Combination immunotherapy intercepts murine pancreatic cancer progression
Aleksandra Popovic1, Nina J. Chu1, Todd Armstrong1, Elizabeth M. Jaffee1 1Johns Hopkins University School of Medicine, USA
Immunotherapy agents that activate T cells are changing clinical practice for some metastatic cancer patients. Vaccines targeting viral oncogenes prevent the development of hepatocellular and cervical cancers. We therefore tested the hypothesis that a vaccine targeting the oncogene that initiates pancreatic cancer (PDA) given in combination with immunotherapy agents that reprogram the earliest procarcinogenic inflammatory changes will slow (intercept) PDA development. The KRAS proto-oncogene is a driver mutation expressed in >90% of pancreatic intraepithelial neoplasms (PanINs) and an ideal candidate target for early vaccination against PDA. However, even early PanINs have immunosuppressive microenvironments highly expressing inhibitory immune checkpoints like CTLA-4 and infiltrating immunosuppressive cells such as regulatory T cells (Tregs).
The overall goal of this research is to reprogram the immunosuppressive environment while specifically activating immunity against early premalignancies to prevent PDA development. We previously reported that early dosing of KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, which endogenously recapitulate human PDA tumorigenesis, with the Treg depleting and inactivating agents cyclophosphamide (cy) and PC61 antibody in combination with an attenuated Listeria monocytogenes vaccine expressing mutant KrasG12D (LM-Kras) can slow tumorigenesis in 40% of mice. In this study we attempted to improve the efficacy of a vaccine approach by specifically inhibiting the immunosuppressive effects of the CTLA-4 immune checkpoint on Tregs with a CTLA-4 blocking antibody.
KPC mice at 4-5 weeks with early PanINs received 2 doses of LM-Kras and cy/PC61 and 8 doses of anti-CTLA-4. Control mice received isotype antibody with LM-Kras and cy/PC61 or no treatment. At 10-11 weeks, pancreata were pathologically evaluated and assigned grades normal, low (1) and high-grade (2 and 3) PanINs, and PDA. Mice receiving LM-Kras + anti-CTLA-4 had a significantly lower incidence of low- and high-grade PanINs and PDA compared to controls. Furthermore, mice receiving LM-Kras, cy/PC61, and anti-CTLA-4 did not develop lesions beyond PanIN 1, indicating that the combination therapy may halt PDA development at early stages.
To investigate the immune profiles of mice receiving treatment, flow cytometry was performed on tumor-infiltrating lymphocytes (TILs) isolated from pancreata to assess abundance of Tregs, infiltration of effector and memory T cell populations, and expression of inhibitory immune checkpoint molecules. Mice receiving combination therapy had decreased numbers of Tregs, increased numbers of effector and memory T cells, and increased CD8+ T cells to Tregs ratio in the microenvironment compared to controls. Further studies using immunohistochemistry and RNA expression profiles are underway to further characterize the immune changes in the microenvironment following treatment.
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