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Supplementary Methods

Supplementary Table S1. Weight changes in different treatment groups for each mouse strain. Supplementary Table S2. Weight change in NTCU treated mice is sex independent. Supplementary Table S3. Survival in NTCU treated mice is sex independent. Supplementary Table S4. Immune phenotype of splenocytes from FVB and NIH Swiss mice treated 4 weeks with 40mM NTCU either subsequent or concomitant to vaccination with IGF1R. Supplementary Table S5. Immune phenotype of splenocytes from NIH Swiss and FVB mice treated with NTCU for 24-32 weeks.

Supplementary Figure S1. Gene expression in mouse SCC is consistent with human SCC data. Supplementary Figure S2. Lung non-neoplastic lesions found in animals treated with NTCU. Supplementary Figure S3. Lymphoid aggregates and PD-L1 expression in the lung of mice treated with NTCU.

In order to translate new treatments to the clinic, it is necessary to use animal models that closely recapitulate human disease. Lung cancer develops after extended exposure to carcinogens. It has one of the highest mutation rates of all cancer and is highly heterogenic. Topical treatment with N-nitrosotris-(2-chloroethyl)urea (NTCU) induces lung squamous cell carcinoma (SCC) with nonsynonymous mutation rates similar to those reported for human non–small cell lung cancer. However, NTCU induces lung cancer with variable efficacy and toxicity depending on the mouse strain. A detailed characterization of the NTCU model is needed. We have compared the effect of three different NTCU doses (20, 30, and 40 mmol/L) in female and male of NIH Swiss, Black Swiss, and FVB mice on tumor incidence, survival, and toxicity. The main findings in this study are (1) NIH Swiss mice present with a higher incidence of SCC and lower mortality compared with Black Swiss and FVB mice; (2) 30 mmol/L NTCU dose induces SCC at the same rate and incidence as the 40 mmol/L dose with lower mortality; (3) female mice present higher grade and incidence of preinvasive lesions and SCC compared with males; (4) NTCU-induced transformation is principally within the respiratory system; and (5) NTCU treatment does not affect the ability to elicit a specific adaptive immune response. This study provides a reference point for experimental designs to evaluate either preventive or therapeutic treatments for lung SCC, including immunotherapies, before initiating human clinical trials.

This year, in the US, an estimated 150,000 people will be diagnosed with colon cancer and ~52,000 will die of the disease. A vaccine approach is well suited to colon cancer prevention. First, a limited number of immunizations can be given over a short period of time to achieve a desired immune response. If immunologic memory develops, antigen specific T-cells will persist in the body for years, ready to be deployed when aberrant cells are detected. Finally, there are several well-defined high-risk populations that would be suitable for testing a preventative colon cancer vaccine including individuals (1) with a hereditary genetic syndrome such as familial adenomatous polyposis, (2) with a history of inflammatory bowel disease such as Crohn’s disease or ulcerative colitis, and (3) those with a history of hyperplastic colonic polyps. Vaccines targeting virally mediated cancers are effective, in part, because T-cells are primed to proteins involved in oncogenesis. The most common type of colon adenoma and invasive cancer associated antigens are non-mutated growth-related proteins that become markedly overexpressed in the disease state. Genes which are aberrantly up-regulated and conserved from adenoma to invasive colon carcinoma may encode overexpressed proteins that are associated with colon cancer initiation. Evaluating gene expression data derived from over 600 colorectal cancers, matched normal tissues, and colon adenomas we identified 160 genes overexpressed in the majority of adenomas and early-stage colon cancers, but not in normal tissues. Using siRNA screening we demonstrated 35 of those genes, when silenced, resulted in apoptosis or inhibition of growth of colon cancer cells and an adenoma cell line but not a non-neoplastic colon cell line. Of those genes, 16 have been shown to encode proteins that are overexpressed in both adenomas and colon cancer and are associated with disease progression and over a third of these proteins are immunogenic. These proteins could serve as potential candidate antigens for inclusion in a multi-antigen vaccine. Recently, we have identified epitopes within the natural sequences of non-mutated tumor antigens that only generate Th1 (Th1 selective) antigen specific T-cells. This discovery and the development of high throughput methods to both identify and screen putative Class II binding peptides for Th selective function has allowed us to develop antigen specific Th1 selective vaccines. A vaccine targeting CDC25B, COX2, and EGFR can inhibit intestinal tumor formation in both APCmin mice and mice with tumor induced by AOM. Concomitant use of NSAIDs potentiates the immune response generated with vaccination. The anti-tumor response observed after vaccination is significantly correlated with elevated CD8 T-cells found in the lesions and induced by immunization as well as high magnitude antigen specific T-cells identified in the spleen. These data validate a genomic approach to antigen discovery and anti-tumor efficacy of Th1 selective vaccines for colon cancer prevention. Citation Format: Mary Disis, Ying Liu, Lauren Corulli, Erin Rodmaker, Denise Cecil. Vaccine approach for the interception of colon cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA016.

