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 (~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 (~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 (~20%), and resistance towards cytotoxic agents and ionizing radiation (~39%). Importantly, in a tumor model using immunocompetent BALB/c mice inoculated with syngeneic CD73pos/EGFRpos 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 CD8pos 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.

A Novel Bispecific Antibody for EpCAM-Directed Inhibition of the CD73/Adenosine Immune Checkpoint in Ovarian Cancer.

PD-1/PD-L1-inhibiting antibodies have shown disappointing efficacy in patients with refractory ovarian cancer (OC). Apparently, OC cells exploit nonoverlapping immunosuppressive mechanisms to evade the immune system. In this respect, the CD73-adenosine inhibitory immune checkpoint is of particular interest, as it rapidly converts pro-inflammatory ATP released from cancer cells to immunosuppressive adenosine (ADO). Moreover, cancer-cell-produced ADO is known to form a highly immunosuppressive extra-tumoral 'halo' that chronically inhibits the anticancer activity of various immune effector cells. Thus far, conventional CD73-blocking antibodies such as oleclumab show limited clinical efficacy, probably due to the fact that it indiscriminately binds to and blocks CD73 on a massive surplus of normal cells. To address this issue, we constructed a novel bispecific antibody (bsAb) CD73xEpCAM that inhibits CD73 expressed on the OC cell surface in an EpCAM-directed manner. Importantly, bsAb CD73xEpCAM showed potent capacity to inhibit the CD73 enzyme activity in an EpCAM-directed manner and restore the cytotoxic activity of ADO-suppressed anticancer T cells. Additionally, treatment with bsAb CD73xEpCAM potently inhibited the proliferative capacity of OC cells and enhanced their sensitivity to cisplatin, doxorubicin, 5FU, and ionizing radiation. BsAb CD73xEpCAM may be useful in the development of tumor-directed immunotherapeutic approaches to overcome the CD73-mediated immunosuppression in patients with refractory OC.