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1084 NTX-0250, a multimodal mRNA-based immunotherapy, eradicates large established tumors in a stringent mouse model of HPV16-driven cancer
  1. Ole Haabeth1,
  2. Diane DaSilva2,
  3. Colin McKinlay1,
  4. Weiqun Liu1,
  5. Edward Lemmens1,
  6. Christopher Rae1,
  7. Adrienne Sallets1,
  8. Daniel Frimannsson1,
  9. Sangeeta Nath1,
  10. Nicole Peck1,
  11. Ou Li1,
  12. Nicole Fay1,
  13. Ruben Prins2,
  14. Meredith Leong1,
  15. W Martin Kast2 and
  16. Samuel Deutsch1
  1. 1Nutcracker Therapeutics, Emeryville, CA, USA
  2. 2Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA


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.



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Abstract 1084 Figure 1

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).

Abstract 1084 Figure 2

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.

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