Splicing Polymorphism rs12459419 Modulates Antibody-Bound Internalization of CD33 Isoforms

Background: While the standard regimen for AML has been intense chemotherapy followed by stem-cell transplantation (SCT), addition of immunotherapeutics such as gemtuzumab ozogamicin (GO) to this has shown to improve outcomes in multiple clinical trials. Since these trials, GO has been added to the National Comprehensive Cancer Network (NCCN) practice guidelines for use in AML therapy (NCCN., 2018). GO is a humanized monoclonal antibody conjugated to calicheamicin and targets the cell surface antigen CD33, observed on most AML blasts. Upon binding to GO, CD33 is internalized and the subsequent release of calicheamicin mediates cytotoxicity. CD33 contains two extracellular domains: IgV and IgC, the IgV domain is coded by CD33 exon 2 and is the binding site for GO. A splicing SNP, rs12459419 (C>T; A14V) in exon 2 results in a shorter isoform (CD33-D2) and has been previously associated with CD33 abundance on cell surface as well as clinical benefit from GO addition to standard chemotherapy. Patients with CC genotype (~50% of the cohort) expressed full length CD33 (CD33-FL) and benefited significantly from GO. However, heterozygous CT and homozygous TT genotype groups did not derive the same benefit. These results were perplexing as CT genotype group had intermediate levels of CD33-FL and CD33-D2 isoforms (Lamba et al., 2017). It is hypothesized that lack of IgV domain in CD33-D2 and putative CD33-FL/CD33-D2 dimerization prevents GO binding and subsequent internalization in rs12459419 CT/TT genotype cells, however this has never been tested. We have generated novel antibodies directed against the IgC domain that recognizes CD33-D2 in AML cell lines as well as primary AML cells (Gbadamosi et al., 2021) thus facilitating testing the above indicated hypothesis. Objective: In this study, we first tested the ability of antibody-bound CD33-D2 to internalize, followed by impact of the internalization status of CD33-FL and CD33-D2 in cells co-expressing both isoforms along with the concomitant impact on GO induced cytotoxicity. Methods: Using pMXs retroviral particles encoding CD33-FL or CD33-D2, we engineered CRISPR/Cas9 mediated CD33 KO in AML cell lines to overexpress either one or both CD33 isoforms to mimic cells expression varying proportions of CD33-FL/CD33-D2. Internalization of either CD33-FL or CD33-D2 over a duration of 4 hours in the presence of antibodies recognizing either of the two isoforms was performed. Cells were labeled with unconjugated mouse ant