Purpose Imiquimod is a toll-like receptor 7 agonist utilized topically to manage genital warts and basal cell carcinoma. We examine the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic HPV vaccination that comprises a naked DNA vector expressing calreticulin fused to HPV16 E7. Experimental Design Using an orthotopic HPV16 E6/E7+ syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we show that combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod results in synergistic immune-mediated tumor clearance. Results Imiquimod induces cervicovaginal accumulation of activated E7-specific CD8+ T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8+ T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract. These chemokines are expressed upon IFNγ receptor activation and attract cells expressing their receptor, CXCR3. In this study, T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as the tissue resident memory T cell (Trm) marker CD49a, a mucosal homing integrin. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3+CD8+ T cells to the genital tract. Conclusions Our study has potential clinical relevance because imiquimod is FDA approved for condyloma accuminata and basal cell carcinoma and intramuscular vaccination with pNGVL4a-CRT/E7(detox) is currently undergoing clinical testing, suggesting potential for their synergistic action to induce strong antigen-specific Trm-mediated immune responses and antitumor effects in genital mucosa.

Substitution of the m,p-chloro groups of bis-benzylidinepiperidone RA190 for p-nitro, generating RA183, enhanced covalent drug binding to Cys88 of RPN13. Treatment of cancer cell lines with RA183 inhibited ubiquitin-mediated protein degradation, resulting in rapid accumulation of high-molecular-weight polyubiquitinated proteins, blockade of NFκB signaling, endoplasmic reticulum stress, an unfolded protein response, production of reactive oxygen species, and apoptotic cell death. High-grade ovarian cancer, triple-negative breast cancer, and multiple myeloma cell lines were particularly vulnerable to RA183. RA183 stabilized a tetraubiquitin-linked firefly luciferase reporter protein in cancer cell lines and mice, demonstrating in vitro and in vivo proteasomal inhibition, respectively. However, RA183 was rapidly cleared from plasma, likely reflecting its rapid degradation to the active compound RA9, as seen in human liver microsomes. Intraperitoneal administration of RA183 inhibited proteasome function and orthotopic tumor growth in mice bearing human ovarian cancer model ES2-luc ascites or syngeneic ID8-luc tumor.