Allogeneic Natural Killer Research Articles

Boosting Natural Killer Cell Therapies in Glioblastoma Multiforme Using Supramolecular Cationic Inhibitors of Heat Shock Protein 90.

Allogeneic natural killer (aNK) cell adoptive therapy has the potential to dramatically impact clinical outcomes of glioblastoma multiforme (GBM). However, in order to exert therapeutic activity, NK cells require tumor expression of ligands for activating receptors, such as MHC Class I peptide A/B (MICA/B) and ULBPs. Here, we describe the use of a blood–brain barrier (BBB) permissive supramolecular cationic drug vehicle comprising an inhibitor of the chaperone heat shock protein 90 (Hsp90), which sustains a cytotoxic effect on GBM cells, boosts the expression of MICA/B and ULBPs on the residual population, and augments the activity of clinical-grade aNK cells (GTA002). First, we identify Hsp90 mRNA transcription and gain of function as significantly upregulated in GBM compared to other central nervous system tumors. Through a rational chemical design, we optimize a radicicol supramolecular prodrug containing cationic excipients, SCI-101, which displays >2-fold increase in relative BBB penetration compared to less cationic formulations in organoids, in vitro. Using 2D and 3D biological models, we confirm SCI-101 sustains GBM cytotoxicity 72 h after drug removal and induces cell surface MICA/B protein and ULBP mRNA up to 200% in residual tumor cells compared to the naked drug alone without augmenting the shedding of MICA/B, in vitro. Finally, we generate and test the sequential administration of SCI-101 with a clinical aNK cell therapy, GTA002, differentiated and expanded from healthy umbilical cord blood CD34+ hematopoietic stem cells. Using a longitudinal in vitro model, we demonstrate >350% relative cell killing is achieved in SCI-101–treated cell lines compared to vehicle controls. In summary, these data provide a first-of-its-kind BBB-penetrating, long-acting inhibitor of Hsp90 with monotherapy efficacy, which improves response to aNK cells and thus may rapidly alter the treatment paradigm for patients with GBM.

Efficient Targeting of CD38 in Mature T-Cell Neoplasms with Daratumumab and Allogeneic NK Cells

