PROJECT SUMMARY/ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype which comprises approximately 20% of newly diagnosed BCs. Although epidermal growth factor receptor (EGFR) is expressed in ~60% of TNBC and 65-72% of basal-like BCs, the anti-EGFR MAb, Cetuximab (Erbitux®), elicits little response as a single-agent or when combined with chemotherapy. Aggressive metastatic behavior of TNBC correlates with the ability to form vascular channels lined by tumor cells themselves, known as Vasculogenic Mimicry (VM), and the formation of cellular protrusions called ‘invadopodia’ that facilitate cellular migration, intravasation and metastasis. Although angiogenesis plays a central role in BC growth and metastasis, combined therapeutic targeting of angiogenesis and VM has largely been untested. We have synthesized an antibody-fusion protein, aEGFR-huEndo-P125A (EEPA125) by linking the heavy chains of an anti-EGFR IgG1 with a highly anti- angiogenic “payload”, huEndoP125A, which is a P125 --> A125 mutant of human endostatin. EEPA125 inhibited both angiogenesis, and VM, which was not seen with cetuximab nor huEndo-P125A alone. A combination of anti-EGFR IgG1 and huEndo-P125A in non-fused form did not inhibit VM suggesting that the delivery of dimeric huEndo-P125A by EEPA125 fusion is essential for VM inhibition. We successfully produced and purified EEPA125 from a stable pool of CHO cells to >95% purity. Testing of EEPA125 in vivo showed inhibition of MDA-MB-468 TNBC tumor growth and metastatic spread than cetuximab. EEPA125 more effective also markedly reduced lung metastasis following intravenous administration of lung tropic MDA-MB-231-4175 TNBC cells, and improved survival of mice compared to cetuximab treatment. EEPA125 also demonstrated ADCC activity, and it markedly inhibited Wnt/b-catenin signaling, TNBC motility and migration, and invadopodia formation. Furthermore, it reduced the secretion of soluble MMP2 and MT1-MMP proteins by TNBC cells compared to treatment with huEndo-P125A, or cetuximab. We also characterized the serum elimination pattern of EEPA125 in immunocompromised mice. We now propose to carry out confirmatory in vivo efficacy testing of EEPA125 using clinically relevant TNBC PDX (patient derived xenograft) models, and to study EEPA125 efficacy in preventing increased VM and “rebound angiogenesis” induced following treatment with the anti-angiogenic VEGFR inhibitor sunitinib. Combination with chemotherapeutic agents such as paclitaxel with EEPA125 will be tested. We will generate a stable CHO cell clone for process engineering and cGMP manufacture of EEPA125, and perform PK/PD and toxicology/safety studies in immunocompromised and immunocompetent rodents and in cynomolgus monkeys. We will also develop an EGFR expressing murine breast cancer model and investigate the immune effects of EEPA125 when combined with of an anti PD-L1 checkpoint inhibitor in immunocompetent mice. Results generated will ena...