Humanized anti-CEACAM6 PEGylated scFv: A promising novel therapy for pancreatic cancer

Abstract

3042 Background: Novel Therapies for pancreatic cancer (PC) based on biological insight are urgently needed. CEACAM6, a member of the carcinoembryonic antigen (CEA) family, is a cell surface oncogene on PC cells. It is a glycoprotein composed of 3 Ig-like domains (Mr ∼35.2kD) and is linked to the plasma membrane via a glycophospholipid linked anchor. It is over-expressed on ≥95% PC patients’ found irrespective of stage of disease and represents a target for antibody therapy. Methods: Murine anti-CEACAM6 monoclonal antibody (Mab) 13–1 was humanized by a structure-based approach and single chain variable fragments (scFv) designed consisting of a Gly/Ser-linker which includes a cysteine residue for PEGylation. Murine scFv and 4 humanized scFv fragments (Version.1, 2, 7, 8) were bacterially expressed, purified, PEGylated and evaluated for activity alone or in combination with gemcitabine in human PC cell lines and mouse xenograft tumors. Cell viability, apoptosis and in-cell Westerns were done for efficacy and binding. Mouse xenograft tumors were treated with scFv or PEGylated scFv or combination with gemcitabine. Tumors (treated Vs untreated) were analyzed by immunohistochemistry for Ki-67, CD31, CEACAM6 and Caspase 3 Results: Mab 13–1, murine and 4 humanized scFvs’ were cytotoxic to CEACAM6 expressing PC cells (BxPC-3, HPAF-2) and not to PC cells that lack CEACAM6 (CaPan-2) with increased PARP-cleavage that was dose dependent (IC50=10μg/mL). Western blotting of culture media and serum from xenograft mice showed that CEACAM6 is not shed. In-cell Western confirmed humanized V.7 to be the best binder (KD=1–10μg/mL) confirming protein-protein interaction studies performed in silico with homology models of murine and humanized scFvs’ and CEACAM6. The murine scFv alone or the humanized scFV (V.8) in combination with gemcitabine delayed tumor growth by >50%. This tumor reduction correlated well with the immunohistochemical markers of response. Conclusions: We have demonstrated that CEACAM6 is a clinically relevant target in PC and that a PEGylated humanized scFv is a novel effective therapy alone and/or in combination with gemcitabine. Humanized scFv V.7 is undergoing mouse xenograft evaluation and expect will be the most effective agent to take forward into clinical development. No significant financial relationships to disclose.