Influence of CD33 Expression Levels and ITIM-Dependent Internalization on Gemtuzumab Ozogamicin-Induced Cytotoxicity.
Authors: Walter Roland B. , Raden Brian W. , Kamikura Darren M. , Cooper Jonathan A. , Bernstein Irwin D. 
Published: April 2020

Abstract
Background: Gemtuzumab ozogamicin (GO; Mylotarg™), a novel immunoconjugate used for treatment of acute myeloid leukemia (AML), contains the humanized anti-CD33 antibody (hP67.6) as a carrier to facilitate cellular uptake of the toxic calicheamicin derivative. However, little is known about the importance of the quantity of CD33 expression for GO-induced cytotoxicity, and it has been argued that cytotoxic effects of GO can be achieved in the absence of CD33 expression. In addition, neither the endocytic process by which CD33 delivers antibody to the cytosol, nor the necessity of CD33 endocytosis for GO-induced cytotoxicity, has been established. We therefore investigated the quantitative relationship between CD33 expression and GO-mediated cytotoxicity, and determined the requirement for CD33 internalization in GO-induced cytotoxicity by identifying and mutating the cytoplasmic domain motif(s) that control internalization of antibody-bound CD33.
Methods: Murine myeloid 32D cells (devoid of CD33) and human myeloid OCI-AML3 and KG-1a cells (weakly CD33+) were transduced with a lentiviral vector expressing either wild-type or mutant CD33 as well as green fluorescent protein (GFP) at a multiplicity of infection (MOI) of 1–100. GFP-positive cells were sorted by flow cytometry, re-cultured, and further analyzed for CD33 expression, internalization of anti-CD33 antibody, and GO susceptibility. CD33 expression was quantified by immunofluorescence staining. Antibody uptake was quantified with a flow-cytometry-based assay, in which cells labeled with unconjugated hP67.6 were allowed to internalize in antibody-free medium for various periods of time, before second and third step reagents were used to measure hP67.6 that remained on the cell surface. To assess GO-induced cytotoxicity, cells were treated continuously with various concentrations of GO for 3 days (+ cyclosporine A in KG-1a sublines), and cytotoxicity then determined by flow cytometry with propidium iodide.
Results: In all 3 cell lines, lentivirus-mediated transfer of wild-type CD33 yielded subpopulations of GFP-positive cells that showed a MOI-dependent increase in expression levels of cell surface CD33. Compared to parental cells, transduced cells were significantly more sensitive to GO. Importantly, GO-sensitivity increased in a MOI-dependent manner, i.e. in a direct correlation with the cell surface expression levels of CD33. We could easily detect internalization of antibody-bound CD33 in cells that were transduced with wild-type CD33. Internalization of hP67.6 was almost completely prevented in cells that expressed a CD33 construct in which the proximal CD33 cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) was disrupted by introduction of point mutations (CD33/Y340F or CD33/L343A), whereas internalization was only partially prevented in cells that expressed a mutated distal ITIM (CD33/Y358F). In addition, disruption of the ITIMs not only prevented effective internalization of antibody-bound CD33, but also significantly reduced GO-induced cytotoxicity, when compared to cells expressing wild-type CD33 at matchable levels.
Conclusion: These data imply a pivotal role of both the number of CD33 molecules expressed on the cell surface and the rate of internalization of CD33 following antibody binding for GO-induced cytotoxicity, and suggest novel therapeutic approaches for improvement of clinical outcome of patients treated with GO.