Introduction: Mature T-cell neoplasms (MTCN) are a heterogeneous and aggressive group of lymphoid neoplasms with very limited options for precision immunotherapy. Targeted immunotherapy with antibodies directed against surface markers on tumor cells has emerged as an effective treatment for B-cell neoplasms, but the development of immunotherapy strategies for MTCN has been much slower. CD38 is expressed at low levels in a subset of normal resting T-cells. Data on the frequency and level of CD38 expression is MTCN are lacking. Our goal was to study the expression and stability of CD38 on a spectrum of neoplastic T-cell populations and assess the potential anti-tumor effect of anti-CD38 monoclonal antibodies in combination with allogeneic natural killer (NK) cells in MTCN.Methods and results: We searched the Thomas Jefferson University Hospital (TJUH) pathology records for all cases of MTCN for which immune-phenotypical characterization of the neoplastic T-cell population by multi-color flow cytometry (FC) was available. CD38 expression was evaluated in cases where an abnormal T-cell population (defined as loss of one on more pan T-cell markers and/or a skewed CD4 to CD8 ratio). A total of 103 unique patients with MTCN were identified. Of these, 51 had at least one biopsy whereby tumor cells had an abnormal immunophenotype which could then be assessed for CD38 expression. CD38 was expressed to some extent in all but 2 cases with expression levels on peripheral T-cell lymphoma-not other specified (PTCL-NOS)(% Mean±SEM = 80.84±10.26, N=11), angioimmunoblastic T-cell lymphoma (AITL) (% Mean±SEM = 80.56±7.34, N=6), nodal PTCL with T follicular-helper (T FH) phenotype (% Mean±SEM = 55.00±12.72, N=7), anaplastic large cell lymphoma (ALCL) (% Mean±SEM = 77.38±10.75, N=3), large granular lymphocytic leukemia (LGLL) (% Mean±SEM = 80.27±7.49, N=4), T-cell prolymphocytic leukemia (T-PLL) (% Mean±SEM = 88.26±4.20, N=7), cutaneous T-cell lymphoma (CTCL) (% Mean±SEM = 49.52±14.77, N=7), adult T‐cell leukemia/lymphoma (ATLL) (% Mean±SEM = 76.68±10.45, N=3), hepatosplenic T-cell lymphoma/monomorphic epitheliotropic intestinal T-cell lymphoma (HSTCL/MEITL) (% Mean±SEM = 75.00±11.37, N=3). The medians and ranges of the MFI of CD38 on CD38+ tumor cells were the following: PTCL-NOS 29.46 (3.1-115.62), AITL 13.64 (2.67-29.41), other PTCL-T FH 5.87 (0-48.67), ALCL 11.13 (3.6-11.28), LGL (13.52 (9.72-18.25), T-PLL 6.49 (3.09-18), CTCL 8.36 (0-116.75), ATLL 27.17 (3.54-60.5), HSTCL/MEITL 15.96 (6.96-167.85). We also measured surface expression of CD38 on the patient-derived MTCN cell lines HuT-78, HuT-102, Jurkat, H9, HH, and MOTN1, all of which expressed CD38, to determine which of these cell lines could be used for in vitro experiments. We next evaluated if the CD38 molecule is an effective target for antibody-mediated therapy in MTCN, by testing the ability of daratumumab (dara) to enhance antibody-dependent cellular cytotoxicity (ADCC) elicited by NK cells. For this, we purified normal NK-cells from TJUH Blood Bank leukoreduction filters and cultured with recombinant IL-15 for 48 hours prior to all experiment. T-cell lines and primary MTCN cells were treated with increasing concentrations (0.1 µg/mL - 2 µg/mL) of dara or isotype control. NK-cells were added at Effector:Target ratio of 5:1 and incubated for 4 hours at 37⁰ Celsius. Cytotoxicity was measured by LDH release assay. Dara induced significant cell lysis starting at doses as low as 0.1μg/mL in both T-cell lines and primary MTCN cells, reaching maximum cytotoxicity at 0.5-2μg/mL (mean±SEM cytotoxicity in isotype vs dara treated cells= 50.0±5.05% vs 97.5±2.5%, N=4, p-value=0.0002). The degree of ADCC induction also correlated with interferon-gamma (IFN-g) release by NK cells in vitro for both T-cell lines and primary MTCN cells.Conclusions: The majority of MTCN analyzed (N=49, 96%) showed any degree of CD38 expression by FC with a wide variation of intensity, including within the same subtype. Allogeneic NK cells efficiently elicited dara-mediated ADCC of tumor cells from all MTCN subtypes and produced abundant IFN-g. These data highlight the potential of targeting CD38 in MTCN with anti-CD38 antibodies and allogeneic NK cells. The strong CD38 expression observed in most tumor cells from ultra-rare and very aggressive subtypes of MTCL opens the door to much needed new treatment strategies. DisclosuresBrammer: Celgene: Research Funding; Kymera Therapeutics: Consultancy; Seattle Genetics: Speakers Bureau. Chakravarti: Kiadis Pharma: Patents & Royalties. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy.

A Pilot Study of MG4101, Allogeneic Natural Killer Cell, in Patients with Relapsed or Refractory Acute Myeloid Leukemia

