NEO-201-based CAR NK cells as a novel immunotherapeutic approach to target truncated core 1 O-glycans in AML

Abstract Introduction: Despite therapeutic advances, acute myeloid leukemia (AML) continues to have poor long-term outcomes, particularly in high-risk and relapsed/refractory patients. Natural killer (NK) cells possess intrinsic cytotoxicity against AML through antigen-independent mechanisms, and adoptive NK cell therapies have shown clinical promise. Combining tumor-targeting monoclonal antibodies (mAbs) or engineering NK cells with chimeric antigen receptors (CARs) can confer antigen specificity and enhance anti-leukemic activity. However, identifying AML-specific antigens remains a major barrier, as many surface targets are shared with hematopoietic stem and progenitor cells (HSPCs), increasing the risk of on-target/off-tumor toxicity. NEO-201 is a humanized IgG1 mAb that recognizes truncated Core 1 O-glycans expressed by several human solid tumors, as well as circulating neutrophils and some human leukemia cell lines, but does not bind most normal tissues or immune cell subsets. In a phase I solid tumor trial (NCT03476681), NEO-201 demonstrated an acceptable safety profile, with transient neutropenia as the primary on-target toxicity, which was reversible with G-CSF given 5–6 days after NEO-201 IV infusion. NEO-201–recognized O-glycans might represent a novel AML antigen amenable to immune targeting. We assessed NEO-201–target antigen expression across AML cell lines and evaluated its potential for antibody-dependent cellular cytotoxicity (ADCC) and CAR-engineered NK cell therapy. Methods: Ten AML cell lines representing multiple molecular subtypes were analyzed for NEO-201 binding by flow cytometry using Pacific Blue–conjugated NEO-201. NK cells were isolated from healthy donor peripheral blood and expanded ex vivo using a clinical-grade protocol involving lymphoblastoid feeder cells and IL-2. For ADCC assays, expanded NK cells were co-cultured with calcein-AM–labeled AML targets and 10 μg/mL NEO-201 for 4 hours across varying effector-to-target ratios. Residual calcein-AM–positive targets were quantified using a Celigo imaging cytometer. CAR NK cells were generated via retroviral transduction with a construct encoding a NEO-201–binding domain, CD8α transmembrane region, 4-1BB and CD3ζ signaling domains, and a truncated CD34 (tCD34) marker. Transduction efficiency was quantified by tCD34 expression. CAR NK cytotoxicity was assessed in 4-hour co-culture assays. Fresh G-CSF–mobilized peripheral blood was analyzed by flow cytometry to assess NEO-201 reactivity across myeloid maturation stages. Results: NEO-201–target antigen was expressed at >40% in 6 of 10 AML cell lines tested, encompassing diverse molecular subtypes including FLT3-ITD mutations, MLL rearrangements, biphenotypic features, and core binding factor abnormalities. Expression levels ranged from 41% to 100%, with the following lines showing NEO-201 positivity: THP-1 (100%), MOLM-14 (88%), MV4-11 (60%), U-937 (45%), HL-60 (44%), and ME-1 (41%). Expanded NK cells mediated ADCC against NEO-201–treated HL-60 and THP-1, but not against MOLM-14 AML. Retroviral transduction achieved ~70% NEO-201 CAR expression in NK cells. In co-culture assays, NEO-201-based CAR NK cells exhibited enhanced killing compared to ADCC mediated by NEO-201 alone in NEO-201–positive AML lines, including MOLM-14, which was resistant to NEO-201–mediated ADCC. At a 10:1 E:T ratio, NEO-201-based CAR NK cells lysed 61% of THP-1 targets vs 29% with ADCC (p=0.001). Similar enhancements were observed in HL-60 (CAR NK: 45% vs ADCC: 22%) and MOLM-14 (CAR NK: 40% vs ADCC: 19%). Analysis of G-CSF–mobilized peripheral blood revealed minimal or absent expression of NEO-201–target antigen on CD34⁺ HSCs (0–1%), low expression in common myeloid and granulocyte-monocyte progenitors (10–15%), and high expression in mature neutrophils (~99%). Conclusion: NEO-201 targets truncated core 1 O-glycans selectively expressed on some AML cells and late-stage myeloid cells, while sparing CD34⁺ HSCs. These findings highlight the NEO-201–recognized antigen as a promising immunotherapeutic target in AML. This is the first study to characterize expression of the NEO-201–target antigen across a broad panel of AML cell lines and assess its targeting using NK-based platforms. While both antibody- and CAR NK-based strategies warrant further investigation, NEO-201-based CAR NK cells demonstrate significantly greater potency in lysing AML targets compared to naked NEO-201's ability to mediate NK cell–driven ADCC against AML cells.