Dual targeting of ROR1 and ROR2 with glycoengineered antibodies elicits potent and selective immunotherapy in hairy cell leukemia

Abstract Receptor tyrosine kinase-like orphan receptors 1 and 2 (ROR1 and ROR2) are oncoembryonic antigens aberrantly expressed on hairy cell leukemia (HCL) B cells and various other cancers, but absent from healthy B cells and postpartum tissues. We generated novel, glycoengineered monoclonal antibodies—GE-zilovertamab (anti-ROR1) and GE-6E6 (anti-ROR2)—by removing core fucose residues from the Fc regions of their parental humanized IgG1 antibodies, zilovertamab (UC-961/cirmtuzumab) and 6E6, respectively. To assess their immunotherapeutic potential, we engineered the CD20+ B cell leukemia MEC1 cell line to stably express either ROR1 or ROR2, generating MEC1-ROR1 and MEC1-ROR2 cell lines. Co-culture of these targets with Jurkat-Lucia™ NFAT-CD16A (V158) reporter cells and antibody treatments revealed that glycoengineered antibodies (GE-zilovertamab and GE-6E6), but not their parental antibodies, robustly triggered luminescence activity in NFAT-CD16 cells in an antigen-specific manner. Chromium-51 (Cr51) release assays further demonstrated that GE-zilovertamab or GE-6E6, in the presence of human peripheral blood mononuclear cells (PBMCs), respectively induced potent and selective lysis of ROR1+ or ROR2+ MEC1 variants that was comparable to that of rituximab against CD20+ parental MEC1 cells, and consistently superior to the non-glycoengineered parental antibodies. These findings were extended to primary HCL cells, which express CD20, ROR1, and ROR2. GE-zilovertamab and GE-6E6 induced greater cytotoxicity against Cr51-labeled HCL cells than their parental antibodies and equaled or surpassed the efficacy of rituximab. Combination treatment with glycoengineered anti-ROR1 and anti-ROR2 antibodies mediated significantly higher killing of Cr51-labeled HCL cells than either agent alone, or rituximab, or the combination of the two parental antibodies. Importantly, in contrast to rituximab, GE-zilovertamab and GE-6E6 spared normal B cells, which lack expression of ROR1 and ROR2. In summary, our findings show that dual targeting with glycoengineered anti-ROR1 and anti-ROR2 antibodies achieves superior, selective ADCC against ROR1/2+ hairy cell leukemia, sparing normal B cells. This approach offers a promising and potentially safer immunotherapeutic strategy for patients with HCL or other ROR-expressing B-cell malignancy.