Background: Prognosis of relapsed or refractory acute myleoid leukemia (AML) is dismal. Several studies demonstrated that allogeneic natural killer (NK) cells have potent anti-tumor effect in AML. In this study, we aimed to investigate the safety and efficacy of allogeneic, ex vivo expanded killer-cell immunoglobulin-like receptor (KIR) B haplotype NK cells (MG4101) in refractory or relapsed AML and to elucidate the association between immunologic characteristics and clinical response.Methods: Patients were scheduled to receive MG4101 for a total of 2 cycles for 28 days in each cycle. After lymphodepletion with fludarabine and cyclophosphamide, MG4101 derived from unrelated healthy donors was administered intravenously for 3 days during the first 3 weeks of the treatment cycle followed by IL-2 for 3 days. Throughout the entire treatment schedule, it was aimed to deliver a total of 2.0 - 5.0 x 10 9 cells to each patient based on actual body weight. NK cell receptor ligand (NKRL) in AML blast and KIR typing of donors and recipients were evaluated and analyzed with the clinical response of the patients.Results: Between Apr 2018 to Feb 2020, a total of 11 patients (male, n=5) with refractory or relapsed AML patients were conesecutively enrolled. 5 patients (45.5%) were intermediate risk group, and 6 patients (54.5%) were adverse risk group. Three patients had antecedent hematologic malignancies (atypical chronic myeloid leukemia [n=1], myelodysplastic syndrome [n=2]). Of 7 evaluable patients, 2 patients (28.6%) showed partial response (PR) and 2 patients (28.6%) showed stable disease (SD). Median overall survival of entire patients was 3.4 months (95% confidence interval, 2.5 - 4.3 months): 3.7 months in responders (PR or SD) and 1.0 months in nonresponders. There was only 1 grade 4 infusion-related hypersensitivity reaction, which resolved with supportive care, and there was no cytokine release syndrome, or any sign of graft-versus-host disease associated with MG4101 treatment.NKRL expression varied widely among the patients treated with MG4101. In patients with favorable clinical outcome, ULBP3, activating NKRL for NKG2D receptor, was highly expressed or increased with treatment. PD-L1 expression, in contrast, was maintained relatively low until the end of the treatment. Besides, activating KIR and its cognate ligand interaction were associated with treatment response. Among the responders including PR and SD, the sum of activating KIR tended to be higher than non-responders.Conclusions: MG4101 in refractory or relapsed AML was well tolerated without significant treatment-related toxicity. NKRL expression in AML blasts was heterogeneous, and treatment response with MG4101 was also highly variable. In our patient cohort, patients with high activating KIR responded favorably to MG4101 treatment. These findings suggests further investigation of MG4101 in patients in relapsed or refractory AML and highlights the importance of immunologic mechanism in treating myeloid neoplasm. [Display omitted] DisclosuresKoh: Pfizer: Consultancy; Jassen: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; GSK: Honoraria; Roche: Honoraria; Takeda: Honoraria. Kim: Seoul National University Hospital: Current Employment.

Phase I Study of FT516, an Off-the-Shelf iPSC-Derived NK Cell Therapy, in Combination with Rituximab in Patients with Relapsed/Refractory B-Cell Lymphoma

Phase I Study of FT516, an Off-the-Shelf iPSC-Derived NK Cell Therapy, in Combination with Rituximab in Patients with Relapsed/Refractory B-Cell Lymphoma

Gda-201, a Novel Metabolically Enhanced Allogeneic Natural Killer (NK) Cell Product Yields High Remission Rates in Patients with Relapsed/Refractory Non-Hodgkin Lymphoma (NHL): 2-Year Survival and Correlation with Cytokine IL7

Gda-201, a Novel Metabolically Enhanced Allogeneic Natural Killer (NK) Cell Product Yields High Remission Rates in Patients with Relapsed/Refractory Non-Hodgkin Lymphoma (NHL): 2-Year Survival and Correlation with Cytokine IL7

PiggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells

Promising progress has been made in adoptive transfer of allogeneic natural killer (NK) cells to treat relapsed or refractory acute myeloid leukemia (AML). In this regard, chimeric antigen receptor (CAR)-modification of NK cells is considered as a compelling approach to augment the specificity and cytotoxicity of NK cells against AML. Using a non-viral piggyBac transposon technology and human peripheral blood-derived primary NK cells, we generated CAR-NK cells to target NKG2D ligands and demonstrated their in vitro activity in lysing cancer cells expressing the ligands and in vivo efficacy in inhibiting tumor growth in a xenograft KG-1 AML model. We further generated CAR-NK cells co-expressing transgenes for the NKG2D CAR and interleukin-15 (IL-15). The ectopic expression of IL-15 improved the in vitro and in vivo persistence of NKG2D CAR-NK cells, leading to enhanced in vivo tumor control and significant prolongation of mouse survival in the KG-1 AML model. Collectively, our findings demonstrate the ectopic expression of IL-15 as an important means to improve the antileukemic activity of NKG2D CAR-NK cells. Our study further illustrates the feasibility of using the piggyBac non-viral platform as an efficient and cost-effective way for CAR-NK cell manufacturing.

