A Phase I Clinical Trial Testing the Safety of IL-21-Expanded, Universally Alloreactive Donor-Derived Natural Killer Cells for Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome
Authors: Vasu Sumithira , Sharma Nidhi , Walker Alison R. , Wall Sarah A , Blachly James S. , Behbehani Greg K. , Choe Hannah , Thakkar Aarohi , Nakkula Robin , Troy Ella , Szuminski Nicole , de Lima Marcos J.G. , O'Donnell Lynn , Lee Dean Anthony 
Published: November 2021

Abstract
Background: Natural Killer (NK) cells are cytotoxic lymphocytes that are able to exert an anti-tumor effect in an MHC-I independent manner, but are dysfunctional and reduced in number in patients with leukemia. Several studies have shown therapeutic potential for related donor NK cells expanded and/or activated ex vivo when administered to cancer patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, personalized, donor-derived cell therapies require time for donor identification, manufacturing and product release, resulting in patient attrition. As NK alloreactivity and NKG2C expression play critical roles in mediating anti-tumor effects, we identified 'ideal' NK cell donors (in partnership with "Be the Match Biotherapies") for collection. Human leukocyte antigen (HLA) and Killer immunoglobulin receptor (KIR) genotyping were done to screen and select donors. Following this, a sample was collected from donors to expand NK cells under small-scale conditions. If donor NK cells showed robust expansion, they proceeded with apheresis. To ensure these therapeutic cells would be readily-available, we established a third-party NK cell bank through scalable, affordable mass-production with membrane-bound IL-21 feeder cells (FC21), and cryopreserved large numbers of these NK cells for immediate 'off-the-shelf' administration to recipients. Here, we report our initial experience in a Phase I trial of these off-the-shelf (OTS) alloreactive NK cells as immunotherapy for patients with relapsed/refractory AML and MDS.
Methods: Patients were treated at Dose level 1, to allow infusion at a dose of 1 x 10e7 NK cells/ kg. Each dose level had two cohorts, stratified by age. Patients were enrolled into Cohort 1 (patients &lt;60 yrs. received Fludarabine (Flu) 30 mg/m2/day & Cytarabine 2 g/m2/day on days -6 to -2) and Cohort 2 (patients ≥ 60 yrs. received Flu 30 mg/m2/day (days -5 to -2) & Decitabine 20 mg/m2/day (days -6 to -2). Patients were evaluated to see if they had any donor-directed antibodies. After chemotherapy, NK cells were infused thrice weekly for 6 doses, to be given over a period of 2 weeks (Clinicaltrials.gov: NCT04220684). Persistence of donor NK cells was determined by flow cytometry using haplotype-discriminating anti-HLA antibodies (Figure 1, 2). NK cells were completely HLA-mismatched between donor and recipients.
Results: 6 patients (3/cohort) were treated at the first dose level of 1x10 7 NK cells/kg/dose. All 6 patients had relapsed/refractory AML and had received at least two prior lines of treatment. One patient (005) withdrew from the study prior to infusion of NK cells. 6 patients received the first NK cell dose as an inpatient and were discharged to receive the remaining 5 doses as an outpatient. 5 patients tolerated infusion of all 6 doses of NK cells administered over 2 weeks. One patient (006) developed a Cytokine response (CRS) - like syndrome in the context of streptococcus bacteremia and received only one dose of NK cells. Symptoms suggestive of CRS responded fully with steroids and patient was able to successfully wean off steroids and discharged to the outpatient setting. One patient proceeded to allogeneic hematopoietic cell transplant. No infusion-related reactions, neurotoxicity or graft versus host disease was observed. In vivo persistence/expansion NK cells were superior with FLU/CY lymphodepletion (cohort 1).
Conclusions: Cryopreserved, third-party donor-derived NK cells are safe and feasible in relapsed/refractory AML patients, and even at this first dose level, completely HLA-mismatched NK cells can persist and expand to high levels in vivo.
Figure 1 Figure 1.

Disclosures
Vasu: Kiadis, Inc.: Research Funding; Seattle Genetics: Other: travel support; Boehringer Ingelheim: Other: Travel support; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Walker: Newave: Other: clinical trial support; Geron: Other: clinical trial support; Novartis: Other: clinical trial support. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. de Lima: Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding. Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.