Natural Killer Cell Infusion Research Articles

TIGIT blockade enhances functionality of peritoneal NK cells with altered expression of DNAM-1/TIGIT/CD96 checkpoint molecules in ovarian cancer

ABSTRACT Advanced ovarian cancer (OC) patients have a poor 5-year survival of only 28%, emphasizing the medical need for improved therapies. Adjuvant immunotherapy could be an attractive approach since OC is an immunogenic disease and the presence of tumor-infiltrating lymphocytes has shown to positively correlate with patient survival. Among these infiltrating lymphocytes are natural killer (NK) cells, key players involved in tumor targeting, initiated by signaling via activating and inhibitory receptors. Here, we investigated the role of the DNAM-1/TIGIT/CD96 axis in the anti-tumor response of NK cells toward OC. Ascites-derived NK cells from advanced OC patients showed lower expression of activating receptor DNAM-1 compared to healthy donor peripheral blood NK cells, while inhibitory receptor TIGIT and CD96 expression was equal or higher, respectively. This shift to a more inhibitory phenotype could also be induced in vitro by co-culturing healthy donor NK cells with OC tumor spheroids, and in vivo on intraperitoneally infused NK cells in SKOV-3 OC bearing NOD/SCID-IL2Rγnull (NSG) mice. Interestingly, TIGIT blockade enhanced degranulation and interferon gamma (IFNγ) production of healthy donor CD56dim NK cells in response to OC tumor cells, especially when DNAM-1/CD155 interactions were in place. Importantly, TIGIT blockade boosted functional responsiveness of CD56dim NK cells of OC patients with a baseline reactivity against SKOV-3 cells. Overall, our data show for the first time that checkpoint molecules TIGIT/DNAM-1/CD96 play an important role in NK cell responsiveness against OC, and provides rationale for incorporating TIGIT interference in NK cell-based immunotherapy in OC patients.

Natural Killer Cells Infusion for Treating Acute Myeloid Leukemia Patients With Minimal Residual Disease

Natural Killer Cells Infusion for Treating Acute Myeloid Leukemia Patients With Minimal Residual Disease

Safety and immune cell kinetics after donor natural killer cell infusion following haploidentical stem cell transplantation in children with recurrent neuroblastoma.

IntroductionUnder the hypothesis that early natural killer cell infusion (NKI) following haploidentical stem cell transplantation (haplo-SCT) will reduce relapse in the early post-transplant period, we conducted a pilot study to evaluate the safety and feasibility of NKI following haplo-SCT in children with recurrent neuroblastoma who failed previous tandem high-dose chemotherapy and autologous SCT.MethodsWe used the high-dose 131I-metaiodobenzylguanidine and cyclophosphamide/fludarabine/anti-thymocyte globulin regimen for conditioning and infused 3 × 107/kg of ex-vivo expanded NK cells derived from a haploidentical parent donor on days 2, 9, and 16 post-transplant. Interleukin-2 was administered (1 × 106 IU/m2/day) subcutaneously to activate infused donor NK cells on days 2, 4, 6, 9, 11, 13, 16, 18, and 20 post-transplant.ResultsSeven children received a total of 19 NKIs, and NKI-related acute toxicities were fever (n = 4) followed by chills (n = 3) and hypertension (n = 3); all toxicities were tolerable. Grade ≥II acute GVHD and chronic GVHD developed in two and five patients, respectively. Higher amount of NK cell population was detected in peripheral blood until 60 days post-transplant than that in the reference cohort. Cytomegalovirus and BK virus reactivation occurred in all patients and Epstein-Barr virus in six patients. Six patients died of relapse/progression (n = 5) or treatment-related mortality (n = 1), and one patient remained alive.ConclusionNKI following haplo-SCT was relatively safe and feasible in patients with recurrent neuroblastoma. Further studies to enhance the graft-versus-tumor effect without increasing GVHD are needed.

Effect of autologous NK cell immunotherapy on advanced lung adenocarcinoma with EGFR mutations.

This study investigated the efficiency of natural killer (NK) cell immunotherapy on non-small cell lung cancer with and without EGFR mutations in order to evaluate the response rate (RR) and progression-free survival (PFS). Among the 48 patients recruited, 24 were clinically confirmed to be EGFR mutation positive. The study group was treated with autologous NK cell immunotherapy. Comparisons of the lymphocyte number, serum tumour-related biomarkers, circulating tumour cells (CTC), Karnofsky Performance Status (KPS) and survival curves were carried out before and after NK cell immunotherapy. The safety and short-term effects were evaluated, followed by median PFS and RR assessments. The serum CEA and CA125 values were found lower in the NK cell therapy group than that of the non-NK treatment group (p < 0.05). The χ2 test showed a 75% RR of the study group A, significantly higher than that of the control group B (16.7%; p < 0.01). The RR of groups C (58.3%) and D (41.7%) were not statistically significant. The p values of the 4 groups were 0.012, 0.012, 0.166 and 1 from group A to group D, respectively. The median PFS was 9 months in EGFR mutation positive group undergoing NK cell infusion interference. By evaluating the changes in immune function, tumour biomarkers, CTC, KPS and PFS, we demonstrated that NK cell therapy had better clinical therapeutic effects on EGFR mutation-positive lung adenocarcinoma.