Ex vivo expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells through different receptor-ligand interactions

BackgroundRecently, allogeneic natural killer (NK) cells have gained considerable attention as promising immunotherapeutic tools due to their unique biological functions and characteristics. Although many NK expansion strategies have been reported previously, a deeper understanding of cryopreserved allogeneic NK cells is needed for specific therapeutic approaches.MethodsWe isolated CD3−CD56+ primary natural killer (pNK) cells from healthy donors and expanded them ex vivo using a GMP-compliant method without any feeder to generate large volumes of therapeutic pNK cells and cryopreserved stocks. After validation for high purity and activating phenotypes, we performed RNA sequencing of the expanded and cryopreserved pNK cells. The pNK cells were used against various cancer cell lines in 7-AAD/CFSE cytotoxicity assay. For in vivo efficacy study, NSG mice bearing subcutaneous cisplatin-resistant A2780cis xenografts were treated with our pNK cells or cisplatin. Antitumor efficacy was assessed by measuring tumor volume and weight.ResultsCompared to the pNK cells before expansion, pNK cells after expansion showed 2855 upregulated genes, including genes related to NK cell activation, cytotoxicity, chemokines, anti-apoptosis, and proliferation. Additionally, the pNK cells showed potent cytolytic activity against various cancer cell lines. Interestingly, our activated pNK cells showed a marked increase in NKp44 (1064-fold), CD40L (12,018-fold), and CCR5 (49-fold), and did not express the programmed cell death protein 1(PD-1). We also demonstrated the in vitro and in vivo efficacies of pNK cells against cisplatin-resistant A2780cis ovarian cancer cells having a high programmed death-ligand 1(PD-L1) and low HLA-C expression.ConclusionsTaken together, our study provides the first comprehensive genome wide analysis of ex vivo-expanded cryopreserved pNK cells. It also indicates the potential use of expanded and cryopreserved pNK cells as a highly promising immunotherapy for anti-cancer drug resistant patients.

Advances in immune therapies in hematological malignancies.

Immunotherapy in cancer takes advantage of the exquisite specificity, potency, and flexibility of the immune system to eliminate alien tumor cells. It involves strategies to activate the entire immune defense, by unlocking mechanisms developed by tumor cells to escape from surrounding immune cells, as well as engineered antibody and cellular therapies. What is important to note is that these are therapeutics with curative potential. The earliest example of immune therapy is allogeneic stem cell transplantation, introduced in 1957, which is still an important modality in hematology, most notably in myeloid malignancies. In this review, we discuss developmental trends of immunotherapy in hematological malignancies, focusing on some of the strategies that we believe will have the most impact on future clinical practice in this field. In particular, we delineate novel developments for therapies that have already been introduced into the clinic, such as immune checkpoint inhibition and chimeric antigen receptor T-cell therapies. Finally, we discuss the therapeutic potential of emerging strategies based on T-cell receptors and adoptive transfer of allogeneic natural killer cells.

Mutant B2M-HLA-E and B2M-HLA-G fusion proteins protects universal chimeric antigen receptor-modified T cells from allogeneic NK cell-mediated lysis.

Recent studies have indicated the antitumor activity and reduced allogeneic response of universal chimeric antigen receptor‐modified T (UCAR T) cells lacking endogenous T cell receptors and beta‐2 microglobulin (B2M) generated using gene‐editing technologies. However, these cells are vulnerable to lysis by allogeneic natural killer (NK) cells due to their lack of human leukocyte antigen (HLA) class I molecule expression. Here, constitutive expression of mutant B2M‐HLA‐E (mBE) and B2M‐HLA‐G (mBG) fusion proteins in anti‐CD19 UCAR T (UCAR T‐19) cells was conducted to protect against allogeneic NK cell‐mediated lysis. The ability of cells expressing mBE or mBG to resist NK cell‐mediated lysis was observed in gene‐edited Jurkat CAR19 cells. UCAR T‐19 cells constitutively expressing the mBE and mBG fusion proteins were manufactured and showed effective and specific anti‐tumor activity. Constitutive expression of the mBE and mBG fusion proteins in UCAR T‐19 cells prevented allogeneic NK cell‐mediated lysis. In addition, these cells were not recognizable by allogeneic T cells. Additional experiments, including those in animal models and clinical trials, are required to evaluate the safety and efficacy of UCAR T‐19 cells that constitutively express mBE and mBG.

Allogeneic CAR Invariant Natural Killer T Cells Exert Potent Antitumor Effects through Host CD8 T-Cell Cross-Priming.

