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In general, AML is susceptible to immunotherapy. One established approach that is based on a functional immune response is the graft-versus-leukemia (GvL) effect of allogeneic HSCT, which is mediated by TCR recognition of foreign antigens.202,207 Also in autologoussettings, the detection of leukemic cells by T cells is essential for a successful immune response.202 Several leukemia- associated antigens (LAAs) have been identified, including gene fusions such as DEK-CAN and neoantigens that evolved from genetic aberrations such as internal tandem duplications (ITD) in the gene of FSM-like tyrosine kinase 3 (Flt3) as well as mutations in the gene of nucleophosmin 1

(NPM1).202,208-211 In addition, antigens have been identified that are overexpressed on the surface

of leukemic blasts compared to healthy tissue. Of these, the most established targets are myeloid differentiation antigen CD33 (i.e. sialic acid-binding immunoglobulin-like lectin-3;Siglec-3) and the α-subunit of the IL-3 receptor, CD123.212 _{These are present on the majority of myeloid blasts} but absent or detected at lower levels on hematopoietic stem cells (HSCs), which qualifies them as promising targets to eliminate leukemic cells while maintaining the capacity for hematopoietic reconstitution.79,213-215 Furthermore, they are expressed on LSCs, which makes them good target antigens to eradicate MRD and to counteract relapse.79,213,216

Since it was discovered that the depletion of CD33+ cells still allows the reconstitution of normal hematopoiesis in vitro, CD33 moved into focus of AML immunotherapy.217 Amongst others, Krupka and coworkers were able to validate CD33 as specific AML target by screening 621 AML

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patients. They reported overexpression in >99% of samples and confirmed CD33 presence on bulk AML cells as well as on CD34+CD38− LSCs. Notably, CD34+CD38− bone marrow (BM) cells from healthy donors demonstrated a comparably lower expression of CD33.79 _Several immunotherapeutic approaches have been tailored to address CD33, including naked mABs and derivatives, ADCsand chimeric antigen receptor (CAR) T cells.75,217-221 The first CD33-targeting mAB was already evaluated in a phase I clinical trial more than 20 years ago.222 Since then, mABs have continuously improved. The mostadvanced unconjugated mAB, lintuzumab (SGN-CD33), was investigated in a phase III clinical trial before it was discontinued due to lack of efficiency.219,223 This might be partially due to the fact that CD33 internalizes upon crosslinking.224 ADCs, however, take advantage of this targeted endocytosis to release their cytotoxic payload within the cell. Thus, the only CD33-targeting agent that hitherto gained market access is gemtuzumab ozogamicin (GO; Mylotarg®), which is a humanized IgG4 mAB conjugated to the cytotoxic agent N-acetyl-γ-calicheamicin dimethyl hydrazide (CalichDMH) via a bifunctional linker.225-227 Upon internalization, CalichDMH is released in the cell to induce DNA double-strand breaks and trigger tumor cell apoptosis.228_{Due to its intriguing success in treating relapsed AML} patients, GO gained accelerated approval by the U.S. FDA in the year 2000.225,229 Ten years later, it was voluntarily withdrawn by Pfizer due to frequent reports of high toxicity and lack of efficiency in post-marketing studies.225,230 However, after careful investigation it was most recently reapproved to treat adults with newly diagnosed as well as r/r AML.226,231,232 _{Aside from therapeutic} molecules that are based on the conventional IgG format, the CD33xCD3 BiTE® _antibody AMG 330 is currently evaluated in a clinical phase I trial (NCT02520427).75,221 In contrast to mABs, AMG 330 is not internalized and has no influence on CD33 surface antigen density.221 It reveals efficient cytolytic activity on AML cell lines and is able to activate T cells ex vivo in an autologous setting of cynomolgus monkey bone marrow aspirates and human patient samples.75,79 Further, in a murine xenograft model, it induces AML regression and prolonged survival.75 Notably, not all patients respond to CD33-targeted therapies.229 One reason is the upregulation of inhibitory immune checkpoints in the tumor microenvironment, including PD-1 and PD-L1.195-197 Expression levels can be particularly increased in response to proinflammatory cytokines, which are released upon immune stimulation by tumor neoantigens or therapeutics such as T cell engagers.189-191,194 In this regard, recent ex vivo studies indicated that AMG 330 treatment leads to upregulation of PD-1 on T cells and PD-L1 on AML cells.194 The coexpression of CD33 and PD-L1 or PD-L2 on AML cell lines decreased the AMG 330-mediated cytolytic activity of T cells, which

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could be reversed by the addition of PD-L1 or PD-L2 blocking mABs.233 Further, it could be shown that the depletion of primary AML samples was enhanced by the combined application of AMG 330 and PD-1/PD-L1 blocking agents.194 _{As preclinical investigations of PD-1/PD-L1} blockade in AML revealed a beneficial effect on disease progression, PD-1 and PD-L1 blocking mABs are currently evaluated in clinical phase I and II trials.234 This includes the administration as monotherapy or the combination with chemotherapy and/or other checkpoint blocking mABs such as ipilimumab.234-236 _{The aforementioned findings, however, provide a strong rationale for} combining PD-1/PD-L1 checkpoint blockade with targeted antibody therapy as well. The efficacy of simultaneous CD33-targeting and PD-1/PD-L1 checkpoint blockade, though successful in vitro, has yet to be shown in vivo in preclinical animal models and in clinical trials.194 In the future, the combination of two different immunotherapeutic strategies might be a highly potent strategy for the treatment of AML.

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