Little is known about optimization of a class II restricted plasmid-based vaccine targeting multiple cancer antigens. We set out to define parameters of an in situ prediction model in vaccine design for optimal immunogenicity. Using previously defined immunogenic class II epitopes from five cancer antigens, we evaluated the effects of using linkers between antigens and varying the antigen order. The construct with linkers was superior to the construct without linkers. The mice receiving the vaccine with linkers developed a greater magnitude (p

Bexarotene and 9cUAB30 are highly selective oral retinoid X receptor (RXR) agonists with anti-proliferative activity in breast cancer. We have demonstrated that bexarotene enhances efficacy of a multi-antigen vaccine to prevent breast cancer in the TgMMTV-neu model and increases CD8 T-cell tumor infiltration. We further have shown that RXRα expression is in 24.9±13% of macrophages, 38.6 ±14% of plasmacytic dendritic cells (pDC), and 33.1 ± 16% of monocytic dendritic cells (mDC). Furthermore, the RXR agonists increased Th1 pDC and mDC. We therefore evaluated whether RXR agonists could enhance the vaccine antigen-specific immunogenicity and polyfunctional T cells in the transgenic mouse mammary tumor model TgMMTV-neu. Vaccination with the 150 µg HER2-IGFBP2-IGF1R vaccine and 5 ug GMCSF adjuvant every 2 weeks for four doses significantly increased antigen-specific IFN-γ T cells, but not antigen-specific IL10 T cells, as compared to control vaccination with empty vector. Interestingly, daily oral administration of 30 mg/kg bexarotene for 5 days prior to the HER2-IGFBP2-IGF1R vaccination series increased the IFN-γ immune responses to HER2, IGFBP2, and IGF1R by 1.2, 2.4 and 2.2 fold, respectively, as compared to the HER2-IGFBP2-IGF1R vaccine alone. Daily administration of a higher dose (200 mg/kg) 9cUAB30 for 5 days prior to the HER2-IGFBP2-IGF1R vaccination series increased the IFN-γ immune responses to HER2, IGFBP2, and IGF1R by 2.0, 2.3 and 1.7 fold, respectively, as compared to the HER2-IGFBP2-IGF1R vaccine alone. Control vaccination with either 9cUAB30 or bexarotene had no impact on antigen-specific IFN-γ T cell response. Type I DCs are important for producing polyfunctional CD4+ T cells that release not only IFN-γ but also TNF-α and IL-2. Polyfunctional T cells induce a longer lasting and more effective immune response in vaccines both for infectious diseases and cancer. We demonstrated the addition of bexarotene or 9cUAB30 increased antigen-specific polyfunctional T cells in the TgMMTV-neu (n=15 mice) transgenic mouse mammary tumor model while vaccination alone did not. There were an average of 1.3±0.2% antigen-specific CD4 polyfunctional T cells and 2.7±0.7 antigen-specific CD8 polyfunctional T cells with empty vector and vehicle control (sesame oil). HER2-IGFBP2-IGF1R vaccination following 30 mg/kg bexarotene treatment increased polyfunctional T cells to an average of 6.1±2.0% antigen-specific CD4 polyfunctional T cells (p=0.07) and 20.3±4.1% antigen-specific CD8 polyfunctional T cells (p=0.0003). HER2-IGBP2-IGF1R vaccination after 200 mg/kg 9cUAB30 increased antigen-specific polyfunctional T cells to 7.6±2.0% (p=0.01) and antigen-specific CD8 polyfunctional T cells to 17.6±4.1% (p=0.003). These data indicate that RXR agonists have an immunostimulatory role with multi-antigen cancer vaccines and may augment the anti-tumor activity of vaccines. Citation Format: Sasha Elizabeth Stanton, Erin Rodmaker, Nicholas Drovetto, Lauren Corulli, Flonia Levy, Venkatram Atigadda, Clinton Grubbs, Romaine Fernando, Shizuko Sei, Mary L. Disis. Retinoid X receptor agonists enhances Th1 antigen-specific and polyfunctional T cells with the HER2-IGFBP2-IGF1R vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1556.

In order to translate new treatments to the clinic, it is necessary to use animal models that closely recapitulate human disease. Lung cancer develops after extended exposure to carcinogens. It has one of the highest mutation rates of all cancer and is highly heterogenic. Topical treatment with N-nitrosotris-(2-chloroethyl)urea (NTCU) induces lung squamous cell carcinoma (SCC) with nonsynonymous mutation rates similar to those reported for human non–small cell lung cancer. However, NTCU induces lung cancer with variable efficacy and toxicity depending on the mouse strain. A detailed characterization of the NTCU model is needed. We have compared the effect of three different NTCU doses (20, 30, and 40 mmol/L) in female and male of NIH Swiss, Black Swiss, and FVB mice on tumor incidence, survival, and toxicity. The main findings in this study are (1) NIH Swiss mice present with a higher incidence of SCC and lower mortality compared with Black Swiss and FVB mice; (2) 30 mmol/L NTCU dose induces SCC at the same rate and incidence as the 40 mmol/L dose with lower mortality; (3) female mice present higher grade and incidence of preinvasive lesions and SCC compared with males; (4) NTCU-induced transformation is principally within the respiratory system; and (5) NTCU treatment does not affect the ability to elicit a specific adaptive immune response. This study provides a reference point for experimental designs to evaluate either preventive or therapeutic treatments for lung SCC, including immunotherapies, before initiating human clinical trials.