The development of allogeneic chimeric antigen receptor (CAR) T-cell therapies for off-the-shelf use is a major goal that faces two main immunologic challenges, namely the risk of graft-versus-host disease (GvHD) induction by the transferred cells and the rejection by the host immune system limiting their persistence. In this work we assessed the direct and indirect antitumor effect of allogeneic CAR-engineered invariant natural killer T (iNKT) cells, a cell population without GvHD-induction potential that displays immunomodulatory properties. After assessing murine CAR iNKT cells direct antitumor effects in vitro and in vivo, we employed an immunocompetent mouse model of B-cell lymphoma to assess the interaction between allogeneic CAR iNKT cells and endogenous immune cells. We demonstrate that allogeneic CAR iNKT cells exerted potent direct and indirect antitumor activity when administered across major MHC barriers by inducing tumor-specific antitumor immunity through host CD8 T-cell cross-priming. In addition to their known direct cytotoxic effect, allogeneic CAR iNKT cells induce host CD8 T-cell antitumor responses, resulting in a potent antitumor effect lasting longer than the physical persistence of the allogeneic cells. The utilization of off-the-shelf allogeneic CAR iNKT cells could meet significant unmet needs in the clinic.

Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-β activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-β signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-β axis as a potentially useful therapeutic target in GBM.

Genome-scale screens identify factors regulating tumor cell responses to natural killer cells.

To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated 'DNA-barcoded' solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell-sensitive tumor cells tend to exhibit 'mesenchymal-like' transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies.

A 11-Year Clinical Summary of DC - CIK / NK Cell Immunotherapy for 152 Patients with Acute Myeloid Leukemia

Objective: To assess the efficacy of immunotherapy based on autologous dendritic cells-cytokine induced killer cells (DC-CIK) and allogeneic natural killer cells (NK) in treating low-risk and intermediate-risk acute myeloid leukemia (AML). The study was conducted over a period of 11 years (April 1, 2006 to April 1, 2017) across the Beijing Ludaopei hospital systems.Methods: DC-CIK cells were derived by culturing patients' peripheral blood mononuclear cells (PBMCs) in vitro with cytokines for 8-12 days. To generate NK cells, allogenic PBMCs were cultured for 3 to 6 days. After patients completed intensified chemotherapy consisting of 4 cycles of high-dose Ara-C within 6 months, DC-CIK or NK cells were infused and given once every 3 months for 2-4 cycles along with chemotherapy of Fludarabine, Cyclophosphamide and Ara-C given few days prior to each cycle of immunotherapy.Results: From April 1, 2006 to April 1, 2017, a total 152 patients in the low and intermediate risk groups of AML (except APL) underwent combined immunotherapy and chemotherapy in our center. Overall survival (OS) rate was 82% and disease-free survival (DFS) rate was 67% for the cohort. Beginning in June 2012, the low and intermediate risk groups were further stratified into very-good risk, good risk, and intermediate risk groups according to the specific AML related gene mutations. The OS rate of the 2012-2017 group was significantly better than that of the 2006-2011 group (91.7% vs. 71.6% p= 0.003). For patients treated prior to 2012, there were no significant differences between the low and intermediate risk group in OS (72.2% vs 71.4%) and DFS (72.2% vs 61.2%). From 2012 to 2017, the OS rates were 94.4%, 86.3%, and 93.3%, and the DFS rates were 83.3%, 81.8%, and 62.2%, (p=0.15) for the very-good risk, good-risk & intermediate risk groups, respectively, again suggesting no statistical differences among the groups. Side effects were mild with some fever, chills and fatigue.Patients who were treated with 2-4 cycles of immunotherapy were further divided into the DC-CIK alone group and the DC-CIK alternating with NK group. Importantly, the OS and DFS rates of 67 patients in the DC-CIK alternating with NK group were significantly better than those of 53 patients in the DC-CIK alone group (OS 95.5% vs 71.4%, p=0.0003), (DFS 85% vs 63.5%, p =0.0099). .Twenty-nine of 48 relapsed patients from both groups underwent allo-hematopoietic stem cell transplantation (allo-HSCT). The long-term OS after HSCT was 65.5%, closed to the OS of AML patients who transplanted in their first complete remission (CR) in our center. OS of 12 patients transplanted after 2012 was significantly better than that of 17 patients transplanted before 2012 (88.2% vs. 33.3%, p = 0.015). This because that after 2012, all patients have been checked minimal residual disease (MRD) by flow cytometry. Once patient was found to have relapse with MRD,immediately pursued HSCT while some patients transplanted prior to 2012 had morphological relapse.Conclusion: When combined with chemotherapy, DC-CIK/NK cells-based immunotherapy significantly improves the long-term OS and DFS rates of good and intermediate risk groups of AML. Auto DC-CIK alternating with allo NK is superior to auto DC-CIK alone. Relapsed patients could be safely and effectively treated with HSCT. If relapse patients with only positive MRD, then the outcome of HSCT will be comparable to those who received the HSCT during their first CR.Figure 1. OS of 152 patients treated with DC-CIK/NK immunotherapy from 2006 to 2017.Figure 2, OS of 67 patients treated from 2006 to 2012 vs 85 patients from 2012 to 2017 (71.6% vs 91.7%, p= 0.003).Figure 3, OS & DFS of DC-CIK+NK vs DC-CIK treated groups from 2012 to 2017 (p= 0.0003, p= 0.0099) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

