observed in tumor fragments from each of the three patients with a total of 12 out of 41 fragments demonstrating TIL expansion. TIL grown out of these fragments were predominantly CD3+ with 32% CD4+T cells and 46% CD8+ T cells and 18.5% NK cells. Seven of the 12 fragments tested were tumor specific as demonstrated by IFN-gamma production in response to autologous tumor. CD8+ T cells within the patient TIL expressed PD-1, BTLA, Lag-3 and Tim-3 and a co-stimulatory molecule, 41BB. In addition, we found high levels of PD-L1 expression on tumors of colorectal patients and human colorectal cell lines. Since we observed high levels of immune checkpoints expression in colorectal patients, we wanted to develop a preclinical colorectal model to investigate the effect of immune checkpoint blockade on tumor growth and TIL function. A previous study from our lab has shown that TIL from our preclinical MC38 colorectal murine model express high levels of co-inhibitory checkpoints and that blockade of PD-L1 leads to enhanced infiltration and anti-tumor specificity of TIL. However, given the dynamic nature of immune responses to tumors and expression of multiple immune checkpoints, it may be necessary to block additional checkpoints to improve clinical efficacy. In this study, we examined whether blockade of the immune checkpoint receptor BTLA had an effect on TIL infiltration and tumor growth. Mice bearing MC-38 tumors treated with anti-BTLA antibody demonstrated a delay in tumor growth (150mm2, p&lt;0.01) on day 28 post tumor injection compared to isotype control (300mm2). We observed an increase in CD8+ T cell infiltration (30%, p&lt;0.001) in the tumors of anti-BTLA antibody treated mice compared to mice treated with isotype control (13%). TIL isolated from the anti-BTLA antibody treated mice demonstrated increased IFN-gamma and granzyme B secretion compared to isotype controls (p&lt;0.001). Dual immune checkpoint blockade with anti-PD-L1 and anti-BTLA antibodies had a synergistic effect in delaying tumor growth. We also measured a significant increase in CD8+CD107a+IFN-gamma+ secreting T cells (37%) compared to TIL from mice treated with anti-PD-L1 (27%) or anti-BTLA antibody alone (25%) (p&lt;0.002). These findings suggest that targeting immune checkpoint molecules can improve T cell infiltration and anti-tumor immunity in a preclinical model of colorectal cancer. In addition, we demonstrate that tumor-specific TIL can be expanded from the tumors of colorectal patients. This study presents a rationale for the potential synergy between blockade of multiple immune checkpoints and adoptive transfer of TIL for the treatment of colorectal cancer.
Citation Format: Krithika N. Kodumudi, James Charles, Carrie Luu, John Mullinax, Julie Li, MacLean Hall, Amy Weber, Amod A. Sarnaik, Shari Pilon-Thomas. Immune checkpoint blockade to improve T cell infiltration and function in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B12.