IRZ-01：一种靶向CEA和MUC1的自佐剂型外膜囊泡疫苗用于结直肠癌免疫治疗

Background: Irazu Oncology developed a tumor vaccine platform using a proprietary, attenuated Salmonella Typhi strain engineered to express human tumor antigens and shed antigen-decorated outer membrane vesicles (OMVs). This flexible system enables rapid development of mono- or multi-valent vaccines. IRZ-01 is an OMV-based vaccine displaying epitopes from two well-characterized and validated tumor-associated antigens (TAAs): Mucin-1 (MUC1) and CEACAM5 (CEA). Here we show that IRZ-01 administration to immunocompetent mice triggers potent antigen-specific humoral and cellular immunity and confers single-agent antitumor efficacy in models expressing CEA and/or MUC1. Methods: Salmonella Typhi CVD911∆ fliC (pPagL-CEA/MUC1) was cultured in animal-free soytone media to produce IRZ-01 OMVs. OMVs were purified by tangential flow filtration and size-exclusion chromatography. Characterization included DLS (size), TRPS (zeta potential), cryo-EM (morphology), immuno-gold EM, and Western blotting for CEA/MUC1 surface display. Toll-like receptor (TLR) activation was tested using HEK-Blue™ cells expressing human TLR2, 3, 4, 5, 7, 8, or 9 (InvivoGen). Immunogenicity was evaluated in C57BL/6 mice given two doses of IRZ-01 (0.25 µg IV or 2 µg IM, days 0 and 7) or 5×10⁹ CFU live bacteria; controls received PBS. Serum IgG was quantified by ELISA (days -1, 6, 20); T-cell responses assessed by IFN-gamma ELISPOT on day 21 splenocytes. Antitumor efficacy was assessed in MC38-CEA and MC38-MUC1 syngeneic models by monitoring tumor growth post-challenge with IRZ-01. Results: Purified IRZ-01 OMVs were 90-100 nm, zeta potential -13.9 mV, with confirmed CEA/MUC1 surface expression by Western blot and immuno-gold EM. IRZ-01 selectively activated TLR2 and TLR4, confirming self-adjuvanting properties due to native microbe-associated molecular patterns (MAMPs) naturally present in their outer membrane.Vaccination elicited rapid, high-titer antigen-specific IgG by day 6, strongly boosted after the second dose, with comparable responses via IV or IM routes and similar to the live vector. IFN-gamma ELISPOT showed significant (p<0.05 vs PBS) CEA- and MUC1-specific T-cell responses that correlated closely with antibody levels.Encouraged by strong humoral and cellular responses, we conducted efficacy studies using a syngeneic C57BL/6 mouse model implanted with MC38-CEA and MC38-MUC1 cells, IRZ-01 induced marked tumor growth inhibition; volumes receded shortly after treatment and remained significantly lower than untreated PBS control groups for all experimental groups. Survival data demonstrated >90% survival in groups treated with IRZ-01 regardless of tumor line. Conclusion: IRZ-01 is a potent, self-adjuvanted OMV vaccine that induces robust CEA/MUC1-specific humoral and cellular immunity and delivers strong single-agent efficacy in syngeneic tumor models.