Backgrounds/AimsFewer 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.MethodsFrom 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.ResultsThe 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.ConclusionsImmunotherapy using ex vivo-expanded allogenic NK cells in hepatectomy patients can be used safely. Further studies should be investigated for efficacy.

Selection, Expansion, and Unique Pretreatment of Allogeneic Human Natural Killer Cells with Anti-CD38 Monoclonal Antibody for Efficient Multiple Myeloma Treatment.

Cellular immunotherapy is becoming a new pillar in cancer treatment after recent striking results in different clinical trials with chimeric antigen receptor T cells. However, this innovative therapy is not exempt from challenges such as off-tumor toxicity, tumor recurrence in heterogeneous tumors, and affordability. To surpass these limitations, we exploit the unique anti-tumor characteristics of natural killer (NK) cells. In this study, we aimed to obtain a clinically relevant number of allogeneic NK cells derived from peripheral blood (median of 14,050 million cells from a single donor) to target a broad spectrum of solid and liquid tumor types. To boost their anti-tumor activity, we combined allogeneic NK cells with the approved anti-cluster of differentiation 38 (CD-38) monoclonal antibody Daratumumab to obtain a synergistic therapeutic effect against incurable multiple myeloma. The combination therapy was refined with CD16 polymorphism donor selection and uncomplicated novel in vitro pretreatment to avoid undesired fratricide, increasing the in vitro therapeutic effect against the CD-38 positive multiple myeloma cell line by more than 20%. Time-lapse imaging of mice with established human multiple myeloma xenografts revealed that combination therapy of selected and pretreated NK cells with Daratumumab presented tumor volumes 43-fold smaller than control ones. Combination therapy with an allogeneic source of fully functional NK cells could be beneficial in future clinical settings to circumvent monoclonal antibodies’ low therapeutic efficiency due to NK cell dysfunctionality in MM patients.

CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide.

There is a strong biological rationale for the augmentation of allogeneic natural killer (NK) cell therapies with a chimeric antigen receptor (CAR) to enhance acute myeloid leukemia (AML) targeting. CD38 is an established immunotherapeutic target in multiple myeloma and under investigation as a target antigen in AML. CD38 expression on NK cells and its further induction during ex vivo NK cell expansion represent barriers to the development of a CD38 CAR-NK cell therapy. We set out to develop a CD38 CAR-NK cell therapy for AML, first by using an NK cell line which has low baseline CD38 expression and subsequently NK cells expanded from healthy donors. To overcome anticipated fratricide due to NK cell CD38 expression when using primary expanded NK cells, we applied CRISPR/Cas9 genome editing to disrupt the CD38 gene during expansion, achieving a mean knockdown efficiency of 84%. The resulting CD38 knockdown expanded NK cells, after expression of an affinity optimized CD38 CAR, showed reduced NK-cell fratricide and an enhanced ability to target primary AML blasts. Furthermore, the cytotoxic potential of CD38 CAR-NK cells was augmented by pretreatment of the AML cells with all-trans retinoic acid which drove enhanced CD38 expression, offering a rational combination therapy. These findings support the further investigation of CD38 knockdown - CD38 CAR-NK cells as a viable immunotherapeutic approach to the treatment of AML.

Safety and efficacy of allogeneic natural killer cell immunotherapy on human immunodeficiency virus type 1 immunological non-responders: a brief report.

