CD33 is expressed by acute myeloid leukemia (AML) cells in >80% of patients but not by normal hematopoietic stem cells, suggesting that elimination of CD33(+) cells may be therapeutically beneficial. A conjugate of a calicheamicin hydrazide derivative attached via hydrazone formation to the oxidized carbohydrates of the anti-CD33 murine antibody P67.6 had been chosen for use in AML prior to humanization of this antibody. However, the CDR-grafted humanized P67.6 could not be used to make the carbohydrate conjugate because of the unexpected sensitivity of this antibody to periodate oxidation. Exploration of a series of bifunctional linkers resulted in a new class of calicheamicin conjugates, termed the hybrid conjugates, that allows for the attachment of the calicheamicin to lysines but incorporates the site of hydrolytic release, a hydrazone, previously shown to be required for activity. The optimized conjugate chosen for clinical trials, gemtuzumab ozogamicin ("gem-ozo", Mylotarg, formerly designated CMA-676), was significantly more potent and selective than the carbohydrate conjugate it replaced. It was selectively cytotoxic to HL-60 leukemia cells in tissue culture with an IC(50) in the low to sub-pg cal/mL range (cal = calicheamicin equivalents). Doses of gem-ozo as low as 50 microg cal/kg given three times to mice bearing HL-60 xenografts routinely resulted in long-term, tumor-free survivors, while a nonbinding control conjugate was relatively inactive. Gem-ozo at a concentration of 2 to 10 ng cal/mL selectively inhibited leukemia colony formation by marrow cells from a significant proportion of AML patients. Gem-ozo has also shown significant activity against AML in Phase II trials and is the first antibody-targeted chemotherapeutic agent approved by the FDA.

Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8(+) cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.

Gemtuzumab ozogamicin (GO) contains an anti-CD33 antibody to facilitate uptake of a toxic calicheamicin-␥ 1 derivative. While recent in vitro data demonstrated a quantitative relationship between CD33 expression and GO cytotoxicity, previous correlative studies failed to identify a significant association between CD33 expression and clinical outcome. Studying patients undergoing GO monotherapy for relapsed acute myeloid leukemia (AML), we now find that AML blasts of responders have a significantly higher mean CD33 level and lower P-glycoprotein (Pgp) activity compared with nonresponders. CD33 expression and Pgp activity are inversely correlated. While both variables are associated with outcome, Pgp remains significantly associated with outcome even after adjusting for CD33, whereas CD33does not show such an association after adjusting for Pgp. The inverse relationship between CD33 and Pgp suggests a maturation-stage-dependent expression of both proteins, and offers the rationale for using cell differentiation-promoting agents to enhance GO-induced cytotoxicity. IntroductionDue to its restricted, maturation stage-dependent expression on normal myeloid cells and its presence on tumor cells of 85% to 90% of adult and pediatric patients with acute myeloid leukemia (AML), CD33 has been exploited as a target for antibody-based AML therapies. [1][2][3][4] The endocytic property of CD33 led to the development of gemtuzumab ozogamicin (GO; Mylotarg; Wyeth Pharmaceuticals, Collegeville, PA), an immunoconjugate consisting of a humanized IgG 4 anti-CD33 monoclonal antibody (hP67.6) joined to a toxic calicheamicin-␥ 1 derivative. 4,5 Encouraging results on 277 patients treated in phase 2 trials revealed that GO monotherapy induces a complete remission (CR) or CR with incomplete platelet recovery (CRp) in 26% of adults with relapsed CD33 ϩ AML. 6,7 hP67.6 itself is largely nontoxic and primarily facilitates uptake of the calicheamicin-␥ 1 derivative, which is then cleaved and eventually induces DNA damage and cell death, 4 implying a critical role of the intracellular calicheamicin-␥ 1 accumulation for GO-induced cytotoxicity. Indeed, drug efflux mediated by Pglycoprotein (Pgp) results in resistance to GO and predicts for adverse outcome after GO monotherapy; conversely, inhibition of Pgp function effectively increases GO-induced cytotoxicity in vitro. [8][9][10][11][12] Furthermore, recent in vitro studies revealed a quantitative relationship between CD33 expression and GO-induced cytotoxicity in human CD33 ϩ AML cell lines, 13 and higher CD33 expression levels on circulating AML blasts were associated with clearance of AML blasts from peripheral blood after one dose of GO. 14 In contrast, an analysis of the first 142 patients treated in the phase 2 trials failed to show a significant impact of CD33 expression levels of bone marrow AML blasts in response to GO. 6 Additional smaller studies similarly did not find a predictive role of CD33 expression. 15,16 To resolve these apparent discrepancies, we re-examined the role of C...

Expression of multidrug resistance (MDR)features by acute myeloid leukemia (AML) cells predicts a poor response to many treatments. The MDR phenotype often correlates with expression of P-glycoprotein (Pgp), and Pgp antagonists such as cyclosporine (CSA) have been used as chemosensitizing agents in AML. Gemtuzumab ozogamicin, an immunoconjugate of an anti-CD33 antibody linked to calicheamicin, is effective monotherapy for CD33 ؉ relapsed AML. However, the contribution of Pgp to gemtuzumab ozogamicin resistance is poorly defined. In this study, blast cell samples from relapsed AML patients eligible for gemtuzumab ozogamicin clinical trials were assayed for Pgp surface expression and Pgp function using a dye efflux assay. In most cases, surface expression of Pgp correlated with Pgp function, as indicated by elevated dye efflux that was inhibited by CSA. Among samples from patients who either failed to clear marrow blasts or failed to achieve remission, 72% or 52%, respectively, exhibited CSA-sensitive dye efflux compared with 29% (P ‫؍‬ .003) or 24% (P < .001) among samples from responders. In vitro gemtuzumab ozogamicininduced apoptosis was also evaluated using an annexin V-based assay. Low levels of drug-induced apoptosis were associated with CSA-sensitive dye efflux, whereas higher levels correlated strongly with achievement of remission and marrow blast clearance. In vitro drug-induced apoptosis could be increased by CSA in 14 (29%) of 49 samples exhibiting low apoptosis in the absence of CSA.