Observation on the Efficacy of Consolidation Chemotherapy Combined with Allogeneic Natural Killer Cell Infusion in the Treatment of Low and Moderate Risk AML

Objective To evaluate the efficacy of consolidation chemotherapy combined with allogeneic natural killer (NK) cell infusion in the treatment of low or intermediate-risk(LIR) AML. Methods 23 cases of LIR AML patients with hematologic complete remission (CR) were enrolled from January 2014 to June 2019 and treated with mIL-21/4-1BBL actived NK cell transfusion combined with consolidation chemotherapy after 3 consolidation courses. Control group were concurrent patients from Department of Hematology and their gender, age, diagnosis, risk stratification of prognosis, CR and the number of courses of consolidate chemotherapy before NK cell transfusion were matched with LIR AML patients. Results A total of 45 times of NK cells were injected into 23 LIR AML patients during 4 to 7 courses of chemotherapy. The median NK cell infusion quantity was 7.5 (6.6~8.64) x 109/L, and the median survival rate of NK cells was 95.4 (93.9~96.9) %. Among them, the median CD3-CD56 + cell number is 5.0(1.4 ~ 6.4) × 109/L, which accounts for 76.8(30.8 ~ 82.9) %; The number of CD3+CD56+ cells was 0.55 (0.24~1.74) x 109/L, accounting for 8.8 (4.9~20.9) %. Before NK cell infusion, the number of patients with positive MRD in the treatment group was 9/23 (39.1%), and the control group was 19/46 (41.3%) (X2=0.03, P=0.862). After NK cell infusion, There was no significant difference in MRD that went from negative to positive between the treatment group and the control group(14.3%vs 22.2%, X2=0.037, P=0.847). In the treatment group, 66.7% (6/9) of the MRD were converted from positive to negative, which was significantly higher than that in the control group10.5% (2/19) (X2=6.811, P=0.009). Morphological recurrence occurred in 1 case of MRD negative in the treatment group and 2 cases of MRD positive in the control group. By the end of the follow-up, the median follow-up was 35 (10~59) months, the number of patients with morphological recurrence in the treatment group was 30.4% (7/23), which is significantly lower than that in the control group (50.2%, 24/46)(X2=2.929, P=0.087),although there was no statistically significant difference between the two groups. There was no significant difference on MRD-negative between the treatment group and the control group(43.5% vs 43.5%, X2=1.045, P=0.307).The 3-year leukemia -free survival were better in the treatment group(65.1±11.1)% than that in the control group (50.0±7.4)%(P=0.047). The 3year overall survival in the treatment and chemotherapy groups were(78.1±10.2)%and (65.8±8.0)%(P=0.212), respectively. Conclusion The consolidation of chemotherapy combined with allogeneic NK cell infusion contributes to the further remission of patients with LMR AML and the reduction of long-term recurrence. Figure Disclosures No relevant conflicts of interest to declare.

Cytokine-Induced Memory-like (ML) NK Cells Persist for > 2 Months Following Adoptive Transfer into Leukemia Patients with a MHC-Compatible Hematopoietic Cell Transplant (HCT)

Natural killer (NK) cells exhibit innate memory or memory-like responses following stimulation with haptens, viruses, or cytokines. Human memory-like (ML) NK cells differentiate following a short-term activation with IL-12, IL-15, and IL-18, and have increased anti-tumor activity against AML and other cancers in vitro, in xenograft models, and in the first-in human phase 1 clinical trial of ML NK cells in AML (PMID27655849). In this trial, CR/Cri was observed in &gt;50% of patients treated, and mass cytometry revealed a unique multi-dimensional phenotype of in vivo differentiated ML NK cells that was confirmed using donor-specific HLA markers. Although adoptively transferred MHC-haploidentical ML NK cells expanded and differentiated over 2-3 weeks, these cells were eliminated by recipient allogeneic immune responses, a challenge observed with all allogeneic lymphocyte therapies. The immune rejection observed in the allogeneic setting precluded following ML NK phenotype, persistence, and function long-term in these patients. We hypothesized that ML NK cells could persist longer than 2-3 weeks in an MHC-compatible setting, and thus be able to assess ML NK cells durability. To test this idea, a phase 2 clinical trial was designed for relapsed/refractory AML patients, who receive a reduced-intensity HLA-haploidentical hematopoietic cell transplant (HCT), followed by same-donor ML NK cell adoptive transfer at day 7, with 2 weeks of IL-15/N-803 support (NCT02782546). Using mass cytometry, ML NK cells were confirmed as distinct from conventional NK cells, CD56hi/NKp30hi/CD62Lhi/KIR+/NKG2A+/CD57+/-, by viSNE analysis, and clearly inconsistent with immature NK cells arising from the HCT graft (CD56bright/KIR-/CD57-). ML NK cells persisted in patients for at least 2 months (n=5) following adoptive transfer, and constituted 20-50% of total NK cells at day 60 (n=3, 206±97 cells/μl; mean±SEM; peak ML NK cells = 751-1106 cells/µl, D21-D28, n=5). These ML NK cells appeared highly functional (56±8% IFN-γ+, 20±3% TNF+, 41±7% CD107a+) when stimulated with tumor targets immediately ex vivo on study day 28 (n=7). Unsupervised clustering of scRNA-seq from patient samples acquired 14-60 days after ML NK cell adoptive transfer identified a subset of NK cells transcriptionally distinct from conventional CD56bright and CD56dim. This NK cell population was the majority of NK cells at study Day 21 and remained identifiable 2 months post-transfer. In agreement with the mass cytometry data, these NK cells expressed high levels of KIRs with scRNA-seq analysis uncovering novel transcriptional changes in granzyme M, perforin, KLRG1, and IFNG suggesting ML NK cells represent a mature, activated NK cell subset distinct from conventional NK cells arising from the graft. In addition, scRNAseq analysis identified high expression of the transcription factor RUNX3, a potential regulator of ML NK cell phenotype in vivo. In conclusion, a single infusion of ML NK cells resulted in a durable population of highly functional NK cells, as evidenced by multi-dimensional analyses using mass cytometry and scRNA-sequencing. These studies provide evidence that ML NK cell therapy in the MHC-compatible setting overcomes persistence barriers and provide a platform for innovation in NK cell therapeutics. Disclosures Cashen: Celgene: Other: Speaker's Bureau; Seattle Genetics: Other: Speaker's Bureau; Novartis: Other: Speaker's Bureau. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).

