Relapse has emerged as the most important cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HSCT). To test the hypothesis that natural killer (NK) cells can decrease the risk of leukemia relapse, we initiated a phase 1 dose-escalation study of membrane-bound interleukin 21 (mbIL21) expanded donor NK cells infused before and after haploidentical HSCT for high-risk myeloid malignancies. The goals were to determine the safety, feasibility, and maximum tolerated dose. Patients received a melphalan-based reduced-intensity conditioning regimen and posttransplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis. NK cells were infused on days -2, +7, and +28 posttransplant. All NK expansions achieved the required cell number, and 11 of 13 patients enrolled received all 3 planned NK-cell doses (1 × 10/kg to 1 × 10/kg per dose). No infusional reactions or dose-limiting toxicities occurred. All patients engrafted with donor cells. Seven patients (54%) developed grade 1-2 acute GVHD (aGVHD), none developed grade 3-4 aGVHD or chronic GVHD, and a low incidence of viral complications was observed. One patient died of nonrelapse mortality; 1 patient relapsed. All others were alive and in remission at last follow-up (median, 14.7 months). NK-cell reconstitution was quantitatively, phenotypically, and functionally superior compared with a similar group of patients not receiving NK cells. In conclusion, this trial demonstrated production feasibility and safety of infusing high doses of ex vivo-expanded NK cells after haploidentical HSCT without adverse effects, increased GVHD, or higher mortality, and was associated with significantly improved NK-cell number and function, lower viral infections, and low relapse rate posttransplant.
Cyclin E and its co-activator, phosphoecyclin-dependent kinase 2 (p-CDK2), regulate G 1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated fulllength from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer. (Am J Pathol 2016 http://dx
Collectively, our data support an arming model of education in which enhanced glycolysis in licensed NK cells supports proliferative and cytotoxic capacity.
Natural killer (NK) cell–based immunotherapy is being explored for treating infectious diseases, including viral infections. Here, we discuss evidence of NK cell responses to different viruses, ongoing clinical efforts to treat such infections with NK cell products, and review platforms to generate NK cell products.
Background: Allogeneic stem-cell transplantation (alloSCT) remains the only curative treatment for patients with advanced AML. However, only a minority of these patients achieve disease control prior to transplantation. Natural Killer (NK) cells have potent anti-leukemic activity but are functionally deficient in AML. Adoptive NK-cell therapy using high-doses of functionally active NK-cells could overcome these limitations. We previously developed an ex vivo NK-cell expansion method based on K562 feeder cells modified to express membrane bound IL-21 (mbIL-21) and 4-1BB ligand, (FC21), which resulted in high numbers of hyperfunctional FC21-NK cells with enhanced cytotoxicity and cytokine production. Here we report outcomes of a phase I clinical trial designed to assess the safety, feasibility and maximum tolerated dose (MTD) of haploidentical FC21-NK cells for patients with relapse/refractory (R/R) AML at MD Anderson Cancer Center. Methods: Eligible patients were ≥18 years, KPS ≥70 with good organ function. Patients with relapsed AML after alloSCT were eligible if they had no active GVHD and did not require immunosuppression. Haploidentical donors were selected based on KIR characteristics, when multiple donors were available. Donor NK cells were expanded over 3 weeks and cryopreserved. Three dose levels between 106-108 cells/kg were planned. Patients received cytoreductive chemotherapy with fludarabine 30 mg/m2/day and cytarabine 2 g/m2/day for 5 days (4 days for age >60) and G-CSF (subsequently eliminated). 3-7 days after chemotherapy, patients received FC21-NK cell infusions 3 times per week, up to 6 infusions. Results: As of 4/14/2020, 15 patients were screened, 12 of whom were eligible and received the FC21-NK cells. Median age was 60 years (range 25-70); 6 (50%) had adverse cytogenetics, 8 (66.7%) had adverse ELN genetic risk, 6 (50%) had primary induction failure, 2 (16.7%) had CNS disease and 4 (33.3%) had secondary AML. Median number of prior treatment regimens was 5 (range 2-8), median blast count at enrollment was 47% (range 7-88). Median time from diagnosis to enrollment and to first NK-cell infusion was 16.6 (range 2.5-98.1) and 17.2 (range 3.1-98.6) months, respectively. Donor-recipient NK-cell alloreactivity was seen in 5 patients (41.7%). Median number of NK-cell infusion was 6 (range 3-6); 8 (66.7%) and 4 (33.3%) patients received NK-cell dose of 1 X106 and 1 X107 cells/kg, respectively. MTD was not reached. Seven patients had ANC recovery post-NK cell infusion with cumulative incidence (CI) of ANC recovery to 500/mm3 at 60 days of 58.3%. Eight patients (66.7%) achieved complete remission (CR) (N=4, 33.3%) or CR with incomplete hematologic recovery (CRi) (N=4, 33.3%) at 30 days post-NK cell infusion. One patient with CR had negative minimal residual disease (MRD). Five patients (41.7%) proceeded to haploidentical alloSCT from the same donor and were transplanted in CR/CRi, all but one with persistent MRD. With a median follow-up of 13 months (range 4.1-42.7), median OS and DFS were 17.6 and 3.3 months, and 28 and 20 months for patients receiving alloSCT, respectively. Other outcomes including 2-year OS, DFS, relapse and TRM are shown in Figure 1 and Table 1. No infusion related toxicity or cytokine release syndrome was observed. Two patients were evaluable for FC21-NK cell persistence with haplotype-specific anti-HLA antibodies. FC21-NK cells were detected 5 and 6 weeks after the last FC21-NK cell infusion, respectively. A progressive decrease of the blast population with progressive expansion of the FC21-NK cell population after repeated NK-cell infusions was noted in samples collected from one pt (Figure 2). Persistence is also being evaluated by STR chimerism. Conclusions: Multiple infusions of FC21-NK cells yielded unprecedented outcomes with 66.7% of patients responding and approximately half proceeding to alloSCT in a heavily pre-treated, ultra-refractory, high-risk patient population. Responses were observed irrespective of dose. FC21-NK cell therapy was very well tolerated with no attributable AEs and were shown to persist for at least 5 weeks after infusion. These encouraging results warrant further clinical evaluation of FC21-NK cells in R/R AML patients. Disclosures Ciurea: Kiadis Pharma: Current equity holder in publicly-traded company, Research Funding. Schafer:Kiadis Pharma: Current Employment. Shpall:Zelluna: Membership on an entity's Board of Directors or advisory committees; Adaptimmune: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Licensing Agreement. Konopleva:Calithera: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Forty-Seven: Consultancy, Research Funding; Sanofi: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Ablynx: Research Funding; AbbVie: Consultancy, Research Funding; Ascentage: Research Funding; Rafael Pharmaceutical: Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Omeros: Consultancy; DKMS America: Membership on an entity's Board of Directors or advisory committees; Cytonus: Consultancy; Genzyme: Speakers Bureau; Takeda: Patents & Royalties.
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.
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