Bossone Research Center, Room 302, located at 32nd and Market Streets. Also on Zoom.
BIOMED Master's Thesis Defense
Title:
Developing Mutant KRAS Targeted Vaccines for Pancreatic Cancer Interception
Speaker:
Ben Barrett, Master's Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University
Advisors:
Neeha Zaidi, MD
Assistant Professor of Oncology
Sidney Kimmel Comprehensive Cancer Center
Johns Hopkins Medicine
Adrian Shieh, PhD
Teaching Professor
School of Biomedical Engineering, Science and Health Systems
Drexel University
Details:
Pancreatic Ductal Adenocarcinoma (PDAC) remains one of the most lethal cancers to date, with a 5-year survival rate of only 12%. The poor survival rate can largely be attributed to an immunosuppressive tumor microenvironment (TME) that is largely devoid of anti-tumor T cells. Approximately 90% of PDACs are driven by mutations in KRAS (mKRAS), with the most common being KRASG12D (~40%). mKRAS serves as an ideal set of candidates for targeting due to their role as a driver mutation and high specificity to precancerous lesions. These shared driver mutations, along with the near decade-long timeframe from pre-cancer to PDAC development, allows for a window-of-opportunity to develop vaccines to activate T cells before cancer develops and immunosuppression evolves. Several considerations are key to vaccine performance including the platform and immunomodulatory adjuvants admixed with neoantigens. Within this thesis, we explore two promising vaccine platforms: a pooled neoantigen peptide vaccine and a bicistronic mRNA vaccine. Each platform contains distinct benefits with peptide vaccines consistently demonstrating immune responses to neoantigens, while mRNA offers the possibility of directly encoding immunomodulatory adjuvants often required to be admixed with peptide vaccines for acceptable responses. The stimulator of interferon genes (STING) pathway offers an increasingly attractive target for optimizing T cell response due to the upregulation of type I interferons.
We thus designed and verified the in vitro functionality of an mRNA vaccine encoding both a KRASG12D vaccine and immunomodulatory adjuvant STINGV155M, a constitutively active STING (caSTING) protein, on the same vector mediated by an EMCV IRES linker. We set 3 requirements that were paramount to design success: 1) Antigenic translation (KRASG12D), 2) Adjuvant Translation (STINGV155M), and 3) Constitutive STINGV155M Activation. Two constructs were tested that alternated the ordering of the cistrons, and thus modified the nature of translation. It was determined that while both encoded proteins were translated from the same vector using Western Blot, there was insufficient caSTING functionality when caSTING was subject to cap-independent translation as measured by a STING reporter cell line. However, STING signaling was rescued when the caSTING was translated via cap-dependent initiation, indicating that bicistronic translation requires improvement before it can be deemed that single construct met all requirements.
In parallel, we characterized the immunogenicity of a pool of mutant KRAS synthetic long peptides (SLPs) (G12C, G12V, G12R, G12A, G12D and G13D) via anti-IFNγ ELISpot and flow cytometry T cell phenotyping in C57BL/6 mice. We demonstrated robust antigen-specific responses when the SLPs were admixed with a STING adjuvant. We also found an increase in mKRAS-specific T cell responses when increasing the number of doses in the vaccination protocol. Interestingly, we found cross-reactive T cell responses in both CD4+ and CD8+ T cells, dependent on the epitopes included in the peptide mixture.
Finally, we characterized the pre-malignant and tumor microenvironment (TME) of an inducible, conditional mouse model of PDAC, commercially available from the Jackson Laboratory, Pdx1-CreERTg/Tg;Trp53fl/fl;KrasG12D/+. This mouse model expands on the Cre-Lox recombination of the traditional KPC mouse model, through the association of an estrogen receptor with the Cre protein. Thus, the estrogen-analog, Tamoxifen, is required for Cre to enter the nucleus and carry out recombination, allowing for the induction of the KRASG12D-driven tumorigenesis. We developed a method of inducing mKRAS, isolating and maintaining tissue, and studying tissue histology. We confirmed the expression of KRASG12D localized to precancerous lesions (PanINs) and PDAC tumors through in situ hybridization (ISH). We further observed a migration of CD3+ and CD68+ cells to sites of PanIN and PDAC via immunohistochemistry (IHC) staining, specifically noting a clear integration of immune cells within PanIN lesions and a clear restriction of CD3+ cells to the border of tumors. Based on the data collected, the tiKPC model serves an ideal candidate for future immunotherapy interception studies using the vaccine formulations outlined above. Throughout the course of this thesis, we demonstrated the potential for mKRAS-targeted vaccine platforms with the intention of intercepting pancreatic cancer, as well as the ideal model to characterize the efficacy of an interception strategy.