Adoptively Transferred Donor-Derived Cytokine Induced Memory-like NK Cells Persist and Induce Remission in Pediatric Patient with Relapsed Acute Myeloid Leukemia after Hematopoietic Cell Transplantation

Acute myeloid leukemia (AML) accounts for 18% of pediatric leukemias. For high-risk AML, standard treatment includes multi-agent chemotherapy followed allogeneic hematopoietic cell transplantation (HCT). Despite a high remission rate, 50% of patients relapse and have a poor prognosis with &lt; 20% of patients surviving more than 3 years. Salvage chemotherapy alone or combined with donor lymphocyte infusions (DLI) have little curative potential, and new treatment strategies are needed for relapsed-refractory AML. Previous studies have shown that natural killer (NK) cells can be stimulated ex vivo with IL-12/15/18 to generate a memory-like phenotype with enhanced anti-leukemia effect. In adults with relapsed-refractory AML, adoptive transfer of MHC-haploidentical cytokine-induced memory-like (CIML or ML) NK cells induced remission in 54% of patients (PMID27655849). The infused donor ML NK cells expand in vivo but are rapidly eliminated following recovery of recipient T cells, providing a window of therapeutic activity of 2-3 weeks. We sought to test the safety and efficacy of ML NK cells for treatment of pediatric/young adult patients with post-HCT relapsed AML. We hypothesized that ML NK cells derived from the HCT donor would be well-tolerated, exhibit anti-leukemia activity, and expand with prolonged persistence following transfer into pediatric AML patients. Here, we report the results of the first pediatric patient treated on a phase I clinical trial using ML NK cell therapy for relapsed AML after allogeneic HCT (NCT03068819). Briefly, patients are treated with FLAG (fludarabine, cytarabine and granulocyte colony stimulating factor) salvage chemotherapy to reduce the bulk of AML and provide lymphodepletion for ML NK cell expansion. Two weeks after chemotherapy, a non-mobilized leukapheresis product is collected from the original HCT donor and processed into a T cell-based DLI and ML NK cells. The T cell DLI (1 x 106 T cells/kg) is immediately infused, and the ML NK cells are generated by stimulation with IL-12/15/18 ex vivo for 12-16 hours and then infused (10x106/kg). An 18-month-old male with infant AML had relapse of his leukemia 3 months after MHC-haploidentical HCT. Treatment with chemotherapy, including mitoxantrone and daunorubicin-cytarabine liposome, failed to induce remission. At the time of enrollment on the phase I trial, he had AML blasts in his bone marrow (Table 1). He was treated with FLAG chemotherapy followed by infusion of DLI and ML NK cells from the original haploidentical HCT donor. Assessment at 30 days, 3 months and 6 months post NK cell infusion showed complete remission with no evidence of leukemia and full donor engraftment. Remarkably, donor-derived ML NK cells expanded to 77% of donor lymphocytes on day 28 and still comprised 24% percent of lymphocytes at 6 months post infusion (Figure 1A-C). The expanded donor NK cell phenotype was consistent with ML NK cells (e.g., NKG2A+KIR+) utilizing CyTOF multidimensional analysis previously confirmed to identify ML NK cells (Figure 1D). The ML NK cells were functional as demonstrated by leukemia-triggered IFN-γ production immediately ex vivo from day 7-28 samples (Figure 1E-F). The patient's clinical course was complicated by mild gastrointestinal graft-versus-host disease that resolved with low-dose steroids and tociluzimab. These early results demonstrate proof-of-principle that adoptive transfer of donor-derived ML NK cells in combination with DLI is feasible and offers a novel immunotherapy option for patients with relapsed AML after HCT. Moreover, in this T and NK cell compatible immune environment post-HCT, donor ML NK cells expand and persist robustly in vivo for &gt; 6 months without exogenous cytokine support and have potent anti-leukemic activity. Thus, ML NK cells are a cellular therapy platform to treat AML that has relapsed after allogeneic HCT. Disclosures Cashen: Celgene: Other: Speaker's Bureau; Seattle Genetics: Other: Speaker's Bureau; Novartis: Other: Speaker's Bureau. Fehniger:Horizon Pharma PLC: Other: Consultancy (Spouse); Cyto-Sen Therapeutics: Consultancy.