Background:Allogeneic natural killer (NK) cell immunotherapy is recognized as a promising anti-tumor strategy, but whether it plays a role in poor CD4 recovery among human immunodeficiency virus type 1 (HIV-1) infected patients is unknown. This study aimed to investigate the safety and effectiveness of allogeneic NK cells immunotherapy on HIV-1 immunological non-responders (INRs) receiving antiretroviral therapy (ART).Methods:From February to April 2018, a prospective, randomized, controlled, open-label clinical trial, which enrolled 20 HIV-1 INRs following specific inclusion criteria, was conducted at Nankai University Second People's Hospital. Participants were randomly allocated (simple randomization 1:1) to either the combined treatment (NK + ART) group (n = 10) or the control (ART) group (n = 10). The allogenic highly activated NK cells from killer cell immunoglobulin-like receptor (KIR)/human leukocyte antigen (HLA)-Cw mismatched healthy donor were prepared (108 cells in each injection) and intravenously infused to each recruited patient of NK+ART group in three courses. Key immune parameters (CD4 count, CD8 count, CD4/CD8 ratio), laboratory tests (count of blood cells, biochemistry panel) and symptoms at baseline and at month 1, 3, 6, 9, 12, and 24 were measured/collected to analyze the safety and efficacy of the therapy. Comparisons were between the seven time-points of both groups using repeated measurement analysis of variance (ANOVA) test. Generalized estimating equations (GEE) model was performed to evaluate the overall effect of the NK+ART group vs. the ART group.Results:From baseline to 24 months, we noted a mean CD4 count augmentation (139 to 243 cells/μL) in the NK + ART group and (144 to 176 cells/μL) in the ART group (difference, 67; 95% CI, 10 to 124; P = 0.024). Our estimations revealed that NK+ART group could improve CD4 level (β = 54.59, P = 0.006) and CD8 level (β = 322.47, P = 0.010) on average among the six measurements compared with the ART group. Only two (2/10, 20%) participants in the NK+ART group developed a transient mild fever after the first course.Conclusions:This preliminary study informs that HIV-1 INRs, allogenic NK cells immunotherapy is safe and could significantly improve CD4 recovery but not CD4/CD8 ratio. The practical effects, however, need long-term follow-up observations. Further study on the potential underlying mechanism is warranted.Registration info:www.chictr.org.cn/showproj.aspx?proj=34912 (No. ChiCTR1900020634).

Expanded and activated allogeneic NK cells are cytotoxic against B-chronic lymphocytic leukemia (B-CLL) cells with sporadic cases of resistance

Adoptive transfer of allogeneic natural killer (NK) cells is becoming a credible immunotherapy for hematological malignancies. In the present work, using an optimized expansion/activation protocol of human NK cells, we generate expanded NK cells (eNK) with increased expression of CD56 and NKp44, while maintaining that of CD16. These eNK cells exerted significant cytotoxicity against cells from 34 B-CLL patients, with only 1 sample exhibiting resistance. This sporadic resistance did not correlate with match between KIR ligands expressed by the eNK cells and the leukemic cells, while cells with match resulted sensitive to eNK cells. This suggests that KIR mismatch is not relevant when expanded NK cells are used as effectors. In addition, we found two examples of de novo resistance to eNK cell cytotoxicity during the clinical course of the disease. Resistance correlated with KIR-ligand match in one of the patients, but not in the other, and was associated with a significant increase in PD-L1 expression in the cells from both patients. Treatment of one of these patients with idelalisib correlated with the loss of PD-L1 expression and with re-sensitization to eNK cytotoxicity. We confirmed the idelalisib-induced decrease in PD-L1 expression in the B-CLL cell line Mec1 and in cultured cells from B-CLL patients. As a main conclusion, our results reinforce the feasibility of using expanded and activated allogeneic NK cells in the treatment of B-CLL.

A Phase 1 Study of NKX101, an Allogeneic CAR Natural Killer (NK) Cell Therapy, in Subjects with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) or Higher-Risk Myelodysplastic Syndrome (MDS)

