CD33 expression and internalization in AML

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Consistent with its physiological expression as myeloid differentiation antigen, 85-90% of adult and pediatric AML patients are considered to have CD33⁺ disease, defined as the presence of CD33 on greater than 20-25% of the leukemic blasts (25, 47). CD33 is not a highly abundant antigen: quantitative flow cytometry studies estimated that AML blasts display an average of ~10⁴ (range: 1x10³ – 5x10⁴) CD33 molecules per cell (28, 48), and expression is typically even lower in immature (e.g. CD34⁺/CD38^-/CD123⁺) cell subsets (49). From a drug development perspective, an important aspect of CD33 is its internalization when engaged with antibodies (23, 50-56). Mechanistic studies indicate that endocytosis of CD33/antibody complexes is largely limited and determined by the intracellular domain of CD33, while the extracellular and transmembrane domains play a minor role (23, 56). Forced tyrosine phosphorylation enhances the uptake of anti-CD33 antibodies, as does depletion of SHP-1 and SHP-2, at least in some cell lines, consistent with a role of tyrosine phosphorylation as regulator of this process (23). Consistently, disruption of the ITIMs by point mutations prevents optimal internalization of antibody-bound CD33 (56) although some internalization of CD33 occurs in an ITIM-independent manner. Furthermore, ubiquitylation of CD33 decreases CD33 cell surface abundance and increases the rate of CD33 internalization (20). Importantly, compared to antigens such as the transferrin receptor, the internalization process of CD33 is relatively slow (56). Together, the low expression of CD33 and the slow internalization of CD33/antibody complexes leads to relatively limited CD33-mediated drug uptake per unit of time; consequently, for an anti-CD33 antibody-drug conjugate to be most successful, a highly potent toxin will be required.