Influence of CD33 expression levels and ITIM-dependent internalization on gemtuzumab ozogamicin–induced cytotoxicity

Abstract

IntroductionCD33 is a 67 kDa type 1 transmembrane sialoglycoprotein and founding member of a rapidly evolving immunoglobulin superfamily subset of sialic acid-binding immunoglobulin-related lectins (siglecs; siglec-3). 1,2 CD33 is characterized by the presence of 2 conserved immunoreceptor tyrosine-based inhibitory motif (ITIM)-like motifs in the cytoplasmic region, a property that is shared with all CD33-related siglecs discovered so far. 2 If phosphorylated upon treatment with pervanadate or anti-CD33 antibody plus crosslinking secondary antibody, these tyrosine motifs recruit and activate Src homology-2 (SH2) domain-containing tyrosine phosphatases (SHP-1 and SHP-2). [3][4][5] Physiologically, CD33 expression is restricted to early multilineage hematopoietic progenitors, myelomonocytic precursors, and more mature myeloid cells, but it is absent on normal pluripotent hematopoietic stem cells. [6][7][8] However, 85% to 90% of adult and pediatric cases of acute myeloid leukemia (AML) express CD33. 8,9 Therefore, CD33 has gained clinical importance as a suitable tumor-associated antigen and target for antibody-based AML therapies. 10 Promising clinical results have been obtained with gemtuzumab ozogamicin (GO; CMA-676; Mylotarg; Wyeth Pharmaceuticals, Philadelphia, PA), an immunoconjugate consisting of a humanized immunoglobulin G 4 (IgG 4 ) anti-CD33 monoclonal antibody (hP67.6) joined to a derivative of the cytotoxic drug calicheamicin-␥ 1 , N-acetyl-␥-calicheamicin dimethyl hydrazide. 11 Although some unconjugated anti-CD33 antibodies may have limited cytoreductive activity when used as monotherapy in patients, 12,13 hP67.6 is believed to function primarily as a carrier to facilitate cellular uptake of the calicheamicin-␥ 1 derivative, which is then cleaved intracellularly in an acid compartment, presumably inside lysosomes, and subsequently induces DNA damage and eventually cell death. 14 This putative mechanism of action of GO still needs firm experimental confirmation but implies a pivotal role of both the number of CD33 molecules expressed on the cell surface and the rate of internalization of CD33 following GO binding for the cytotoxic effect. However, little is known about the importance of the quantity of CD33 expression for GO-induced cytotoxicity. Although multiple studies have confirmed that binding of bivalent and multivalent anti-CD33 antibodies results in CD33 internalization in both CD33 ϩ hematopoietic cell lines and primary AML blast cells, 15-18 some authors have argued that cytotoxic effects of GO are achieved in the absence of CD33 expression. 19 In addition, neither the endocytic process by which CD33 delivers antibody to the cytosol nor the necessity of CD33 endocytosis for GO-induced cytotoxicity has been established.The primary objective of the present study was therefore to investigate the quantitative relationship between CD33 expression and the requirement for CD33 internalization in GO-mediated cytotoxicity. To accomplish this, lentivirus-mediated gene transfer was used to manipulate...

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

“…Values for the non-specific clearance (CL = 4.92 mL/day/kg) and volume of central compartment (V 1 = 36.7 mL/kg) are comparable to the values historically observed for other human IgG1 antibodies in monkeys 1 . The value of intrinsic clearance (CL INT ), defined as the ratio V max /K M , 35 was 372 mL/kg/day. The target-mediated clearance for low concentrations (i.e., when C 1 <<K M ) is thus roughly 75-fold the non-specific clearance (CL).…”

“…In addition, heterogeneity in levels of CD33 expression is anticipated in patients. 33-35 In the PK-RO model, the V max parameter represents the amount of antibody that is cleared by target when the target is fully saturated and can be associated with the target expression level. Based on this, a range of values for V max was used to assess the impact of possible differences in antigenic burden in patients 36 .…”

Fludarabine, Cytarabine and Gentuzumab Ozogamicin (FLA-GO) as Salvage Therapy and Bridge to Transplant in Adult Relapsed Acute Myeloid Leukemia (AML) Patients

“…Preclinical data indicate that GO is more effective in AMLs with a high CD33 expression [28]. In a study of GO single-agent for unfit elderly patients with previously untreated AML, Amadori et al confirmed this observation [29].…”

Efficacy and resistance of gemtuzumab ozogamicin for acute myeloid leukemia

“…This mechanism is central to the efficacy of 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].…”

“…Delivery of GO to bone marrow may be important for enhancing the effect of GO [28,38]. An excess of circulating CD33-positive cells decreased the effect of GO, and resulted in worse outcomes [27,64].…”