Tyrosine Phosphorylation Enhances ITIM-Dependent Internalization of CD33, the Target for the Anti-AML Immunoconjugate, Gemtuzumab Ozogamicin.

Background: CD33, the target for the anti-AML immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). We have previously shown that these motifs control uptake of antibody-bound CD33 and GO-induced cytotoxicity. In this study, we determined which phosphorylation state favors uptake of antibody-bound CD33, identified proteins that bind to CD33 in an ITIM-dependent manner, and assessed their importance for CD33 internalization by siRNA-based gene silencing.

Methods: Internalization of anti-CD33 antibodies was measured by flow cytometry in the presence or absence of the tyrosine phosphatase inhibitor, pervanadate, in human CD33⁺ AML cell lines (ML-1, HL-60, NB4, U937, TF-1) and CD33⁻ Jurkat T cells infected with wild-type and mutant CD33. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33, using human myeloid cell lysates. Co-immunoprecipitations were performed with myeloid cell lines expressing HA-tagged wild-type CD33. Lentivirus-based siRNA constructs were generated for gene silencing, and expressed in human CD33⁺ AML cell lines.

Results: Pervanadate significantly increased uptake of anti-CD33 antibodies in human AML cell lines; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2, suggesting that Src family kinase-dependent phosphorylation of the ITIMs critically controls uptake of antibody-bound CD33, possibly by altering which proteins binds to CD33 or by facilitating binding of adaptor-proteins required for endocytosis. We identified several proteins, including the tyrosine phophatases, SHP-1 and SHP-2, and the non-receptor tyrosine kinase, Syk, which bound to phosphorylated wild-type and mutant CD33 in a manner that paralleled the endocytic properties of the corresponding CD33 protein. Since these three proteins have been implicated in endocytic processes of other cell surface proteins, we assessed their role in uptake of antibody-bound CD33 by siRNA-mediated gene silencing. Simultaneous depletion of SHP-1 and SHP-2, but not SHP-1 or SHP-2 alone, significantly increased internalization of antibody-bound CD33 in the two AML cell lines with the highest cell surface expression of CD33, whereas no effect was seen in two other cell lines with lower CD33 expression levels. In contrast, depletion of Syk, whose expression has previously been correlated to the inhibitory effect of anti-CD33 antibodies on AML cell growth, failed to affect antibody internalization in the cell lines assessed.

Conclusion: These studies indicate that the phosphorylation status of the ITIMs controls uptake of antibody-bound CD33. In line with this model, SHP-1 and SHP-2, which have been shown to dephosphorylate CD33 in vitro, can affect this endocytic process. Thus, our data imply manipulation of the phosphorylation state of CD33, e.g. by activating Src family kinases or interfering with phosphatases as a novel means to increase uptake of anti-CD33 antibody-based therapeutics such as GO. Finally, the variable effect of SHP-1 and SHP-2 depletion suggests that there are significant cell-type specific differences in the response to anti-CD33 antibody ligation, for example differences in tyrosine phosphorylation levels and/or activation/recruitment or redundancies of tyrosine phosphatases.