CD73 functions as an ecto-enzyme that converts extracellular ATP (eATP), released under stress by tumor cells, into immunosuppressive adenosine (ADO). Many solid tumors exploit CD73 overexpression to evade immune responses, positioning it as an immune checkpoint target. Antibodies such as oleclumab are currently being tested in clinical trials. However, standard monospecific CD73 antibodies may affect normal tissues due to widespread CD73 expression, limiting tumor selectivity. Hence, strategies that focus checkpoint inhibition specifically on tumor cells are needed. To achieve targeted inhibition, we developed a tetravalent bispecific antibody, bsAb CD73xEGFR, and tested its anticancer effects in both in vitro carcinoma models and in vivo tumor-bearing mice. Treatment of carcinoma cells with bsAb CD73xEGFR reduced CD73 activity by approximately 71% in an EGFR-directed manner. The antibody promoted rapid internalization of CD73/EGFR complexes, causing prolonged concurrent removal of both antigens from the tumor cell surface. BsAb CD73xEGFR also enhanced the efficacy of cytotoxic chemotherapy and decreased tumor cell proliferation and migration by roughly 40%. Surprisingly, oleclumab treatment alone enhanced certain pro-tumor processes, including EGFR phosphorylation, increased proliferation (~20%), and heightened resistance to cytotoxic drugs and radiation (~39%). In immunocompetent BALB/c mice carrying syngeneic CD73^pos/EGFR^pos CT26 tumors, bsAb CD73xEGFR achieved superior tumor reduction compared with oleclumab (65% vs. 31%) and increased the presence of intratumoral CD8^pos T cells and M1 macrophages. BsAb CD73xEGFR demonstrates superior antitumor activity relative to oleclumab, selectively inhibiting CD73 in an EGFR-targeted fashion while simultaneously mitigating oncogenic signaling from EGFR and CD73. These findings highlight the potential of bsAb CD73xEGFR for the treatment of hard-to-treat solid tumors.