Natural Killer Cells in Myeloid Malignancies: Immune Surveillance, NK Cell Dysfunction, and Pharmacological Opportunities to Bolster the Endogenous NK Cells.

Natural killer (NK) cells are large granular lymphocytes involved in our defense against certain virus-infected and malignant cells. In contrast to T cells, NK cells elicit rapid anti-tumor responses based on signals from activating and inhibitory cell surface receptors. They also lyse target cells via antibody-dependent cellular cytotoxicity, a critical mode of action of several therapeutic antibodies used to treat cancer. A body of evidence shows that NK cells can exhibit potent anti-tumor activity against chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and myelodysplastic syndromes (MDS). However, disease-associated mechanisms often restrain the proper functions of endogenous NK cells, leading to inadequate tumor control and risk for disease progression. Although allogeneic NK cells can prevent leukemia relapse in certain settings of stem cell transplantation, not all patients are eligible for this type of therapy. Moreover, remissions induced by adoptively infused NK cells are only transient and require subsequent therapy to maintain durable responses. Hence, new strategies are needed to trigger full and durable anti-leukemia responses by NK cells in patients with myeloid malignancies. To achieve this, we need to better understand the interplay between the malignant cells, their microenvironment, and the NK cells. This review focuses on mechanisms that are involved in suppressing NK cells in patients with myeloid leukemia and MDS, and means to restore their full anti-tumor potential. It also discusses novel molecular targets and approaches, such as bi- and tri-specific antibodies and immune checkpoint inhibitors, to redirect and/or unleash the NK cells against the leukemic cells.

Observation on the efficacy of consolidation chemotherapy combined with allogeneic natural killer cell infusion in the treatment of low and moderate risk acute myeloid leukemia

目的评价巩固化疗联合异基因NK细胞输注治疗低中危急性髓系白血病（AML）的疗效。方法回顾性分析2014年1月至2019年6月北京大学人民医院诊治的23例经化疗达血液学完全缓解（CR），且巩固治疗3个疗程后，应用巩固化疗联合异基因NK细胞回输治疗的低中危AML患者，对照组为1∶2配对的46例AML患者，配对因素为患者的性别、年龄、诊断、预后危险分层、诱导CR及NK细胞输注前巩固化疗的疗程数。比较两组微小残留病（MRD）转阴率、复发率、无白血病生存（LFS）及总生存（OS）。结果治疗组23例低中危AML患者于巩固化疗第4~7个疗程共输注NK细胞45次，中位NK细胞输注数量7.5（6.6~8.6）×109/L，NK细胞中位存活率95.4%（93.9%~96.9%），中位扩增倍数141.1（86.1~171.1），其中中位CD3−CD56+细胞数5.0（1.4~6.4）×109/L，占回输细胞总数的76.8%（30.8%~82.9%）；CD3+CD56+细胞数0.55（0.24~1.74）×109/L，占回输细胞总数的8.8%（4.9%~20.9%）。NK细胞输注前，治疗组MRD阳性患者为9例（39.1%），对照组MRD阳性患者为19例（41.3%），两组间差异无统计学意义（χ2=0.030，P=0.862）。治疗组输注NK细胞前后疗效评估与同期对照组对比，MRD阴性转阳性比例两组差异无统计学意义[14.3%（2/14）对22.2%（6/27），χ2=0.037，P=0.847]。治疗组MRD由阳性转为阴性的患者占66.7%（6/9），明显高于对照组的10.5%（2/19）（χ2=6.811，P=0.009）。其中治疗组有1例MRD阴性患者发生了形态学复发，对照组有2例MRD阳性患者形态学复发。截至末次随访，中位随访35（10~59）个月，治疗组与对照组MRD阴性患者比例差异无统计学意义[43.5%（10/23）对43.5%（20/46），χ2=1.045，P=0.307]。在形态学复发方面，虽然两组差异无统计学意义，但是治疗组形态学复发患者比例明显低于对照组[30.4%（7/23）对50.2%（24/46），χ2=2.929，P=0.087]。治疗组预计3年LFS率为（65.1±11.1）%，明显高于对照组的（50.0±7.4）%（P=0.047）。治疗组、对照组的预计3年OS率分别为（78.1±10.2）%、（65.8±8.0）%（P=0.212）。结论巩固化疗联合异基因NK细胞输注可能有助于低中危AML患者达到更深层次的缓解，减少远期复发。

Strategies to Augment Natural Killer (NK) Cell Activity against Solid Tumors

The immune system plays a crucial role to prevent local growth and dissemination of cancer. Therapies based on activating the immune system can result in beneficial responses in patients with metastatic disease. Treatment with antibodies targeting the immunological checkpoint axis PD-1 / PD-L1 can result in the induction of anti-tumor T cell activation leading to meaningful long-lasting clinical responses. Still, many patients acquire resistance or develop dose-limiting toxicities to these therapies. Analysis of tumors from patients who progress on anti-PD-1 treatment reveal defective interferon-signaling and antigen presentation, resulting in immune escape from T cell-mediated attack. Natural killer (NK) cells are innate lymphocytes that can kill tumor cells without prior sensitization to antigens and can be activated to kill tumor cells that have an impaired antigen processing and presentation machinery. Thus, NK cells may serve as useful effectors against tumor cells that have become resistant to classical immune checkpoint therapy. Various approaches to activate NK cells are being increasingly explored in clinical trials against cancer. While clinical benefit has been demonstrated in patients with acute myeloid leukemia receiving haploidentical NK cells, responses in patients with solid tumors are so far less encouraging. Several hurdles need to be overcome to provide meaningful clinical responses in patients with solid tumors. Here we review the recent developments to augment NK cell responses against solid tumors with regards to cytokine therapy, adoptive infusion of NK cells, NK cell engagers, and NK cell immune checkpoints.

