Abstract P166: The rs35112940 CD33 polymorphism reduces CD33 internalization and efficacy of CD33-directed gemtuzumab ozogamicin

Abstract

Abstract CD33 is a myeloid-specific cell surface protein widely expressed on acute myeloid leukemia (AML) cells making it an excellent immunotherapeutic target. Current CD33-directed immunotherapeutic treatment strategies include gemtuzumab ozogamicin (GO), an antibody-drug conjugate (ADC) approved in 2017 for treatment AML and other ADCs are various stages of development. Previously, studies from our group have identified germline variations in CD33 that are associated with CD33 cell surface expression levels and clinical outcomes in response to GO. Among these germline variations is rs35112940 (G>A; Arg304Gly), a missense polymorphism located in exon five of CD33 which is associated with lower CD33 expression and reduced benefit from treatment using GO. Herein, we functionally validated the effect of the rs35112940 variant by treating CD33KO-HL60 engineered to overexpress wildtype CD33 (HL60-CD33FL) or CD33 encoding the rs35112940 variant (HL60-CD33FL-rs35112940) with GO. After 48-hour treatment with 250 ng/mL of GO, we observed that HL60-CD33FL-rs35112940 cells were more resistant to GO than HL60-CD33FL cells (46.5% vs 66.4% cell viability, P = 0.02). Of note, all engineered cells expressed CD33 with less than 1-log fold difference in MFI (HL60-CD33FL median fluorescence intensity (MFI) vs HL60-CD33FL-rs35112940 MFI: 22536 vs 24882). One critical aspect of the mechanism of action of GO is the internalization of the GO-CD33 complex which allows free calicheamicin to induce DNA damage leading to cellular apoptosis. Given the proximity of the rs35112940 loci to the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) domain of CD33, which is critical for CD33 internalization, we hypothesized that the rs35112940 variation may impact CD33 internalization as well. To assess this, we performed a flow cytometry-based internalization assay to capture the remaining amounts of CD33 present on the cell surface after 4 hours allowing us to determine the internalization of CD33 over time. Over a 4-hour window, we observed that that HL60-CD33FL-rs35112940 cells had an approximate 10% reduction in CD33 internalization in comparison to HL60-CD33FL cells. Taken together these results provide insight into the effect of the rs35112940 variant on GO efficacy and CD33 biology, corroborating our previous findings, and support the use of CD33 polymorphisms to guide patient selection for treatment with GO. Citation Format: Mohammed O. Gbadamosi, Vivek M. Shastri, Soheil Meshinchi, Jatinder K. Lamba. The rs35112940 CD33 polymorphism reduces CD33 internalization and efficacy of CD33-directed gemtuzumab ozogamicin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P166.