Background: Patients with R/R AML, or intermediate-, high-, and very high-risk MDS have a poor prognosis with, respectively, a 5-year survival of <10%, or median overall survival of 0.8 to 3.0 years (https://seer.cancer.gov/, Greenberg 2012). Novel chimeric antigen receptor (CAR) T cell products have had limited clinical success in patients with myeloid disorders due to absence of clear AML-specific target antigens and a hostile microenvironment for T cell-based therapy (Gill 2019, Epperly 2020). Non-engineered allogeneic haplomatched NK cells have shown activity in AML, and are therefore an attractive cell source to develop novel targeted cellular therapies to treat AML/MDS. Complete remission with or without hematologic recovery from 20% to 50% has been noted in patients with R/R AML with non-engineered, allogeneic haplomatched NK cells administered after lymphodepletion with minimal cytokine release syndrome or neurotoxicity (Miller 2005, Bachanova 2014, Rubnitz 2015, Romee 2016). The cytolytic activity of NK cells arises from a balance of activating and inhibitory receptors; key among the activating receptors is NKG2D. NKX101 is composed of CAR NK cells engineered to express a chimeric NKG2D receptor fused to co-stimulatory (OX40) and signaling (CD3ζ) domains to enhance their intrinsic anti-AML activity. NKX101 also expresses a membrane-bound interleukin-15 to serve as an autocrine growth factor and thereby increase persistence. Preclinical characterization showed that NKX101 has 4- to 8-fold greater cytotoxicity, superior intrinsic longevity, and enhanced anti-tumor activity in AML xenograft models relative to non-engineered NK cells. Hence clinical evaluation of NKX101 is being undertaken in this Phase 1 study in subjects with R/R AML or higher-risk MDS. Methods: This is a multicenter, open-label, Phase 1 study of NKX101 (Figure). The study will be conducted in 2 parts: Part 1 (dose finding) to determine the recommended Phase 2 dose (RP2D) of NKX101 utilizing a modified "3+3" enrollment schema. The study will enroll subjects with either R/R primary or secondary AML or R/R higher-risk MDS. Building on existing clinical data referenced above with non-engineered haplomatched NK cells, NKX101 manufactured from NK cells obtained from haplo-identical-related donors (H) will be used in Part 1. Part 2 (dose expansion) will further evaluate safety and tolerability, pharmacokinetics (PK), immunogenicity, pharmacodynamics (PDn), and anti-tumor activity of NKX101 from unrelated, HLA-mismatched donors with separate expansion cohorts for patients with AML or MDS. Each dose is defined by the total number of viable CAR NK cells. The starting dose of NKX101 is 1 × 108 viable CAR NK cells where NKX101 will be administered on Days 0, 7, and 14 of a 28-day cycle following standard fludarabine/cyclophosphamide lymphodepletion. Three dose levels are planned (Table). The primary endpoint is incidence of adverse events, dose-limiting toxicities, clinically significant laboratory abnormalities, and determination of the RP2D. Secondary endpoints include evaluation of standard cellular PK parameters, PDn, immunogenicity, and anti-tumor responses. AML patients will be assessed for efficacy using updated ELN criteria (Döhner 2017) including measurable residual disease assessment, and MDS using modified IWG criteria (Cheson 2006). Exploratory endpoints are correlation of various degrees of HLA and KIR ligand match/mismatch between donor and recipient with primary and secondary safety, PK, and efficacy measures. Enrollment across multiple US sites is expected to start in late 2020. Disclosures Bachier: CRISPR: Honoraria; Juno Therapeutics, a Bristol-Myers Squibb Company: Honoraria; AlloVir: Honoraria; Sanofi: Speakers Bureau. Borthakur:Novartis: Research Funding; FTC Therapeutics: Consultancy; Curio Science LLC: Consultancy; BioTherix: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; Incyte: Research Funding; AstraZeneca: Research Funding; Cyclacel: Research Funding; Argenx: Consultancy; Abbvie: Research Funding; Jannsen: Research Funding; PTC Therapeutics: Research Funding; GSK: Research Funding; Oncoceutics: Research Funding; Xbiotech USA: Research Funding; BioLine Rx: Research Funding; Polaris: Research Funding; BioLine Rx: Consultancy; PTC Therapeutics: Consultancy; BMS: Research Funding. Hosing:NKARTA Inc.: Consultancy. Blum:Syndax: Membership on an entity's Board of Directors or advisory committees; Celyad: Research Funding; Leukemia and Lymphoma Society: Research Funding; Xencor: Research Funding; Amerisource Bergen: Honoraria. Rotta:Merck: Speakers Bureau; Jazz Pharma: Speakers Bureau. Ojeras:Nkarta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barnett:Nkarta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rajangam:Nkarta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Majhail:Mallinckrodt: Honoraria; Anthem, Inc.: Consultancy; Incyte: Honoraria; Nkarta Therapeutics: Honoraria. Nikiforow:Kite/Gilead: Honoraria; Nkarta Therapeutics: Honoraria; Novartis: Honoraria.

Triple Gene-Modified iPSC-Derived NK Cells Combined with Daratumumab for Targeted Immunotherapy Against AML

Triple Gene-Modified iPSC-Derived NK Cells Combined with Daratumumab for Targeted Immunotherapy Against AML