Abstract CT216: Phase I study of intraventricular infusions of autologous ex vivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system

Abstract Scientific background/rationale: The prognosis of recurrent/refractory medulloblastoma (MB), ependymoma (EP) and atypical teratoid/rhabdoid neoplasms (AT/RT) which constitutes the majority of malignant posterior fossa tumors (MPFT) in children, remains dismal with no defined effective therapy. Current treatment strategies have significant toxicities, thus profiling newer trials with clinical efficacy and minimal side-effects are imperative. Advantages of direct infusion of Natural Killer (NK) cells into the ventricles could enhance therapeutic efficacy by maximally concentrating them in the brain in close proximity to the tumor area, bypassing the blood brain barrier and alleviating toxicity often seen with systemic chemotherapy. We have shown efficacy of activated and propagated NK cells to lyse and kill the MPFT in vitro and tumor explants in mice models. We demonstrated in a pilot study for the first time in humans, the feasibility of infusions of biologic agents into the fourth ventricle. Infusing biologic agents into the lateral ventricles are well established in neurooncology. The current ongoing study is the first-in-human, loco-regional infusion of NK cells directly into the brain evaluating safety and efficacy in the context of a phase I trial with an intent to avoid the significant side-effects of systemic therapy. This novel trial, will hopefully be able to provide therapeutic efficacy and a meaningful increase in the survival of these children. Correlative studies: Cerebrospinal fluid (CSF) withdrawn during infusions of NK cells will be evaluated for the immunophenotype, function, persistence of NK cells and influx or presence of any other immune cells. Methods: NK cells are directly infused into the lateral or fourth ventricles, after placement of a catheter at that location. Recurrent or refractory MB, AT/RT and EP involving the brain and/or spine at original diagnosis or relapse with histological verification are eligible. Three to six evaluable patients, are entered at a dose level in this study for determination of maximum tolerated dose (MTD). A minimum of 9 patients and a maximum of 24 patients are planned to be enrolled. After autologous expanded NK cell product have been manufactured as per good manufacturing guidelines (GMP) and released, patients receive 3 cycles of NK cell infusions over 12 weeks. Each cycle consists of 1 infusion per week for 3 weeks followed by a rest week (week 4). Dosing is based on recipient body surface area (BSA) and 3 dose levels will be evaluated. Currently the trial is at the final cohort (third dose level), with no dose limiting toxicity (DLT) noted with the previous dose levels. 80% of the initial minimal accrual have been met. Also as a secondary aim we will evaluate the anti-tumor activity of the NK cell infusions within the confines of a phase I study. Trial (NCT02271711-recruiting) Citation Format: Soumen Khatua, Dean Lee, Laurence Cooper, Judy Moyes, David Sandberg, Zsila Sadighi, Heather Meador, Leena Ketonen, Michael E. Rytting, Jason M. Johnson, Dristhi Raagonanan, Diane D. Liu, Vidya Gopalakrishnan, Wafik Zaky. Phase I study of intraventricular infusions of autologous ex vivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT216.

Efficacy and peripheral immunity analysis of allogeneic natural killer cells therapy in patients with hepatocellular carcinoma

To evaluate the safety and efficacy of allogeneic natural killer (NK) cells in the treatment of primary hepatocellular carcinoma (HCC), and to elucidate the mechanism of NK cells therapy. Twenty-one patients with primary HCC treated with allogeneic NK cells at the Fifth Medical Center of the PLA General Hospital were followed up for 1 year. Peripheral blood mononuclear cells (PBMCs) were isolated from patient-related donors and cultured in vitro for 15 days and infused to the patients in two consecutive days. Clinical data and laboratory data were collected and analyzed, including survival, clinical features, imaging changes, hematology, immunology, and biochemical indicators to evaluate the safety and efficacy of allogeneic NK cell therapy. The changes of peripheral blood lymphocyte subsets after treatment were also analyzed to explore the possible anti-tumor mechanisms. (1) Of the 21 patients with primary HCC, 11 patients were treated once, 5 patients were treated twice, and 5 patients were treated 3 times. After allogeneic NK cells infusion, 10 patients had fever, 1 patient had slight hepatalgia and 1 patient had slight headache, no other adverse events occurred including acute and chronic graft-versus-host disease (GVHD). They resolved spontaneously within 8 hours without other treatment. (2) The total disease control rate was 76.2% during one-year follow-up. Among them, the patients with Barcelona clinic liver cancer (BCLC) stage A had a disease control rate of 100%, stable disease (SD) in 10 cases; BCLC stage B patients had a disease control rate of 60%, partial response (PR) in 1 case, and SD 2 in cases; BCLC stage C patients had a disease control rate of 50%, complete response (CR) in 1 case, and 2 cases of PR. (3) The frequencies of NK cells and CD8+ T cells in peripheral blood were significantly lower than that before at 24 hours after treatment, and the frequencies of CD4+ T cells and CD4/CD8 were significantly higher than the baseline. Allogeneic NK cells have good safety and efficacy in the treatment of primary HCC. The anti-tumor effect of the allogeneic NK cells may play an important role in the activation of the patient's natural immune system and delay disease progression, suggesting that allogeneic NK cells combined with sorafenib may be a very effective treatment for advanced HCC, and further large-sample multicenter randomized controlled clinical trials are needed to validate this result.

