Adjuvant therapy using ex vivo-expanded allogenic natural killer cells in hepatectomy patients with hepatitis B virus related solitary hepatocellular carcinoma: MG4101 study.

BACKGROUNDS/AIMS: Fewer reports have been published regarding hepatectomy patients with solitary hepatocellular carcinoma (HCC) who received immunotherapeutic agents as adjuvant therapy. We evaluated the safety and efficacy of ex vivo-expanded allogenic natural killer (NK) cells in those patients with modified International Union Against Cancer (UICC) stage T3. METHODS: From August 2014 to October 2015, five patients who underwent hepatic resection received ex vivo-expanded allogenic NK cells. Patients received five rounds of NK cells (2-3×107 cells/kg) at postoperative 4, 6, 8, 12, and 16 weeks. This study is registered with ClinicalTrials.gov, number NCT02008929. RESULTS: The median age of the five patients (three men and two women) was 44.8 years (range, 36-54 years). All had hepatitis B virus-related HCC, and the median tumor size was 2.2 cm (range, 2.1-8.2 cm). None of the patients had any adverse events. HCC recurrence developed in two patients at one year after hepatic resection, but four patients were alive at 3 years. The two recurrence-free patients showed a higher ratio of CD8+ T lymphocyte populations before and after administration of ex vivo-expanded allogenic NK cells compared with the three patients who experienced recurrence. CONCLUSIONS: Immunotherapy using ex vivo-expanded allogenic NK cells in hepatectomy patients can be used safely. Further studies should be investigated for efficacy.

Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy.

As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell's HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.

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

INTRODUCTION: Under 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. METHODS: We 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. RESULTS: Seven 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. CONCLUSION: NKI 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.

Phase I Study of Random Healthy Donor-Derived Allogeneic Natural Killer Cell Therapy in Patients with Malignant Lymphoma or Advanced Solid Tumors.

Natural killer (NK) cells with mismatched killer cell immunoglobulin-like receptor-ligand pairs have shown efficacy and been proven safe in treatment of cancer patients. Ex vivo-expanded and highly activated NK cells (MG4101) had been generated under good manufacturing practice conditions, which demonstrated potent anticancer activity in vitro and in vivo in preclinical studies. The current phase I clinical trial was designed to evaluate safety and possible clinical efficacy of repetitive administrations of MG4101 derived from random unrelated healthy donors into patients with malignant lymphoma or advanced, recurrent solid tumors. The maximum dose (3 × 10(7) cells/kg, triple infusion) was tolerable without significant adverse events. Of 17 evaluable patients, 8 patients (47.1%) showed stable disease and 9 (52.9%) showed progressive disease. We also evaluated the capacity of MG4101 to influence host immune responses. Administration of MG4101 augmented NKG2D expression on CD8(+) T cells and upregulated chemokines that recruit T cells. In contrast, administration of MG4101 reduced regulatory T cells and myeloid-derived suppressor cells and suppressed TGFß production. In conclusion, administration of a large number of MG4101 cells was not only safe and feasible, but also exhibited efficacy in maintaining the effector arm of the host immune response.

Present and Future of Allogeneic Natural Killer Cell Therapy.

Natural killer (NK) cells are innate lymphocytes that are capable of eliminating tumor cells and are therefore used for cancer therapy. Although many early investigators used autologous NK cells, including lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation revealed the antitumor effect of allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor ligand-mismatched allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells from non-HLA-related healthy donors have been recently used in cancer therapy. The use of allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK cells with higher flexibility and to prepare expanded, cryopreserved NK cells for instant administration without delay for ex vivo expansion. In cancer therapy with allogeneic NK cells, optimal matching of donors and recipients is important to maximize the efficacy of the therapy. In this review, we summarize the present state of allogeneic NK cell therapy and its future directions.

GMP-compliant, large-scale expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells in vitro and in vivo.

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3(+) T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3(-)CD16(+)CD56(+) NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.