Cytokine-Induced Memory-like NK Cells Exhibit Massive Expansion and Long-Term Persistence after Infusion Post-Haploidentical Stem Cell Transplantation: A Report of the First Three Cases in a Phase I Trial
Authors: Shapiro Roman M , Nikiforow Sarah , Rambaldi Benedetta , Vergara Juliana , Daley Heather , Kim Haesook T. , Cutler Corey , Ho Vincent T. , Koreth John , Gooptu Mahasweta , Antin Joseph H. , Brock Jennifer , Wu Catherine J. , Soiffer Robert J. , Ritz Jerome , Romee Rizwan 
Published: February 2021

Background
Relapse of acute myeloid leukemia (AML) after allogeneic stem cell transplant has a poor prognosis with limited treatment options. Cytokine-induced memory like natural killer (CIML NK) cells are a novel therapy with enhanced cytotoxicity independent of KIR ligand interactions able to induce remission of relapsed/refractory AML (Romee et al, Science TM 2016). We are evaluating the safety and potential efficacy of donor-derived CIML NK cells in patients with relapsed myeloid malignancies after haploidentical donor transplant (haploSCT) in a phase 1/1b study (clinicaltrials.gov: NCT040247761). Here we report on manufacturing, safety, and in vivo correlative biology of the adoptively transferred CIML NK cells in the first 3 enrolled patients.
Methods
The primary endpoint is to identify the maximum-tolerated dose (MTD) of CIML NK cells in patients with MDS, MPN, or AML who relapsed after haplo-SCT. NK cells were enriched from donor-derived non-mobilized leukapheresis product via two-step CD3 depletion followed by selection of CD56+ cells using the CliniMACS reagent system (Miltenyi Biotec). The enriched NK cell product was cultured for 12-16 hours in X-VIVO 15 media containing rhIL-12, rhIL-15, and rhIL-18 to generate CIML NK cells (Figure 1A). Patients in the current cohort were lymphodepleted with fludarabine 25mg/m2 daily for 3-5 days and cyclophosphamide 60mg/kg daily for 2 days followed by CIML NK cells at a dose of 5-10 x 106 cells/kg and IL-2 106 IU/m2 QOD for 7 doses. Dose-limiting toxicities were evaluated for 6 weeks following NK cell infusion. Response to therapy was assessed at day+28 following CIML NK cell infusion.
Results
Patient #001 has FLT3-ITD AML that relapsed 5 months after a reduced intensity (RIC) haploSCT. She received 7.4x106 NK cells/kg followed by IL-2 106 IU/m2 QOD for 7 doses. Her day+28 bone marrow had no leukemia blasts although FLT3-ITD mutation remained detectable. Two months post-CIML NK the leukocyte and granulocyte chimerism were 88% and 89%, respectively. Patient #002 has AML with multiple pathogenic variants, including a potentially pathogenic variant in TP53. His disease relapsed 15 months post haplo-SCT with repeat marrow showing all the original mutations, including the TP53 variant (VAF 50.5%). He received 9.5x106 NK cells/kg followed by IL-2 106 IU/m2 QOD for 7 doses. His day+28 marrow had trilineage hematopoiesis without any mutations, including no TP53 mutation (Figure 1B). Two months post-CIML NK the leukocyte and granulocyte chimerism were both 99%. Patient #003 has MDS whose disease relapsed 8 months post RIC haploSCT with persistence of all her diagnostic pathogenic mutations. She received approximately 9.2x106 NK cells/kg and has only just completed IL-2 106 IU/m2 QOD for 7 doses. Among the 3 patients, the main toxicity was prolonged cytopenia requiring stem cell boost in one case.
Donor NK cells demonstrated a dramatic shift from a predominantly CD56dimCD16hi (88% of NK cells) to a CD56dimCD16lo phenotype (49% of NK cells) as CIML NK cells. Infused CIML NK cells expanded massively, with approximately 50-fold, 10-fold, and 15-fold maximum in vivo expansion in the first three patients, respectively (Figure 1C). CIML NK cells were the major population in the day+28 marrows in both patients #001 and #002, with CD56+CD7+ cells constituting 89% of the cellularity in the former and 48% in the latter (Figure 1C). CIML NK cells persisted for 3 and 6 months post-infusion in the first two patients. The NK cells were predominantly mature, with most expressing CD16 and low levels of the inhibitory receptor NKG2A. PD-1 expression was much lower on the expanded NK cells vs the pre-infusion donor-derived NK cells. There was minimal concurrent expansion of CD4+CD25+ T-regulatory cells (Figure 1D).
Conclusion
We show with the first 3 patients in this trial that CIML NK cells can be generated and infused safely, can expand massively in the peripheral blood and bone marrow within the first 30 days post-infusion, and can persist for several months. In addition, CIML NK cell infusion can reduce the burden of pathogenic variant alleles to below the limit of detection, including the burden of high-risk mutations such as in TP53. Though our results are preliminary, the massive in vivo expansion and long-term persistence of adoptively transferred CIML NK cells underscores the unique biology of these cells that makes them an attractive option for cellular therapy protocols.

Disclosures
Nikiforow: Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Rambaldi:Equillium: Research Funding. Cutler:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees. Koreth:Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; Clinigen: Other; Miltenyi: Other: Research Support; BMS: Other: Research Support; Therakos: Membership on an entity's Board of Directors or advisory committees; Equillium: Consultancy; EMD Serono: Consultancy; Biolojic Design Inc: Consultancy; Amgen: Consultancy; Moderna Therapeutics: Consultancy. Wu:Pharmacyclics: Research Funding; BionTech: Current equity holder in publicly-traded company. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; alexion: Consultancy; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Ritz:TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Falcon Therapeutics: Consultancy; Infinity Pharmaceuticals: Consultancy.