Abstract P18: Splicing polymorphism rs12459419 modulates antibody-bound internalization of CD33 isoforms
Authors: Shastri Vivek M , Lamba Jatinder K 
Published: March 2024

Abstract
Gemtuzumab ozogamicin (GO) is a humanized monoclonal antibody conjugated to calicheamicin targeting cell surface antigen CD33, which is observed on most AML blasts. CD33 is internalized upon binding GO and the subsequent release of calicheamicin mediates cytotoxicity. CD33 contains two extracellular domains: IgV and IgC, with the IgV domain coded by exon 2 being the binding site for GO. Splicing SNP rs12459419 (C&gt;T; A14V) in exon 2 results in a shorter isoform (CD33-D2), which is associated with CD33 cell surface abundance. Patients with CC genotype (~50% of the cohort) express full length CD33 (CD33-FL) and benefit significantly from GO. However, patients with heterozygous CT and homozygous TT genotypes did not derive the same benefit. Lack of IgV domain in CD33-D2 and putative CD33-FL/CD33-D2 dimers in cells expressing both isoforms are hypothesized to prevent GO binding and subsequent internalization in rs12459419 CT/TT genotype cells. We used our previously generated antibody HL2541 directed against the IgC domain to recognize CD33-D2 in AML cell lines and primary AML cells to test: the ability of antibody-bound CD33-D2 to internalize, internalization status of CD33FL and CD33-D2 in cells co-expressing both isoforms, and any concomitant impact on GO cytotoxicity. Briefly, CRSIPR/Cas9 mediated CD33 KO AML cell lines (HL-60, Kasumi-1, and THP-1) were engineered to overexpress either or both CD33 isoforms. Cells were labeled with unconjugated mouse anti-CD33-FL P67.6 or anti-CD33-D2 HL2541 antibody on ice and incubated at 37°C for 0-4 hours. Flow cytometry was performed after staining with biotinylated anti–mouse IgG antibody and streptavidin-FITC conjugate to detect surface CD33. Percentage antibody-bound CD33 internalization was expressed as change in FITC MFI between each time point incubated and cells kept at 0°C post antibody labeling. We show for the first time that HL2541-bound-CD33-D2 internalizes upon antibody binding in cell lines expressing CD33-D2 isoform only. In AML cell lines co-expressing both CD33 isoforms, a &gt;2.5-3 fold decrease in antibody-bound internalization was observed for each isoform. Upon GO exposure for 48 hours, cells co-expressing both CD33 isoforms had 7-9.5-fold higher IC50 values compared to cells expressing only CD33-FL. As expected, cells expressing only CD33-D2 did not respond to GO. Our finding that antibody-bound CD33-D2 internalizes like CD33-FL adds critical evidence in understanding the biology of this isoform while opening opportunities to develop ADCs using this CD33-D2 antibody, which could provide a treatment option in patients with TT genotypes. Our results also provide a mechanistic insight on the previous clinical observation showing lack of benefit from GO in patients heterozygous for this CD33 splicing SNP. Our ongoing studies are focused on validating this result using live cell imaging and efforts towards design of new ADC or T cell engagers using CD33-D2 antibody. The study was supported by Leukemia Lymphoma Society, UFHealth Cancer Center, and the Frank A. Duckworth Endowment.
Citation Format: Vivek M Shastri, Jatinder K Lamba. Splicing polymorphism rs12459419 modulates antibody-bound internalization of CD33 isoforms [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P18.