ABSTRACT Full-length CD33 (CD33FL) is a transmembrane glycoprotein consisting of a membrane-distal V-set and a membrane-proximal C2-set immunoglobulin-like domain in its extracellular portion. As a myeloid differentiation antigen, CD33FL is displayed on at least a subset of malignant myeloblasts in almost all patients with acute myeloid leukemia (AML), and possibly leukemia stem cells in some. Improved survival in defined patient subgroups with the CD33FL antibody-drug conjugate gemtuzumab ozogamicin (GO) validates CD33FL as the first and thus far only immunotherapeutic target in AML. However, many patients with CD33FL+ AML do not benefit from GO, and the need for improved CD33-targeted therapeutics remains undisputed. In the COG- AAML0531 trial, improved outcome with GO was limited to the ~50% of patients homozygous for the major allele in the CD33 single-nucleotide polymorphism rs12459419C>T (CC genotype) but not in those heterozygous or homozygous for the minor allele (CT or TT genotypes). The minor (T) allele results in reduced expression of CD33FL and preferential translation of a splice isoform that lacks exon 2 and only contains the C2-set domain in the extracellular portion (CD33∆E2). Since all current CD33 antibodies, including GO, recognize the immune-dominant V-set domain that is encoded by exon 2, CD33∆E2 is therefore not targeted with any available CD33 antibody. We hypothesize that C2-set domain antibodies will provide a novel approach to CD33-targeted immunotherapy that is superior to current therapeutics and could benefit patients regardless of the rs12459419 genotype because 1) these antibodies recognize all known CD33 isoforms, including CD33∆E2, and 2) the membrane-proximal binding enhances the effector functions of CD33 antibodies, as indicated by our preliminary studies. As a first step toward development of this new form of CD33 immunotherapy, we have very recently raised murine antibodies against the human C2-set domain of CD33 and identified 5 antibody clones that recognize both CD33FL and CD33∆E2 (i.e. are CD33FL+∆E2 [CD33PAN] antibodies). We now propose detailed preclinical studies to determine the therapeutic potential of CD33PAN antibodies, the format in which these antibodies might be particularly useful, and identify subsets of AML patients who might be particularly good candidates for this type of immunotherapy based on CD33FL+∆E2 expression. Results from our research are expected to guide the further development strategies of CD33PAN antibodies for use in patients with AML and other myeloid neoplasms and hence may lead to a new treatment option for patients for whom outcomes continue to be unsatisfactory.