Abstract 2696: Polymeric structure and host Toll-Like Receptor 4 dictate the immunogenicity of NY-ESO-1 in vivo

Field: Immunology and Microbiology (Life Sciences)

Abstract Introduction: NY-ESO-1 is a tumor-associated Ag (TAA) with distinctively strong immunogenicity. Our recent investigation of the specific interaction between NY-ESO-1 and complement C1q receptor (or Calreticulin, CRT) on the surface of immature DC, macrophages, and monocytes, indicated a unique interaction between NY-ESO-1 and the innate immune system (The Journal of Immunology, Zeng et al 2006). We further explore the molecular mechanism of the link between NY-ESO-1 and immature DC. Experimental Procedures: Cysteine-to-serine (C-to-S) substitutions were introduced into NY-ESO-1 proteins. Wild type NY-ESO-1 and mutant proteins were expressed in bacterial and mammalian cells and detected by western blot. The binding properties of wild type and mutant NY-ESO-1 with human and mouse immature DC were characterized. Bone marrow derived immature DC from the wild-type, TLR2-/-, and TLR4-/- mice were used for binding to NY-ESO-1. Mouse prostate cancer myc-cap cells with NY-ESO-1 and variants expression were co-cultured with human immature DC and mouse macrophage cell RAW264, and Th1/Th2 cytokines secretion were measured. Normal and the TLR4-/- mice were immunized with gene-gun delivery of plasmids encoding the wild type and variants of NY-ESO-1. Plasmids encoding NY-ESO-1 fused with a major mugwort pollen allergen Art v1 and a TAA carbonic anhydrase 9 were also delivered by gene gun to balb/c mice for immunization. Results: Wild Type NY-ESO-1 showed clear formation of dimers, trimers and even polymers due to inter-molecular disulfide bonds. Mutant NY-ESO-1 with all cysteines substituted by serines appeared only as monomer. C-to-S mutations significantly abolished the ability of NY-ESO-1 binding to human and mouse immature DC in vitro. The absence of TLR4 but not TLR2 partially impaired the binding of NY-ESO-1 to mouse bone marrow derived DC in vitro. Similarly, binding of recombinant NY-ESO-1 to human immature DC was blocked by anti-TLR4 monoclonal antibody. Mouse TLR4 co-precipitated with NY-ESO-1 by western blotting confirming the direct interaction between NY-ESO-1 and TLR4. Disrupting polymeric structure of NY-ESO-1 led to diminished immunogenicity and altered TLR4 dependence in the induced antibody responses. NY-ESO-1 expression in myc-cap cells impacted the immature DC inflammatory responses in co-culture system. Myc-cap cells bearing membrane-anchored NY-ESO-1 also affected tumor growth in vivo, correlated with in vitro studies. Gene gun-delivered plasmid DNA encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Conclusion: NY-ESO-1 binds to immature DC depending on its polymeric architectural structure and its interaction with immature DC surface receptor TLR4. The unique interaction between NY-ESO-1 and immature DC renders NY-ESO-1 serve as a molecular adjuvant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2696. doi:10.1158/1538-7445.AM2011-2696