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Abstract
Background Human papillomavirus (HPV) is a contagious cause of anogenital and oropharyngeal cancers developing from persistently infected and subsequently transformed basal keratinocytes of mucosal epithelium. More than 90% of cervical cancers and pre-cancerous cervical intraepithelial neoplasia (CIN) are linked to infections with high-risk HPV, with more than 50% of cancers linked to HPV16.1,2 At least 25% of women with high-grade CIN lesions progress to in situ or invasive cancer, if untreated.3 Current treatments for high-grade CIN can remove abnormal tissue but do not address underlying HPV infection, and 15% of women treated develop residual or recurrent high-grade CIN or cervical cancer.4 Long-term efficacy may require induction of tumor-specific T cell responses combined with alleviated local immune suppression and increased tumor immune cell infiltration. Multimodal mRNA-based immunotherapies that deliver both antigens and immunomodulators in a single drug product represent a promising new approach for treatment of CIN and cervical cancer that can address current disease as well as the underlying cause (HPV infection). Here we report on pre-clinical efficacy of NTX-0250, a nanoparticle-formulated, multi-component mRNA drug that co-delivers a novel HPV16 antigen design with two potent immunomodulators.
Methods To test efficacy, we utilized the well-established, clinically relevant, C3.43 tumor model (5). C3.43 is a progressive subclone of C3, HPV16-transformed B6 mouse embryo cell line that expresses HPV16 E6 and E7 antigens under the natural promoter.5 Therapeutic efficacy of NTX-0250 was assessed in mice with large (>120mm3) C3.43 tumors. HPV16-specific T cells were assessed by flow cytometry on peripheral blood mononuclear cells (PBMCs). Mechanistic studies were performed by post-treatment tumor microenvironment characterization. To assess translational potency of NTX-0250, induction of HPV-specific T cell responses in cynomolgus monkeys was measured by flow cytometry and IFNg ELISpot on PBMCs
Results In tumor challenged mice, administration of NTX-0250 induces complete regression of large tumors resulting in long-term, tumor-free survival of 100% of treated animals (figure 1A). Complete responses are accompanied by strong tumor immune infiltration of CD8+, CD4+ APCs and NK cells and upregulation of IFNγ in the tumor microenvironment (figure 1B). In cynomolgus monkeys, administration of NTX-0250 induces strong HPV16-specific responses (figure 2).
Conclusions Here we report for the first time robust pre-clinical efficacy of a multimodal, mRNA-based therapeutic combining antigen- and immunomodulator-encoding mRNAs in a novel nanoparticle formulation. NTX-0250 treatment resulted in complete regression of large established murine tumors and robust induction of HPV-specific T cell responses in non-human primates.
References
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NTX-0250 induces complete regression of large tumors in miceA) 10 C57/BL6 mice per group were inoculated with 1x106 C3.43 cells in the subcutaneous compartment. NTX-0250 or Mock-treatment, was initiated when tumors were >120mm3 (18 after tumor inoculation). Mice received 3 doses of 1.5µg NTX-0250 with 7 days interval. Tumor growth was monitored for 90 days. B) Representative immunohistochemistry staining of C3.43 tumors 3 days post treatment with vehicle control (Mock treated) or NTX-0250. Slides were stained for infiltrating CD8+ T cells (Brown).
NTX-0250 induces robust HPV16-specific responses in NHPs.Cynomolgus monkeys (n=2) were immunized three times with low or high dose of NTX-0250. Induction of HPV16 E6 and E7-specific T cells responses were measured by IFNγ ELISpot after one and three doses.