Bispecific antibody CD73xEGFR more selectively inhibits the CD73/adenosine immune checkpoint on cancer cells and concurrently counteracts pro-oncogenic activities of CD73 and EGFR

Background CD73 is an ecto-enzyme that is involved in the conversion of pro-inflammatory extracellular ATP (eATP) excreted by cancer cells under stress to anti-inflammatory adenosine (ADO). A broad variety of solid cancer types was shown to exploit CD73 overexpression as a suppressive immune checkpoint. Consequently, CD73-antagonistic antibodies, most notably oleclumab, are currently evaluated in several multicenter trials for clinical applicability. However, the efficacy of conventional monospecific CD73-inhibiting antibodies may be limited due to on-target/ off-tumor binding to CD73 on normal cells. Therefore, a novel approach that more selectively directs CD73 immune checkpoint inhibition towards cancer cells is warranted. Methods To address this issue, we constructed a novel tetravalent bispecific antibody (bsAb), designated bsAb CD73xEGFR. Subsequently, the anticancer activities of bsAb CD73xEGFR were evaluated using in vitro and in vivo tumor models. Results In vitro treatment of various carcinoma cell types with bsAb CD73xEGFR potently inhibited the enzyme activity of CD73 (similar to 71%) in an EGFR-directed manner. In this process, bsAb CD73xEGFR induced rapid internalization of antigen/antibody complexes, which resulted in a prolonged concurrent displacement of both CD73 and EGFR from the cancer cell surface. In addition, bsAb CD73xEGFR sensitized cancer to the cytotoxic activity of various chemotherapeutic agents and potently inhibited the proliferative/migratory capacity (similar to 40%) of cancer cells. Unexpectedly, we uncovered that treatment of carcinoma cells with oleclumab appeared to enhance several pro-oncogenic features, including upregulation and phosphorylation of EGFR, tumor cell proliferation (similar to 20%), and resistance towards cytotoxic agents and ionizing radiation (similar to 39%). Importantly, in a tumor model using immunocompetent BALB/c mice inoculated with syngeneic CD73(pos)/EGFR(pos) CT26 cancer cells, treatment with bsAb CD73xEGFR outperformed oleclumab (65% vs 31% tumor volume reduction). Compared with oleclumab, treatment with bsAb CD73xEGFR enhanced the intratumoral presence of CD8(pos) T cells and M1 macrophages. Conclusions BsAb CD73xEGFR outperforms oleclumab as it inhibits the CD73/ADO immune checkpoint in an EGFR-directed manner and concurrently counteracts several oncogenic activities of EGFR and CD73. Therefore, bsAb CD73xEGFR may be of significant clinical potential for various forms of difficult-to-treat solid cancer types.