Adaptive single-KIR⁺NKG2C⁺NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia

Abstract

BackgroundNatural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+adaptive NK cells to maximize missing-self reactivity.MethodsWe developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML.ResultsADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dimblast subtypes.ConclusionsThese preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

Donor selection for adoptive immunotherapy with NK cells in AML patients: Comparison between analysis of lytic NK cell clones and phenotypical identification of alloreactive NK cell repertoire

“…If KIR/HLA genetics are primarily relevant to understanding if a haploidentical donor can be NK alloreactive, the actual frequency of this subset in the total NK cell repertoire should be evaluated by phenotypic and/or functional assays. These studies allow the choice of the most suitable donor, including the identification of “superdonors” ( 59 ), whose NK repertoire reveals the highest frequency of alloreactive NK cells, and also the quantification of the “functional NK cell dose” to be infused into the patient. In conclusion, the systematic parallel study of NK alloreactivity and the critical view of the two approaches add new insights into the criteria for donor selection in the haploidentical setting.…”

Harnessing novel strategies and cell types to overcome immune tolerance during adoptive cell therapy in cancer

“… 81 Moreover, a recent report demonstrated the use of a GMP-compliant expansion protocol that confers NK cells’ sustainable adaptive features with robust tumor-killing capacity. 82 Gang et al also demonstrated CAR-transduced memory-like NK cells had better anti-tumoral responses compared with conventional CAR-NK cells in the treatment of such NK-resistant lymphoma. 83 Additionally, the targeting of cytokine-inducible SH2-containing protein (CIS) which is a potent negative regulator of IL-15 signaling pathway, would also enhance NK cell functions.…”

Viral escape from NK‐cell‐mediated immunosurveillance: A lesson for cancer immunotherapy?

“…Recapitulating the HCMVinduced expansion of NKG2A − NKG2C + NK cells to overcome viral immune evasion and translating this naturally occurring process into a tool for therapeutic application is an intriguing approach. Leveraging the inherent response pattern during coculture with HLA-E-expressing feeder cells allows for the selective expansion of NKG2A − NKG2C + NK cells in vitro [29][30][31][32]. These NKG2A − NKG2C + NK-cell products combine the absence of inhibition through the lack of NKG2A with robust activation through the presence of NKG2C and thus represent optimal candidates for the treatment of tumors enriched for HLA-E expression.…”

“…Prevention of binding between NKG2A and HLA-E to avoid inhibition of infused cells [26,27] Infusion of NK cells with blocked NKG2A expression Prevention of binding between NKG2A and HLA-E to avoid inhibition of infused cells [28] Infusion of NKG2A − NKG2C + NK cells Selective growth of NKG2A − NKG2C + NK cells in vitro to preclude NKG2A-mediated inhibition and simultaneously enables NKG2C-driven activation upon binding to HLA-E [32] Potential modulation of the tumor HLA-E peptidome Window of opportunity to adjust the pool of peptides presented on HLA-E with the goal to interfere with the binding between HLA-E/peptide complexes and NKG2A…”