Synthetic Transcriptional Activators for Cancer Immunotherapy

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Cancer immunotherapy is a promising approach to treat cancer by activating a patient’s immune system against tumor cells and even providing long-term remission. These clinical benefits have led to recent FDA approvals for immune-checkpoint blockade (ICB), adoptive cell transfer (ACT), and molecular vaccine technologies, and have also motivated substantial commercial investments for their continued development. Nonetheless, these therapies are also associated with nonselective immunogenicity—which limits efficacy and harms healthy tissue. For these treatments to reach their full promise, new strategies are desperately needed to enhance immunotherapy responses while also minimizing off-target effects. One key challenge with developing immunotherapies is achieving robust anti-tumor immune responses. Strategies to promote responses include administering peptide or DNA-encoded T-cell antigens, designed to expand T-cell populations against tumor cells. These vaccines have demonstrated a potential to activate T cells, but often provide limited efficacy due to immune evasion by tumor cells. Recently, engineered transcription factor (ETF) proteins were shown to induce protein expression of latent HIV-infected cell reservoirs by targeting the HIV-1 promoter. Inspired by this approach, we envisioned developing new ETF proteins that induce expression of tumor antigens and, in turn, facilitate immune recognition of cancer cells. Although CRISPR technologies are also used for gene activation, therapeutic applications remain limited due to insufficient safety and intracellular delivery properties. Our overarching goal in this proposal is to demonstrate using an ETF to induce expression of tumor antigens in vitro. Over time, we will establish the efficacy of ETFs, which will guide designs of future in vivo experiments (not included in this proposal) and future grant applications. We will begin these studies by developing ETF activators that induce expression of antigenic proteins associated with human papilloma virus (HPV)-18, which is associated with cervical carcinoma. We selected HPV-18 because it is an ideal starting point for developing ETFs in our group for several reasons. One reason is that the Upstream Regulatory Region (URR) and reporter cell line (HeLa) are well-characterized. Another reason is that these studies build upon the prior work with HIV. If successful, we plan to develop additional ETF proteins as a facile approach for enhancing tumor cell immunogenicity in the clinic. We will develop ETFs through the design, preparation, and characterization of ZF building blocks, which we will assemble to recognize promoter region genes that are 18 nucleotides in length. We will also incorporate a ‘transcriptional activator’ to induce protein transcription and translation. We will evaluate ETF-mediated activation by encoding these constructs into a mammalian expression vector, followed by measuring expression of the protein antigen in cancer cells. We will also prepare synthetic ETFs as therapeutic proteins to enable DNA-binding studies and to enhance intracellular delivery properties.