Table 1. Advantages and disadvantages of AML-associated antigens for antibody development (for a cartoon representation of each bispecific antibody format, please see Figure 1).

Antigen	Advantages	Disadvantages	Bispecific formats
CD33	Expressed on the majority of myeloid blasts and leukemic stem cells (LSC)6, 117 Not expressed or expressed at lower levels on normal HSCs6, 117 Not expressed outside of the hematopoietic system118 High CD33 expression is associated with high-risk mutations and inversely associated with low-risk mutations6, 119 High CD33 expression correlated with inferior disease features and outcome119 Expressed on immunosuppressive myeloid derived suppressor cells that are associated with extramedullary infiltration and detection of minimal residual disease120	Not expressed on all myeloid blasts or myeloid stem cells117 Expressed on HSCs in some studies121 Expressed on activated natural-killer (NK) cells and T cells5, 9 Modulation (decreased surface expression) of surface CD33 expression upon bivalent anti-CD33 antibody treatment10, 122 Circulating CD33 might interfere with the anti-CD33 antibodies123 Treatment-related myelosuppression (neutropenia and thrombocytopenia)124 Relatively low abundance on cell surface125	Tandem double and triple scFv (BiTE, ScBsTaFv, bsscFv, TandAb, BiKE, TriKE, sctb), chemical conjugates
CD123	Expressed on the majority of myeloid blasts and LSCs126 Not expressed or expressed at lower levels on normal HSCs38, 121 Absent on T cells127 Associated with lower complete remission and poorer survival rates39 Associates with higher proliferation and more resistance to apoptosis of AML cells39	Expressed on HSCs121, 128 Not expressed on all myeloid blasts or myeloid stem cells39 A chimeric antigen receptor that was made based on CD123 antibody significantly reduced B cells, platelets and myeloid cells in an animal model128	Tandem double and triple scFv (sctb), DART, BIf
WT1	Expressed on the majority of myeloid blasts and LSCs44, 129 Is not expressed or expressed at extremely low levels in a small population of CD34(+)cells in bone marrow129 Higher WT1 gene expression is associated with lower complete remission and decreased survival45, 130	HLA-restricted expression of WT1 limits the application of each anti-WT1 antibody to one special HLA haplotype46 Not expressed on all myeloid blasts or myeloid stem cells129 Low cell surface density of HLA-WT1-peptide complexes46 Expressed in some normal tissues131	Tandem double scFv (BiTE)
CD13	Expressed on the majority of myeloid blasts and LSCs132 Expressed at higher levels on AML stem cells than on normal HSCs Anti-CD13 monoclonal antibodies can induce apoptosis in AML cells	Expressed in some normal tissues and cells including monocytes, granulocytes, capillary endothelium, nephron convoluted tubules, bile ducts, pancreas, skin, small intestine and liver133 Not expressed on all myeloid blasts132 Expressed on HSCs121	Chemical conjugation (Fab′ fragments)
CD15	Expressed on the majority of AML cells134	Expressed on some NK cells, T cells, monocytes, neutrophils, eosinophils and neurons52, 135 Expressed on more than 50% of activated T cells136	Chemical conjugation (Fab conjugated with whole IgM)
CD30	Associated with FLT-3-ITD mutations, leucocytosis and possibly poorer prognosis57	Expressed on respiratory epithelial cells, glandular cells of gallbladder, colon, rectum and uterus137, 138 Expressed on activated T cells55 Not expressed on all AML cells56, 57 Secreted upon cleavage of the extracellular domain58, 59	T and Ab
CD45	Not internalized upon antibody ligation139 Expressed on most AML cells140 Not expressed outside hematopoietic system	Not expressed on LSCs141 Expressed on all hematopoietic cells except mature red blood cells and platelets61
CD47	Is a universal target in human cancers CD47 upregulation on leukemic cells allows them to evade macrophage killing64 Overexpressed on AML stem cells than on their normal hematopoietic counterparts65 Overexpression of CD47 on AML cells is associated with shortened survival65	Expressed on the majority of normal tissues63 CD47 expression on normal tissues may generate an antigen sink preventing the therapeutic antibody to reach its target on AML cells66	DVD-Ig
CLL1	Expressed on the majority of myeloid blasts and LSCs142 Not expressed on normal tissues142 Not expressed on normal hematopoietic stem cells67, 142	Expressed on peripheral blood monocytes, dendritic cells and granulocytes142 Relatively low abundance on cell surface142 Antigen modulation upon ligation with anti CLL1 antibody142 Not expressed on all AML cells142	Chemical conjugation (Fab fragments)
FLT-3	Expressed on the majority of AML samples143 Expression on LSCs is higher than on normal hematopoietic cells70, 73 Expressed on LSCs143	Expressed on hematopoietic stem and progenitor cells and on dendritic cells70, 71 Not expressed on all AML cells143	Tandem double scFv (BiTE), Fabsc
VEGF-A, Ang-2	VEGF-A and Ang-2 are overexpressed on the majority of AML bone marrow samples144 Anti-angiogenic therapy might control disease in patients with relapsed AML144 Expression of VEGF-A and Ang-2 on AML is associated with negative outcome74, 75, 144	Not expressed on all AML cells144 Are secreted from the cells so T-cell responses cannot be redirected against AML cells	CrossMab, chemical conjugation