Safety and Feasibility of Administration of High Doses of Ex Vivo Expanded NK Cells for Prevention of Disease Relapse after Transplantation for Patients with Myeloid Malignancies - Final Results of a Phase I Clinical Trial
Authors: Ciurea Stefan O. , Lee Dean A. , Denman Cecele , Schafer Jolie , Bassett Roland L , Cao Kai , Rondon Gabriela , Chen Julianne , Soebbing Doris , Willis Dana B. , Ahmed Sairah , Bashir Qaiser , Kebriaei Partow , Khouri Issa F. , Oran Betul , Parmar Simrit , Popat Uday R. , Hosing Chitra , Yvon Eric , Rezvani Katayoun , Shpall Elizabeth J. , Champlin Richard E. 
Published: October 2019

Abstract
Background: Disease relapse remains the most common cause of treatment failure after transplantation. To enhance anti-tumor effect of the graft we explored the use of high doses of donor-derived ex vivo expanded NK cells administered after haploidentical stem cell transplantation (HaploSCT) with the ultimate goal to decrease relapsed rate post-transplant.
Methods: We aimed to study safety and determine the maximum tolerated dose (MTD) of high doses of mbIL-21 ex vivo expanded NK cells in a phase I clinical trial (clinicaltrials.gov NCT01904136) administered post-transplant in patients with myeloid malignancies (AML, CML, MDS). We hypothesized that infusion of mature NK cells would compensate for the lower NK-cell numbers and poor function previously observed by our group in the first month post-transplant. Patients received conditioning with melphalan 140mg/m2, fludarabine 160mg/m2 and 2GyTBI, and GVHD prophylaxis with post-transplant cyclophosphamide, tacrolimus and mycophenolate (Gaballa S, et al. Cancer. 2016). All patients had a bone marrow graft. NK cells were generated from peripheral blood mononuclear cells of the same donor with infusions on Days -2, +7 and on/after +28. The first infusion was with fresh and the other two were with cryopreserved NK cells. Dose escalation was planned in cohorts of 2 patients starting at 1x105 to 1x109 NK cells/Kg. Having an NK-cell alloreactive donor or certain KIR genotype was not a requirement to participate in this study, although these characteristics were evaluated in all patients.
Results: Thirteen patients were enrolled. Eight patients had AML (7 in CR1, 4 with high-risk cytogenetics, 3 had primary induction failure AML and 3 had +minimal residual disease (MRD) at transplant, and one FLT3+ AML in CR2 with persistent MRD by flow cytometry), and 5 had CML (4 in second chronic phase, 2 with prior CNS disease, one had associated MDS with monosomy 7, and 2 who failed multiple TKIs). The median age was 41.5 years (range 18-60). Five patients were males and 8 females. The NK-cell dose escalation was as follows: 1x105/kg (N=2), 1x106/kg (N=3), 1x107/kg (N=3), 3x107/kg (N=2) and 1x108/kg (N=2). One patient was treated with 1x104/kg (Dose -1). All patients received the 3 planned infusions except one. Median purity of the NK-cell product was 98.98%. All patients achieved primary engraftment (100%) with 100% donor chimerism except one patient (who received 1x104/kg) who had secondary graft failure with concurrent parainfluenza pneumonia and died of treatment-related mortality (TRM). The median time to neutrophil and platelet engraftment was 18 and 26 days, respectively. Of 12 patients evaluable for aGVHD, the maximum grade was II in 7 patients. No grade III-IV aGVHD or cGVHD was observed. Only 5/12 patients had CMV reactivation (41.6% compared with 71% in retrospective data with the same treatment without NK cells), while none developed BK virus hemorrhagic cystitis. All patients (N=12) achieved remission with negative MRD after transplant. One patient treated NK cells at 1x105/kg/dose relapsed, received salvage treatment and was alive at last follow-up. All other patients are alive and in complete remission (N=11) after a median follow-up of 12.8 months (range 6-25.1). Compared with patients treated on a previous clinical trial with the same conditioning without NK cells (AML in CR1/2 and CML in CP), the patients who received NK cells had marked improvement in NK-cell function and cytotoxicity, as well as a significantly increase in INF gamma and TNF alpha production. A lower relapse rate and improved survival was observed compared with the retrospective cohort of patients treated without NK cells, although not statistically significant (p=0.21) (Figure 1).
Conclusions: Doses up to 3x108/kg total dose of mbIL-21 ex vivo expanded NK cells obtained from the same donor can be safely administered in the early post-transplant period after HaploSCT. This was the maximum feasible dose to be manufactured in the current system, while no toxicity or increase in GVHD was observed. Three infusions of 1x108 NK cells per kg will be administered in the phase II study
Figure 1 Relapse and survival for patients treated with the same conditioning regimen with and without NK cells. Figure 1. Relapse and survival for patients treated with the same conditioning regimen with and without NK cells.

Disclosures
Ciurea: Spectrum Pharmaceuticals: Other: Advisory Board; Cyto-Sen Therapeutics: Equity Ownership. Lee:Sanofi: Consultancy; Cyto-Sen Therapeutics: Equity Ownership, Other: Board of Directors; Intellia Therapeutics: Other: Advisory Board; Courier Therapeutics: Other: Advisory Board; Intrexon: Consultancy, Patents & Royalties; Shire: Other: Advisory Board; Ziopharm: Consultancy, Patents & Royalties. Bashir:Spectrum: Consultancy; Celgene: Research Funding; Takeda: Research Funding; Takeda: Consultancy. Champlin:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties.