Abstract PR03: Characterizing the immune context of responses to immunotherapy in humanized patient derived xenograft models of CRC

Abstract

Introduction: Inhibiting the activity of the epidermal growth factor receptor (EGFR) with monoclonal antibodies has been utilized as a therapeutic strategy for patients with metastatic colorectal cancer (CRC), leading to improved clinical results alone and in combination with standard chemotherapy. Many systematic reviews and metanalyses were performed to better understand the role of EGFR inhibition in CRC, revealing that KRAS exon 2 mutations and furthermore exons 3 and 4 and NRAS exons 2, 3, and 4 were predictive of non-responsiveness to these agents. Concurrent with these results has been the development of immunotherapy targeting immune regulatory checkpoints such as CTLA-4 and PD-1 that have initiated a new era in the treatment of cancer. In order to gain a better biological understanding of the context of immune responses and facilitate preclinical evaluation of cancer immunotherapy, we developed a hematopoietic humanized mouse model utilizing patient-derived CRC xenograft tumor models to assess immune therapy for RAS mutant CRC. Not only could evaluation of humanized RAS mutant PDX models provide additional information on the potential for clinical activity of immune therapies, but could also improve the understanding of immune responses to RAS mutant cancers. We therefore hypothesize that humanized RAS mutant colorectal PDX models can be used to evaluate the preclinical activity of immune targeted agents for treatment of RAS mutant colorectal cancer. Methods: Humanized BRG mice developed from the BALB/cRag2-/-IL2Rγc-/- (BRG) strain which is known to accept human hematopoietic stem cells, have been used to enhance engraftment. BRG newborn pups were humanized through transplantation of approximately 1x105 CD34+ cells purified from umbilical cord blood. The mice were evaluated for chimerism at 8 and 12 weeks. At 14 weeks, tumor tissue from established PDX models were implanted on the right and left flank of humanized mice. The tumor was selected among a cluster within the “immune-enriched” subtype (C2) based upon the RNAseq characterization of the models. When the average tumor size reached a volume of approximately ~150-300 mm3, the mice were randomized into either vehicle or nivolumab treatment groups. Mice were monitored daily for signs of toxicity and weighed twice weekly. They were treated with nivolumab (30 mg/kg) twice a week by intraperitoneal injection for 15 days. Tumor size was evaluated twice weekly by caliper measurements using the following equation: tumor volume= (length × width2) × 0.52. At the end of the treatment, mice were euthanized while sera, lymph nodes, spleen, bone marrow and tumors were collected for further investigation. Results: Humanized RAS mutant CRC PDX models were successfully established in vivo. While no differences were observed in tumor growth among the control and treated arms, we were able to detect differences in PD1 expression among treated versus control mice, with lower expression in the nivolumab treated group. We also observed higher numbers of T cells in the lymph nodes of nivolumab treated mice, suggesting T cell expansion. Interestingly, we also observed an increase of T cells in the spleen and blood and late occupancy of T cells in the bone marrow. Two of the treated mice exhibited identifiable TILs that were comprised of a majority of CD4+ T cells with an activated phenotype (CD69+). Conclusions: Humanized KRAS mutant CRC PDX models were successfully established and tumor engraftment occurred in all humanized mice with nivolumab-treated mice demonstrating the development of lymph nodes that were populated by activated T cells. These preliminary results demonstrate that human immunity and PD-1 expressing T cells exist in these models and provide the basis for planned immunotherapy combination studies. This abstract is also being presented as Poster A23. Citation Format: Anna Capasso, Julie Lang, Todd M. Pitts, S. Lindsey Davis, Chris H. Lieu, Stacey M. Bagby, Aik Choon Tan, John J. Tentler, Jill E. Slansky, Roberta Pelanda, S. Gail Eckhardt. Characterizing the immune context of responses to immunotherapy in humanized patient derived xenograft models of CRC. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr PR03.

Current and Future Horizons of Patient-Derived Xenograft Models in Colorectal Cancer Translational Research

“…It has been shown that tumors with MSI and hypermutation display high degrees of immune infiltration with cytotoxic T lymphocytes, activated helper T cells, and natural killer cells [158,159], and these subsets of CRC selectively have high expression levels of multiple immune checkpoints, including PD-1, PD-L1, CTLA-4, LAG-3, and IDO [160,161]. Preliminary studies demonstrated that human immune cells and PD-1-expressing T cells are present in humanized PDX models of CRC [162,163]. Furthermore, in vivo MSI-high and microsatellite-stable (MSS) humanized CRC PDXs have been successfully established [164], and, as expected, characterization of these models confirmed higher levels of immune infiltrates and improved response to the PD-1 inhibitor, nivolumab, in MSI-high PDX models compared to MSS PDX models.…”