Internalization and cell cycle-dependent killing of leukemic cells by Gemtuzumab Ozogamicin: rationale for efficacy in CD33-negative malignancies with endocytic capacity

Abstract

Multicenter phase II trials with Gemtuzumab Ozogamicin (GO/ Mylotarg s ), consisting of a CD33 antibody linked to the cytotoxic drug calicheamicin, have shown a 30% overall response rate in relapsed acute myeloid leukemia patients. However, no clear correlation was observed between CD33 expression on leukemic blasts and response to GO therapy. We analyzed the CD33 specificity of GO-induced cell death and the effect of GO on CD33-negative malignancies. We demonstrate that lysis induced by clinically relevant GO concentrations is partially CD33 mediated, and that efficient non-CD33-mediated GO uptake can occur via endocytosis. In agreement with these results, we observed GO-mediated death of human CD33-negative acute lymphoblastic leukemia cells both in vitro and in vivo in an NOD/SCID mouse model. Finally, sensitivity to GOinduced cell death was at least partially determined by the activation status of leukemic cells, with cells in activated phases of the cell cycle being most effective in both CD33-specific GO internalization, renewed expression of CD33 molecules, and non-CD33-mediated GO uptake via endocytosis. In conclusion, these data provide mechanistic insight into the efficacy of GO in CD33-positive as well as in CD33-negative malignancies with endocytic capacity, and provide a rationale for the use of GO in the treatment of malignancies with endocytic capacity.

Leukemic stem cells: identification and clinical application

“…Although targeting CD33 was originally not meant as an anti-LSC therapy, it turned out that CD33 was overexpressed in LSC compared to HSC [6]. Treatment with Gemtuzumab ozogamicin (GO) treatment was associated with reduced relapse risk and improved overall survival in patient subgroups [49,50]. Whether GO targets CD33+ LSC, causing the reduction in relapse risk, remains unclear [50] as higher numbers of CD34+/CD38−/CD33+ cells and high CD33 expression levels decreased GO sensitivity in vitro [51].…”

Efficacy and resistance of gemtuzumab ozogamicin for acute myeloid leukemia

“…Several studies have sought to explain the efficacy of GO on CD33-negative leukemia. One proposed explanation is that GO is partially moved into cell by CD33-independent endocytosis [39]. In their study, anti-CD33 blocking antibodies prevented the death of CD33-positive cells at low concentrations of GO, but not at higher concentrations.…”

“…However, the synergistic effect of GO with these agents has not been well elucidated in clinical studies. Other resistance mechanisms have been suggested by several groups, including the alternative GO pharmacokinetics, and the reduction of CD33 on leukemia cells [36,39,43,44]. In fact, multiple mechanisms may be at work in the development of resistance to GO.…”

“…It explains that cells expressing higher levels of CD33 are more susceptible to GO [38]. However, several patients with CD33-negative leukemia have also responded to GO [39]. Several studies have sought to explain the efficacy of GO on CD33-negative leukemia.…”

The role of Gemtuzumab Ozogamicin in the treatment of acute myeloid leukemia patients

“…Furthermore, disruption of the CD33 cytoplasmic ITIMs, which are crucial for the internalization of antibody bound to CD33, by introduction of point mutations not only prevents internalization of Abbound CD33, but also reduces GO-induced cytotoxicity. It must be also mentioned that efficient non-CD33-mediated GO uptake can occur via endocytosis in human CD33neg acute lymphoblastic leukemias (Jedema et al, 2004), providing a potential rationale for the use of GO in the treatment of CD33neg malignancies with endocytic capacity.…”