Dual-mechanism anti-CD73 antibodies CR201 and CR202 targeting distinct domains for cancer immunotherapy.

Background: CD73 is a key enzyme in the adenosine-mediated immunosuppressive pathway and represents an attractive target for cancer immunotherapy. Here, we aimed to develop novel anti-CD73 antibodies with dual mechanisms of action by simultaneously inhibiting enzymatic activity and promoting receptor internalization to more effectively disrupt the adenosine barrier within the tumor microenvironment. Methods: Two monoclonal antibodies, CR201 and CR202, were generated and characterized for cross-species reactivity, inhibition of both membrane-bound and soluble CD73 enzymatic activity, and domain-specific epitope recognition. Functional assays were performed to evaluate reversal of AMP-mediated T-cell suppression, restoration of IFN-γ secretion, and induction of CD73 internalization. Results: CR201 and CR202 specifically bound human and cynomolgus CD73 and recognized distinct structural domains, with CR202 targeting the N-terminal domain and CR201 binding the C-terminal domain. Functionally, CR202 demonstrated greater potency against membrane-bound CD73, whereas CR201 achieved complete inhibition of soluble CD73 activity. Both antibodies effectively restored T-cell proliferation and IFN-γ production and promoted internalization of surface CD73. Conclusion: CR201 and CR202 are domain-specific, dual-mechanism anti-CD73 antibodies that integrate potent enzymatic blockade with receptor internalization, thereby enhancing antitumor immune responses. These findings highlight the therapeutic potential of targeting distinct functional domains of CD73 to overcome adenosine-mediated immunosuppression in cancer.