Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed Immunotherapy

There is increasing interest in targeting CD33 in malignant and non-malignant disorders. In acute myeloid leukemia, longer survival with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this strategy. Still, GO benefits only some patients, prompting efforts to develop more potent CD33-directed therapeutics. As one limitation, CD33 antibodies typically recognize the membrane-distal V-set domain. Using various artificial CD33 proteins, in which this domain was differentially positioned within the extracellular portion of the molecule, we tested whether targeting membrane-proximal targeting epitopes enhances the effector functions of CD33 antibody-based therapeutics. Consistent with this idea, a CD33(V-set)/CD3 bispecific antibody (BsAb) and CD33(V-set)-directed chimeric antigen receptor (CAR)-modified T cells elicited substantially greater cytotoxicity against cells expressing a CD33 variant lacking the entire C2-set domain than cells expressing full-length CD33, whereas cytotoxic effects induced by GO were independent of the position of the V-set domain. We therefore raised murine and human antibodies against the C2-set domain of human CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain (“CD33(PAN) antibodies”). These antibodies internalized when bound to CD33 and, as CD33(PAN)/CD3 BsAb, had potent cytolytic effects against CD33(+) cells. Together, our data provide rationale for further development of CD33(PAN) antibody-based therapeutics.