Relationship between CD33 expression, splicing polymorphism, and in vitro cytotoxicity of gemtuzumab ozogamicin and the CD33/CD3 BiTE® AMG 330

Abstract

The CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) improves outcomes in some persons with acute myeloid leukemia (AML). Because GO is ineffective in many patients and/or may have adverse effects, there is interest in understanding factors, including CD33 expression, that possibly predict benefit and/or risk. A quantitative relationship between cell surface CD33 molecules and GO efficacy was demonstrable in engineered AML cell lines. However, validating this relationship clinically has been difficult. Still, CD33 expression, alternately estimated by mean fluorescence intensity [MFI] or the percentage of CD33 blasts, has been associated with response to GO in several randomized trials. Recent efforts have centered on the relationship between GO benefit and CD33 genotypes. Most attention thus far has been paid to a single nucleotide polymorphism (SNP), rs12459419 (C>T), present in exon 2 close to the intron/exon junction. The minor (T) allele results in reduced expression of full-length CD33 and preferential transcription of a variant missing exon 2 (CD33∆E2) which is predicted to encode for a protein containing the C2-set but not V-set domain. This is important because the V-set domain in the full-length CD33 protein contains the immune-dominant epitope(s) typically recognized by CD33 antibodies, including GO. In a large pediatric AML trial (COG-AAML0531), subjects with the rs12459419 CC genotype (about 50% of study-entrants) had a significantly lower risk of relapse and better eventand disease-free survival when randomized to addition of GO whereas this benefit was not seen in patients with CT or TT genotypes, suggesting value of this SNP as response biomarker. However, uncertainties about the role of the rs12459419 genotype for GO-based therapy have remained, both with regard to prognostic significance (there was no evidence this SNP was associated with response to GO in younger adults with AML treated on MRC/NCRI trials) and mechanistically (we previously found forced overexpression of CD33D in AML cells does not impact GOinduced cytotoxicity in vitro). It is also unknown how the splicing polymorphism relates to the cytotoxic activity of other CD33-targeted drugs. These uncertainties prompted our studies examining the relationship of CD33 expression, rs12459419 genotype, and in vitro cytotoxic effects of GO and the CD33/CD3 Bispecific T-cell Engager (BiTE®) AMG 330, which also recognizes an epitope in CD33’s V-set domain. Frozen aliquots of Ficoll-isolated mononuclear cells from pretreatment (“diagnostic”) peripheral blood or bone marrow specimens from adults with AML were obtained from institutional AML cell repositories and cultured in vitro in short-term assays as described previously. We used the 2016 WHO criteria to define AML and the 2010 MRC/NCRI criteria to assign cytogenetic risk. Patients provided written informed consent for the collection and use of their specimens for research purposes under protocols approved by the Fred Hutchinson Cancer Research Center Institutional Review Board. Clinical data were de-identified in compliance with the Health Insurance Portability and Accountability Act. CD33 expression on primary AML samples was quantified with a PE-Cy7-labeled antibody (clone P67.6) as described. CD33 rs12459419 genotyping followed the methodology described by Gale et al. Specifically, gDNA was purified using Qiagen AllPrep DNA/RNA mini kit (Qiagen, Valencia, CA) and, typically, 200 ng of gDNA amplified per reaction using the following primers (adapted from Gale et al.): For 5’-CTGGAAGCTGCTTCCTCAGACATG-3’ and Rev 5’-GAACCAGTAACCATGAACTGGGGAG-3’. The resulting 266 bp CD33 amplicon was digested overnight with HaeIII (New England Biolabs, Ipswich, MA, USA) and separated on a 2% agarose gel. To quantify drug-induced cytotoxicity, AML cells were incubated in 96-well round bottom plates at 5-10 x 103 cells/well with various concentrations of 1) GO (Pfizer,