Abstract
With the demonstration of improved survival of some acute myeloid leukemia (AML) patients with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), CD33 has been validated as a target for antigen-specific immunotherapy. Since previous studies identified a CD33 splice variant missing exon 2 (CD33∆E2) and, consequently, the immune-dominant membrane-distal V-set domain, we investigated the expression and functional characteristics of CD33 transcript variants in AML. In primary AML specimens, we not only found full-length CD33 (CD33FL) and CD33∆E2 but also corresponding variants containing an alternate exon 7 predicted to encode a CD33 protein lacking most of the intracellular domain (CD33E7a and, not previously described, CD33∆E2,E7a) in almost all cases. In acute leukemia cell sublines engineered to express individual CD33 splice variants, all splice variants had endocytic properties. CD33FL and CD33E7a mediated similar degrees of GO cytotoxicity, whereas CD33∆E2 and CD33∆E2,E7a could not serve as target for GO. Co-expression of CD33∆E2 did not interfere with CD33FL endocytosis and did not impact CD33FL-mediated GO cytotoxicity. Together, our findings document a greater-than-previously thought complexity of CD33 expression in human AML. They identify CD33 variants that lack exon 2 and are not recognized by current CD33-directed therapeutics as potential target for future unconjugated or conjugated antibodies.
Highlights
CD33, a member of the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family, is a 67 kD single pass transmembrane glycoprotein with endocytic properties that is primarily expressed on normal multipotent myeloid precursors, unipotent colony-forming cells, and maturing granulocytes and monocytes [1]
To survey the pattern of CD33 splice variant expression in human acute myeloid leukemia (AML), we took advantage of whole transcriptome RNA sequencing (RNAseq) data from 61 pre-treatment bone marrow and 7 peripheral blood specimens collected from pediatric patients with newly diagnosed AML
Having demonstrated the presence of CD33 splice variant missing exon 2 (CD33∆E2), CD33E7a, and CD33∆E2,E7a mRNA in primary human AML cells, we studied the functional characteristics of encoded proteins
Summary
CD33, a member of the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family, is a 67 kD single pass transmembrane glycoprotein with endocytic properties that is primarily expressed on normal multipotent myeloid precursors, unipotent colony-forming cells, and maturing granulocytes and monocytes [1]. CD33 is comprised of an amino-terminal variable (V)-set Ig-like domain mediating sialic acid binding followed by a C2-set Ig-like domain in its extracellular region, a transmembrane domain, and a cytoplasmic tail that contains 2 conserved tyrosine-based inhibitory signaling motifs. Upon phosphorylation, the latter provide docking sites for the recruitment and activation of the Src homology-2 (SH2) domain-containing tyrosine phosphatases SHP-1 and SHP-2 [1]. GO was withdrawn from the commercial market in most countries in 2010, these results have validated CD33 as therapeutic target in AML and have sparked renewed interest in CD33-directed immunotherapies [1]
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