1113 Engineering vaccines against S100A9 to treat cancer co-morbidities

<h3>Background</h3> Chronic inflammation is linked to various diseases associated with cancer co-morbidities, which negatively impact cancer prognosis. One major regulator of inflammation is calcium-binding protein S100A9. It can broadly regulate myeloid and monocyte-derived immune populations.^{1 2} Further, high S100A9 expression has been implicated in multiple cancers and metastasis, suggesting its potential role in cancer-associated inflammatory dysregulation. Interestingly, higher S100A9 is also a potential marker for cardiovascular risk.³ Engineered vaccines against S100A9 reduced the onset of lung metastasis⁴ and atherosclerosis independently,⁵ yet the benefit of S100A9 inhibition in a co-morbidity setting is not yet known. Here, we aim to evaluate immune-engineered vaccines against S100A9 in a pre-clinical co-morbidity model as a potential therapeutic target for cancer patients presenting co-morbidities. <h3>Methods</h3> We engineered peptide vaccines by chemically conjugating S100A9 B-cell epitope peptide onto different virus-like particles (VLPs) to elicit anti-S100A9 titers. After vaccinating C57BL/6 mice, anti-S100A9 titers were evaluated to determine S100A9 affinity, avidity, and Th-bias using ELISA. Vaccinated mice were then intravenously challenged with B16F10 melanoma cells to examine the vaccine-induced protection against lung metastasis. Lung nodules were manually counted. Splenocytes from vaccinated mice were evaluated via IFN-γ ELISpot to assess break of immune-tolerance. <h3>Results</h3> In the B16F10 lung metastasis model, vaccination with VLP2-S100A9 demonstrated trending significance in reducing lung nodules compared to VLP1-S100A9 (figure 1). Antibody evaluation demonstrates that while both vaccine candidates generate a Th₁-biased response after the first two vaccines, VLP1-S100A9 maintains Th₁-bias after the third vaccine, while VLP2-S100A9 switches to a Th₂-bias (figure 2A). However, VLP2-S100A9 generates significantly more titers and with higher avidity than VLP1-S100A9 (figure 2B), suggesting VLP2 is a promising adjuvant. VLP2, as a stimulant, stimulated comparable activation in splenocytes from all groups, while VLP1 only activated splenocytes derived from VLP1-S100A9 and Mixture mice. Splenocytes co-incubated with B16F10 cells showed similar activation levels, regardless of group. Thus, the vaccines likely did not induce tumor killing but rather modulated the lung microenvironment to reduce tumor seeding, similarly to what our group reported⁴. Future work using ApoE^-/- mice on high-fat diet will assess if our engineered vaccines concurrently reduce metastasis and plaque formation. Critical evaluation in a cancer-cardiovascular model will provide important insight into treating cancer co-morbidities. <h3>Conclusions</h3> In this study, our goal is to inhibit an inflammatory protein associated with cancer and cancer-morbidities. We envision the innovative engineering of vaccines against markers associated with cancer and commonly reported co-morbidities will provide an encompassing therapeutic approach for cancer co-morbidity cases. <h3>Acknowledgements</h3> We would like to acknowledge the following funding sources: NIH award R01-CA224605, R01-CA253615, and T32CA121938. We want to thank the UCSD Chancellor’s Fellowship and SD IRACDA Program for supporting Dr. Ledezma. We also appreciate the technical support provided by the UCSD veterinarians for this study. <h3>References</h3> Markowitz J, Carson WE. Review of S100A9 biology and its role in cancer. <i>Biochim. Biophys. Acta</i> 2013;<b>1835</b>(1):100–109. Cheng P, Corzo CA, Luetteke N, Yu B, Nagaraj S, Bui MM, Ortiz M, Nacken W, Sorg C, Vogl T, Roth J, Gabrilovich DI. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. <i>J. Exp. Med.</i> 2008;<b>205</b>(10):2235–2249. Healy AM, Pickard MD, Pradhan AD, Wang Y, Chen Z, Croce K, Sakuma M, Shi C, Zago AC, Garasic J, Damokosh AI, Dowie TL, Poisson L, Lillie J, Libby P, Ridker PM, Simon DI. Platelet expression profiling and clinical validation of myeloid-related protein-14 as a novel determinant of cardiovascular events. <i>Circulation</i>. 2006;<b>113</b>:2278–2284. Chung YH, Ortega-Rivera OA, Volckaert BA, Jung E, Zhao Z, Steinmetz NF. Viral nanoparticle vaccines against S100A9 reduce lung tumor seeding and metastasis. <i>Proc Natl Acad Sci U S A</i>. 2023 Oct 24;<b>120</b>(43):e2221859120. Ortega-Rivera OA, Shin MD, Moreno-Gonzalez MA, Pokorski JK, Steinmetz NF. A single-dose Qβ VLP vaccine against S100A9 protein reduces atherosclerosis in a preclinical model. <i>Adv Ther (Weinh)</i>. 2022 Oct;<b>5</b>(10):2200092. <h3>Ethics Approval</h3> The present study was approved by the institutional IACUC (protocol number S18021) and all institutional guidelines were followed.