Enhanced Bone Marrow Homing of Natural Killer Cells Following mRNA Transfection With Gain-of-Function Variant CXCR4R334X.

Adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory leukemia and myeloma. However, to date, clinical efficacy of NK cell immunotherapy has been limited to a sub-fraction of patients. Here we show that steps incorporated in the ex vivo manipulation/production of NK cell products used for adoptive infusion, such as over-night IL-2 activation or cryopreservation followed by ex vivo expansion, drastically decreases NK cell surface expression of the bone marrow (BM) homing chemokine receptor CXCR4. Reduced CXCR4 expression was associated with dampened in vitro NK cell migration toward its cognate ligand stromal-derived factor-1α (SDF-1α). NK cells isolated from patients with WHIM syndrome carry gain-of-function (GOF) mutations in CXCR4 (CXCR4R334X). Compared to healthy donors, we observed that NK cells expanded from WHIM patients have similar surface levels of CXCR4 but have a much stronger propensity to home to BM compartments when adoptively infused into NOD-scid IL2Rgammanull (NSG) mice. Therefore, in order to augment the capacity of adoptively infused NK cells to home to the BM, we genetically engineered ex vivo expanded NK cells to express the naturally occurring GOF CXCR4R334X receptor variant. Transfection of CXCR4R334X-coding mRNA into ex vivo expanded NK cells using a clinically applicable method consistently led to an increase in cell surface CXCR4 without altering NK cell phenotype, cytotoxic function, or compromising NK cell viability. Compared to non-transfected and wild type CXCR4-coding mRNA transfected counterparts, CXCR4R334X-engineered NK cells had significantly greater chemotaxis toward SDF-1α in vitro. Importantly, expression of CXCR4R334X on expanded NK cells resulted in significantly greater BM homing following adoptive transfer into NSG mice compared to non-transfected NK cell controls. Collectively, these data suggest up-regulation of cell surface CXCR4R334X on ex vivo expanded NK cells via mRNA transfection represents a novel approach to improve homing and target NK cell-based immunotherapies to BM where hematological malignancies reside.

Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy.

Natural killer (NK) cells are attractive within adoptive transfer settings in cancer immunotherapy due to their potential for allogeneic use; their alloreactivity is enhanced under conditions of killer immunoglobulin-like receptor (KIR) mismatch with human leukocyte antigen (HLA) ligands on cancer cells. In addition to this, NK cells are platforms for genetic modification, and proliferate in vivo for a shorter time relative to T cells, limiting off-target activation. Current clinical studies have demonstrated the safety and efficacy of allogeneic NK cell adoptive transfer therapies as a means for treatment of hematologic malignancies and, to a lesser extent, solid tumors. However, challenges associated with sourcing allogeneic NK cells have given rise to controversy over the contribution of NK cells to graft-versus-host disease (GvHD). Specifically, blood-derived NK cell infusions contain contaminating T cells, whose activation with NK-stimulating cytokines has been known to lead to heightened release of proinflammatory cytokines and trigger the onset of GvHD in vivo. NK cells sourced from cell lines and stem cells lack contaminating T cells, but can also lack many phenotypic characteristics of mature NK cells. Here, we discuss the available published evidence for the varying roles of NK cells in GvHD and, more broadly, their use in allogeneic adoptive transfer settings to treat various cancers.

Tumor pathological and immunological changes in HER2+ metastatic breast cancer (MBC) following trastuzumab combined with expanded and activated autologous natural killer (NK) cell infusion.

