Abstract: More than one million cases of cutaneous squamous cell carcinoma (cSCC) are diagnosed annually in the US and approximately 4% of patients develop metastases and 2% die of cSCC; thus, a similar number of people die each year from cSCC as melanoma. Immune checkpoint inhibitors (ICI) are a new class of drugs that have transformed the therapy of multiple cancer types, but only half of cSCC patients respond to ICI treatment. ICI target receptors on T cells, such as PD-1, that are expressed after activation and function to turn off T cell responses. The response of cSCC patients to ICI demonstrates the ability of T cells to constrain cSCC growth. However, it remains unclear the extent to which CD8 and, in particular, CD4 T cells contribute to immune-mediated control of cSCC. While the focus of anti-tumor T cell responses has been on MHC class I neoantigens that elicit cytotoxic CD8 T cell responses, there is growing evidence that MHC class II neoantigens eliciting CD4 T cell responses are critical in constraining tumor growth and enhancing response to ICI. Thus, there is a critical need to understand the role of CD8 and CD4 T cells, especially the role of neoantigen-specific T cells, in controlling cSCC growth. We generated a novel physiologic cSCC transplantable model on the BALB/c background from a solar simulated light-induced invasive cSCC tumor. Preliminary data supports that T cells constrain the in vivo tumor growth in the cSCC model and that this model is sensitive to anti-PD-1 treatment. Using bioinformatic approaches with whole exome and RNA sequencing data, we have identified immunogenic MHC class I and II neoantigens predicted to elicit a T cell response based on the binding affinity and presentation of the neoantigen:MHC complex and neoantigen expression. Using melanoma patient data, our lab has previously demonstrated that these characteristics accurately predict the ability of a neoantigen to elicit a T cell response. The central hypothesis is that both neoantigen- specific CD8 and CD4 T cells contribute to immune-mediated control of cSCC growth and response to treatment with vaccination with immunogenic neoantigens alone or in combination with anti-PD-1. To address this hypothesis, we will determine the role of CD8 and CD4 T cells in controlling tumor growth, identify MHC class I and II neoantigens that elicit in vivo T cell responses, and evaluate the expression of functional and inhibitory neoantigen-specific CD8 and CD4 T cells throughout cSCC tumor growth. Then, we will vaccinate mice with dendritic cells loaded with irradiated tumor cells or immunogenic MHC class I and/or II neoantigens and compare the efficacy of these vaccination strategies in inducing CD8 and/or CD4 T cells to prevent cSCC growth and treat cSCC alone or in combination with anti-PD-1. We will demonstrate the requirement for CD8 and/or CD4 T cells through antibody depletion and adoptive transfer. The impact of this project is to 1) identify the contributions of neoantigen-specific CD8 and CD4 T cells in control of cSCC growth and 2) advance the application of personalized neoantigen vaccines to treat cSCC alone or in combination with anti-PD-1.

PROJECT NARRATIVE Non-melanoma skin cancer, including cutaneous squamous cell carcinoma (cSCC), is the most common cancer. This proposal uses a novel physiologic cSCC murine model to address the critical need to understand the role of CD8 and CD4 T cells, especially the role of neoantigen-specific T cells, in controlling cSCC growth. Completion of this proposal will determine the features of neoantigen-specific T cell responses that constrain cSCC growth and identify features of cancer vaccination strategies that improve outcomes in cSCC and enhance response to anti-PD-1.