PRECLINICAL OBSERVATIONS WITH GO

Initial preclinical studies showed that GO exerted selective cytotoxicity against CD33⁺ AML cells, effectively inhibiting colony-forming cells in pediatric and adult AML specimens in vitro whereas a control immunoconjugate did not, and caused regression of CD33⁺ AML cell line xenografts in athymic mice (99). Subsequent in vitro studies have confirmed these findings and provided insight into the cellular characteristics that are relevant for the clinical GO efficacy (Table 1).

In contrast to other anti-CD33 antibodies (68, 69), both P67.6 and hP67.6 are largely non-toxic against CD33⁺ AML cell lines and human AML specimens (87, 97, 102). It is thus thought that the antibody component primarily functions as a carrier to facilitate cellular uptake of the calicheamicin-g₁^I derivative into CD33⁺ cells (Figure 5). The contribution of non-receptor mediated endocytic drug uptake, a possibility suggested by limited in vitro studies with CD33^- acute lymphoblastic leukemia cell lines (103), for clinical GO efficacy is unknown. Internalized CD33/GO complexes are routed to lysosomes, where the toxic moiety is presumably released (54, 104). The free calicheamicin-g₁^I derivative can then enter the nucleus and initiate DNA damage. This putative mechanism of action implies a critical role for the intracellular accumulation of the calicheamicin-g₁^I derivative as well as the cellular response to the toxin’s DNA damaging effect for GO-induced cytotoxicity (Table 1). Conceptually, the intracellular load of activated calicheamicin-g₁^I is impacted by the amount of GO uptake, the efficacy of toxin release from the antibody and subsequent activation via cellular thiols, as well as toxin inactivation/metabolism or expulsion. However, while induction of DNA damage appears to be a prerequisite for GO-induced cytotoxicity (105, 106), it is not sufficient, indicating that the toxicity of the calicheamicin-g₁^I moiety is modulated by the cell’s ability to repair DNA damage and the activity of downstream pro- and anti-apoptotic pathways. Overall, the sensitivity to the toxic moiety varies over 100,000-fold between individual primary AML cells samples (107), an observation that emphasizes the importance of patient-specific factors for the clinical efficacy of GO.

Several patient-specific factors have been identified: most importantly, studies have repeatedly shown that drug efflux mediated by members of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily of proteins, predominantly P-glycoprotein (ABCB1) and to a lesser degree multidrug resistance protein 1 (MRP1; ABCC1) but not breast cancer resistance protein (BCRP; ABCG2), mediate resistance to GO; conversely, inhibition of drug efflux effectively increases GO-induced cytotoxicity in vitro (87, 108-113). Taken together, these investigations have identified drug efflux as a major determinant of GO’s anti-AML activity. Experimental studies also revealed a striking, quantitative relationship between CD33 expression and GO efficacy in engineered human AML cell lines and demonstrated the requirement of GO/CD33 complex internalization for GO-induced cytotoxicity (56). Thus, the amount of GO uptake is a limiting factor for GO efficacy. In support of this notion, CD33 expression levels directly correlate with the in vitro sensitivity of immature AML cell fractions to GO (102, 114). While the role of DNA repair and downstream signaling pathways for GO efficacy has not yet been examined in detail, Bcl-2 family proteins modulate GO cytotoxicity against AML cell lines (110). Furthermore, recent studies found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML

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cells, whereas the investigational AKT inhibitor, MK-2206, sensitizes various human AML cells to GO or free calicheamicin-g₁^I (106). Likewise, limited data suggest that MEK1/2 activity may be involved in resistance to GO (115). Curiously, the presence of an FLT3/ITD mutation has been associated with increased sensitivity of immature AML cells to GO (114) but, so far, studies have not identified this marker to predict clinical response to GO. Finally, some studies have suggested Syk expression to be a biomarker of response to GO, and depletion of Syk to result in unresponsiveness to GO (116). However, in our experience, depletion of Syk by lentivirus-mediated siRNA expression did not affect GO cytotoxicity in engineered AML cell lines (R.B.W. unpublished observation).

A number of studies have indicated that the sensitivity of AML cells to GO can be enhanced through the use of other agents. Such chemosensitizing effects have been observed with histone deacetylase inhibitors (117), DNA methyltransferase I inhibitors (116, 118), the farnesyl transferase inhibitor, tipifarnib (119), heat shock protein-90 inhibitors (40), anti-CD45 antibodies (120), as well as mitoxantrone (121). Furthermore, the combination of GO with other conventional chemotherapeutics such as cytarabine, daunorubicin, idarubicin, doxorubicin, etoposide, or 6-mercaptopurine has additive cytotoxic effects in vitro, whereas methotrexate and vincristine may antagonize the effects of GO (121, 122). Interestingly, no rigorous studies have attempted to define the optimal timing when GO should be given with other agents. The timing, especially with respect to the cell cycle status, is likely of some importance, however, in view of data from in vitro studies suggesting that resting cells are relatively less susceptible to GO while treatment with granulocyte colony-stimulating factor (G-CSF) sensitizes to GO (103, 123). Finally, an interesting yet unstudied aspect of CD33-targeted immunotherapy is the question whether shorter isoforms lacking the extracellular V-set Ig-like domain and antibody epitope (16) could modulate the efficacy of anti-CD33 antibody-based drugs.