132 A novel anti-PDL1 antibody-based bifunctional protein with enhanced immunological activity

Zfra is a 31-amino-acid zinc finger-like protein, which participates in tumor necrosis factor signaling. Here, we determined that synthetic full-length Zfra1−31 peptide selfpolymerized in degassed buffers without catalytic enzymes. When nude mice and BALB/c mice were pre-injected with micromolar levels of Zfra1−31 or truncated Zfra4−10 via tail veins, these mice became resistant to the growth, metastasis and stemness of prostate, breast, and lung cancer cells, melanoma cells, and many malignant cancer cells. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra polymerization and cancer suppression in vivo. Injected Zfra was deposited mainly in the spleen. Transfer of Zfra-stimulated spleen cells to naive mice conferred resistance to cancer growth. Mechanistically, Zfra bound membrane hyaluronidase Hyal-2 and suppressed the TGF-b/Hyal-2/WWOX/Smad4 signaling, via down-regulation of Hyal-2 and activated WWOX (with Y33 phosphorylation), in the spleen for generating novel non-T/non-B memory cells, designated Hyal2+ CD3−CD19− cells. Transfer of these cells to naive mice also induced anticancer response. Similarly, agonist anti-Hyal-2 antibody mimicked the effect of Zfra in causing cancer suppression. In conclusion, Zfra self-polymerizes in the spleen to activate Hyal2+ CD3− CD19− cells for blocking cancer growth, stemness and metastasis in vivo. Supported in part by NSC and NHRI, Taiwan, and DoD, USA