e12511 Background: A central mechanism for the anti-tumor activity of Trastuzumab, a HER2 monoclonal antibody, against HER2+ tumors is induction of antibody dependent cell cytotoxicity (ADCC) mediated by NK cells. We are conducting a first-in-human trial of Trastuzumab followed by infusion of expanded, activated autologous NK cells in refractory HER2+ MBC (ClinicalTrials.gov: NCT02030561) to test the hypothesis that NK cell infusions will augment Trastuzumab-mediated ADCC and increase immune cell infiltration in tumor. Pre- and post-infusion tumor biopsies were obtained in a subset of patients to determine degree of NK cell infiltration in tumor and histopathological and immunological effects after infusion. Methods: HER2+ MBC patients underwent apheresis to harvest NK cells for ex vivo expansion and activation. NK cells (107/kg), expressing high levels of the antibody receptor CD16, were infused 24 hours post-Trastuzumab. Histology analysis and immunohistochemistry with CD56, CD3, CD20 and cleaved caspase-3 to identify NK, T, B, and apoptotic cells respectively, was performed in pre- and post-infusion biopsies. Studies of CD4 and CD8 to further classify T cell infiltrates, and CD16 to assess NK cell functionality are underway. Results: Analysis of 7 paired tumor biopsies collected before and 7-14 days after NK cell infusion showed absolute increase in lymphocyte infiltration (mean cell count/5 HPF: 204 vs 265 in pre- vs post- biopsy, p = 0.109). Most infiltrating lymphocytes were CD3+ T cells (74.28±12.72% vs 80 ±10% in pre vs post) and CD56+ NK cell number in the immune infiltrate were increased post infusion (mean cell count/5 HPF: 4.57±3.46 vs 20.57±13.83 in pre vs post; p = 0.009). Increased tumor apoptosis was observed post NK cell infusion (mean apoptotic cell count/5 HPF: 3.14±2.48 vs 5.86±6.72 in pre vs post, p = 0.27). Conclusions: Histopathology analysis of tumors from HER2+ MBC patients demonstrated significant increase in NK and T cells in tumor following Trastuzumab and NK cell infusion, suggesting that this combination might also augment recruitment of T lymphocytes into tumor, further enhancing anti-tumor activity. Clinical trial information: NCT02030561.

MRI based radiomic signature to predict treatment response to intraventricular natural killer (NK) cell infusion therapy for recurrent/refractory pediatric brain tumors.

e21511 Background: NK cell therapy is a novel immunotherapeutic strategy used in cancer therapy. We conducted first-in-human, loco-regional infusion of autologous ex-vivo expanded NK cell directly into the brain in children with recurrent medulloblastoma (MB) and ependymoma (EP). Radiomics is considered an emerging method to predict treatment response as well as evaluate prognosis and tumor milieu. This is the first study evaluating the ability of radiomics to predict response to intraventricular infusions of NK cell in children with recurrent/refractory posterior fossa malignant brain tumors. Methods: We evaluated 7 patients (5 males: 2 females; mean age 11.8 years) with refractory/recurrent MB or EP (MBs = 3, EPs = 4), who were enrolled on a Phase-1 trial (NCT02271711) and received 3 cycles of intraventricular NK cell infusions. Patients were categorized based on their clinical response into responders (N = 2) and non-responders (N = 5). After semiautomatic segmentation of the tumor, 3,660 radiomic features were extracted from the baseline (before treatment) MRI scan. The least absolute shrinkage and selection operator regression was used for feature selection, and the radiomics signature was built using the unsupervised anomaly detection algorithm. The performance of the radiomics model was assessed using leave-one-out cross-validation. Results: The radiomic signature/model was comprised of 6 radiomic features and demonstrated high discriminatory performance in predicting response to NK cell therapy with an area under the curve, sensitivity, and specificity of 100% ( P = 0.09486). In addition, patients clustered into responders and non-responders using unsupervised hierarchical clustering. Conclusions: To our knowledge, this is the first study to identify the ability of radiomics to predict those who will likely benefit from NK cell therapy. Though this small study did not attain statistical significance, the robust discriminatory results warrant larger prospective studies, evaluating radiomics as a potential tool to identify responders to NK cell therapy at diagnosis.

Fc engineered anti-CD33mAb potentiates cytotoxicity of mbIL-21 expanded NK-cells against primary AML pre-treated with decitabine

Background & Aim Natural killer (NK) cells are limited in number and require cytokines for in vivo expansion. Recipient NK cells are shown to have both qualitative and quantitative defects in acute myeloid leukemia (AML) patients, providing rationale for infusion of donor-derived NK cells. We showed that decitabine (DAC) enhances expression of NKG2D ligands in AML blasts and renders the blasts susceptible to killing by NK cells (Vasu et al. 2016). We conducted a phase I trial giving 5-day DAC in relapsed, refractory AML patients followed by haploidentical NK cells on day 0 and six doses of IL-2, where we demonstrated short term engraftment of donor-derived NK cells (up to 48 hours) can be achieved by fludarabine and DAC. Here, we report preclinical studies using membrane bound IL-21 (mbIL-21) expanded NK cells, which can be given without extraneous cytokine support. Methods, Results & Conclusion NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying mbIL-21 and 4-1BBL (CSTX002) to derive mbIL-21 NK cells. These cells have minimal senescence despite robust proliferation with the feasibility to infuse multiple NK cell doses of 1 × 108/kg (Denman et al. 2012). We investigated if mbIL-21 NK cells exhibit natural killer activity alone or in the presence of Fc-engineered anti-CD33 antibody [antibody-dependent cellular cytotoxicity (ADCC)] against primary AML samples from patients who received DAC, pre-NK cell infusion (day-1) and at 24 days post haplo-NK infusion (Post-NK infusion). Upon incubation with primary AML blasts, mbIL-21 NK cells displayed an activation profile as evidenced by surface CD107a induction (p=0.0051) and variable donor-dependent intra-cellular IFNγ production, which was further increased with CD33mAb coated AML cells (p=0.0018). ADCC assays using chromium-51 (Cr51) labelled primary AML blasts revealed mbIL-21 NK cells lysed primary AML blasts alone which was significantly enhanced with CD33mAb coated target cells (p=0.0004). Importantly, CD33mAb dependent enhanced-cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post DAC treatment. Further, in-vivo infusion of mbIL-21 NK cells in AML patient derived xenograft (AML PDX) mice, treated with CD33mAb, reduced the tumor burden. These data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of Fc engineered anti-CD33 antibody in AML.

Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells.

A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA-E in tumor cells, which suppresses natural killer (NK) cell activity via ligation of the NK inhibitory receptor CD94/NKG2A. Gene expression data from approximately 10,000 tumor samples showed widespread HLAE expression, with levels correlating with those of KLRC1 (NKG2A) and KLRD1 (CD94). To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum-retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A Protein Expression Blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E-expressing tumor cells. Transduction of anti-NKG2A PEBL produced more potent cytotoxicity than interference with an anti-NKG2A antibody and prevented de novo NKG2A expression, without affecting NK cell proliferation. In immunodeficient mice, NKG2Anull NK cells were significantly more powerful than NKG2A+ NK cells against HLA-E-expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune-checkpoint, and increases the anti-tumor activity of NK cell infusions. Because this strategy is easily adaptable to current protocols for clinical-grade immune cell processing, its clinical testing is feasible and warranted.

Overcoming Resistance to Natural Killer Cell Based Immunotherapies for Solid Tumors

Despite advances in the diagnostic and therapeutic modalities, the prognosis of several solid tumor malignancies remains poor. Different factors associated with solid tumors including a varied genetic signature, complex molecular signaling pathways, defective cross talk between the tumor cells and immune cells, hypoxic and immunosuppressive effects of tumor microenvironment result in a treatment resistant and metastatic phenotype. Over the past several years, immunotherapy has emerged as an attractive therapeutic option against multiple malignancies. The unique ability of natural killer (NK) cells to target cancer cells without antigen specificity makes them an ideal candidate for use against solid tumors. However, the outcomes of adoptive NK cell infusions into patients with solid tumors have been disappointing. Extensive studies have been done to investigate different strategies to improve the NK cell function, trafficking and tumor targeting. Use of cytokines and cytokine analogs has been well described and utilized to enhance the proliferation, stimulation and persistence of NK cells. Other techniques like blocking the human leukocyte antigen-killer cell receptors (KIR) interactions with anti-KIR monoclonal antibodies, preventing CD16 receptor shedding, increasing the expression of activating NK cell receptors like NKG2D, and use of immunocytokines and immune checkpoint inhibitors can enhance NK cell mediated cytotoxicity. Using genetically modified NK cells with chimeric antigen receptors and bispecific and trispecific NK cell engagers, NK cells can be effectively redirected to the tumor cells improving their cytotoxic potential. In this review, we have described these strategies and highlighted the need to further optimize these strategies to improve the clinical outcome of NK cell based immunotherapy against solid tumors.

In Vivo Persistence and Function of Adaptive NK Cell Infusions (FATE-NK100) from CMV Seropositive Haploidentical Related Donors

Adoptive transfer of peripheral blood natural killer (NK) cells from related haploidentical donors incubated in IL-2 persist and expand in vivo and induce clinical responses in patients with AML or ovarian cancer. Individuals who have been exposed to human cytomegalovirus (CMV) have a unique subset of CD57+/NKG2C+ NK cells that are long lived with properties of immunologic memory, potent mediators of antibody dependent cellular cytotoxicity (ADCC), and resistant to tumor suppressive mechanisms. We developed a 7 day culture process (including GSK3b inhibition and IL-15) to produce a purified adaptive NK cell product (FATE-NK100) from CMV seropositive related haploidentical donors. We are testing this product in two phase 1 clinical trials, one for refractory ovarian cancer (NCT03213964) and one for refractory AML (NCT03050216). After intermediate dose Cy/Flu, 4 patients with ovarian cancer have safely received FATE-NK100 cells delivered intraperitoneally with 6 doses of 6 million units of IL-2 given every other day. Ascites samples demonstrate persistence of the donor NK cell product based on flow cytometric analysis of donor vs. recipient HLA on NK cells. At dose cohorts 2 and 3 (maximum), all subjects had intraperitoneal NK cells of donor origin at Day 5-7 (60-90%), persisting at Day 9 (10-40%) and up to 21 days after infusion in one patient. Importantly, functional assays of the in vivo samples from the patients demonstrate high proliferation based on Ki67 and superior function compared to endogenous patient NK cells based on CD107a degranulation and IFNg production when stimulated with K562 (Figure 1). Clinical activity has been observed and the patient with NK persistence at Day 21 had stable disease and was eligible for re-treatment. The in vivo persistence and function of the FATE-NK100 product was maintained after redosing. Similarly, in 4 evaluable patients with AML treated after higher dose Cy/Flu with IV FATE-NK100 and 6 doses of subcutaneous IL-2 6mu, NK cells of donor origin were detected in the peripheral blood at Day 7 (55-100%), persisting up to 28 days in one patient. All patients at dose cohort 2 achieved clearance of their refractory AML at Day 14 (morphologic leukemia free state) and we await enrollment to the 3rd (maximum) dose cohort (3-10 × 107 NC/kg). In summary, FATE-NK100 cell infusions persist in vivo in both the peritoneum and peripheral blood and exhibit potent functional activity.