Summary Triple negative breast cancers (TNBC) are devastating diseases with a median survival of less than 1-year for patients with metastatic disease. TNBC patients with tumor of high fibrotic stroma have even worse prognosis. There are no specific targeted therapies available for TNBC, and the only treatment option is broadly cytotoxic chemotherapy drugs, despite less effective and strong unwanted side effects of such drugs. One major barrier to efficacy of anti-tumor therapeutics is the dense fibrotic stromal and dysregulated tumor blood vessels which contribute to failure of therapies. Evidence suggests that cancer associated fibroblasts (CAF) produce the stromal collagen. The ECM laid down by CAF is considered to be one of the major contributors of resistance to established therapies of the diseases. TNBC has high angiogenic activity. Dense tumor vasculature associated with a shorter time from diagnosis to relapse and from relapse to death. The dysregulated vessel structure in TNBC tumor often leads to resistance to blood flow into tumor, which is another important barrier for drug delivery. Depleting CAF and abrogating tumor angiogenesis could significantly improve efficacy of existing TNBC cancer treatments. However, currently, there are no approved therapies that are able to deplete CAF and tumor angiogenesis in TNBC cancer. We have developed a novel therapeutic protein (ProAgio) using rational protein design. ProAgio is designed to target integrin v3 at a novel site (not the ligand binding site). ProAgio specifically induces apoptosis of integrin v3 expressing cells with high efficacy by a novel mechanism of drug action (recruiting & activating caspase 8 at cytoplasmic domain of). We reasoned that, since both CAF and angiogenic endothelial cells (aEC) express high levels of integrin v3, and since ProAgio is very effective in inducing apoptosis of integrin v3 expressing cells, ProAgio should both deplete CAF, eliminate intratumoral angiogenic blood vessels in and around TNBC tumors. This unique strategy may prove advantageous in treatment of TNBC. The main objective of this direct phase II SBIR application is to generate a definitive dataset to enable the development of ProAgio as a viable therapeutic option for TNBC patients. Characterization of the toxicity and tolerability and determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of ProAgio as a single agent is on-going. Aim 1 will characterize the toxicity and tolerability and determine the recommended phase II dose (RP2D) of ProAgio in combination with gemcitabine (Gem). Aim 2 will obtain preliminary anti-cancer activity data of ProAgio and ProAgio + Gem in TNBC patients. Aim 3 will analyze the effects of ProAgio in patient tumor to validate the mechanism of drug action in patients. This clinical study project will explore new therapeutic avenue for TNBC patients. Our goal is that, through our study, we will introduce a new treatment approach for ...