SYNERGYS BIOTHERAPEUTICS, INC. — Department of Health and Human Services SBIR Phase I: 102

Description

PROJECT SUMMARY ABSTRACT Triple Negative Breast Cancer TNBC is a highly aggressive subtype of breast cancer which exhibits both enhanced angiogenesis and vasculogenic mimicry or VM i e the direct formation and lining of vascular channels by tumor cells While the expression of epidermal growth factor receptor EGFR HER is reported in greater than of TNBC and is similarly expressed in to of basal like tumors no proven targeted therapy is currently available for the treatment of TNBC Cetuximab Erbitux a chimeric monoclonal antibody targeting EGFR elicits little response as single agent therapy in the setting of advanced TNBC In the present study the combined inhibition of angiogenesis and VM is proposed as a potentially superior therapeutic strategy for inhibiting TNBC growth and metastasis Our approach utilizes a fusion protein that was generated by linking an anti EGFR MAb as a targeted delivery entity of two molecules of a mutant form of human endostatin huEndoP A where alanine substitution of Proline significantly increases the anti angiogenic potency of endostatin The Anti EGFR antibody coupled huEndo P A fusion anti EGFR huEndo P A containing bivalent or dimeric huEndo P A thus generated exhibits potent inhibition of angiogenesis by endothelial cells More interestingly it also demonstrates profound inhibition of VM not seen with parental antibody nor with wild type endostatin or huEndo P A alone or when combined with cetuximab without fusion suggesting that the bivalent or dimeric endo P A in fusion is highly essential for VM inhibition The fusion is differentiated from anti VEGF antibody bevacizumab which did not inhibit TNBC VM suggesting the fusion also has mechanisms of action distinct from targeting of the VEGF pathway We also observed a consistent decrease in total catenin levels following fusion treatment of MDA MB TNBC cells which is consistent with inhibition of Wnt signaling pathway while endostatin had minimal effects at similar molar concentrations Inhibition of VM by the anti EGFR huEndo P A fusion proteins was also associated with significantly reduced tumor cell motility We have now generated a stable CHO cell pool with fusion production yield of andgt mg liter and have optimized the purification methods that show andgt purity of monomeric fusion protein that is stable at both and for several months In an initial experiment the fusion has shown am markedly enhanced inhibitory effect on inhibition of TNBC lung metastasis compared to cetuximab These promising preliminary results provide the basis for accelerated testing of the potential therapeutic utility of dual inhibition of both angiogenesis and VM on TNBC growth and metastasis in vivo to generate pre clinical data that will lead to therapeutic testing in human TNBC patients Due to the striking in vitro and encouraging preliminary in vivo results and with the availability of cell line for production of large quantity of the fusion protein we will focus on TNBC as an unmet need and extend our work to optimize the effects of the fusion by itself or in combination with approved anti angiogenics such as bevacizumab and or chemotherapy agent s on TNBC growth and metastasis The fusion will also be tested for its ability to mediate effective ADCC We anticipate that the multifunctional effects of anti angiogenesis VM inhibition and ADCC will render EGFR IgG huEndo P A a breakthrough therapeutic for TNBC and other EGFR malignancies PROJECT NARRATIVE Despite the development of new therapeutic agents Triple Negative Breast Cancer TNBC a basal like subtype of breast cancer which is prevalent among young women remains a critical health care issue and new treatments are desperately needed Since TNBC elicits enhanced angiogenesis as well as vasculogenic mimicry i e the formation of fluid conducting channels by highly invasive and genetically dysregulated tumor cells in the present study we are exploring the combined inhibition of VM and angiogenesis as a potentially superior therapeutic strategy for inhibiting TNBC growth and metastasis To this end we have generated a novel recombinant fusion protein using an anti EGFR antibody and a mutant form of human endostatin endostatin P A where alanine substitution of Proline significantly increases the anti angiogenic potency of endostatin Anti EGFR huEndo P A fusion thus generated contains bivalent or dimeric endo statin P A which exhibited excellent anti angiogenic activity and profound inhibition of TNBC vasculogenic mimicry in vitro More interestingly no inhibition of VM was observed with parental antibody nor with huEndo P A alone or when huEndo P A was present in conjunction with the parent antibody without being fused to it The fusion was further differentiated from the market approved anti angiogenic therapeutic bevacizumab Avastin in that it did not show any inhibition of VM and was significantly less effective in demonstrating anti angiogenic properties in our in vitro assay system In initial experiment an IgG isotype of the fusion protein anti EGFR IgG huEndo P A has shown substantial inhibition of in vivo growth of TNBC xenografts the anti EGFR IgG huEndo P A fusion has demonstrated excellent lung metastasis inhibition in TNBC mouse model With the availability of largescale CHO production method and purification methods in place we aim to carry out more systematic evaluation of dual inhibition of both angiogenesis and VM on TNBC growth and in order to generate compelling pre clinical data in anticipation of its therapeutic testing in TNBC patients The fusion will also be tested for its ability to mediate ADCC Antibody Dependent Cell mediated Cytotoxicity Finally the impact of using the anti angiogenic drug bevacizumab Avastin in combination with chemotherapeutic agent s on the fusion as regards to inhibition of angiogenesis and tumor growth and vascularity will be determined If successful the proposed studies involving a multi functional antibody fusion having immune anti angiogenic and anti VM therapeutic activities could establish new treatment options for the highly unmet needs of TNBC and possibly for other EGFR malignancies as well including lung head and neck bladder esophageal and glioblastoma Folberg R and Maniotis AJ Vasculogenic mimicry APMIS