Allogeneic Natural Killer Cell Therapy Research Articles

Phase I study of safety and efficacy of allogeneic natural killer cell therapy in relapsed/refractory neuroblastomas post autologous hematopoietic stem cell transplantation

Despite low incidence, neuroblastoma, an immunologically cold tumor, is the most common extracranial solid neoplasm in pediatrics. In relapsed/refractory cases, the benefits of autologous hematopoietic stem cell transplantation (auto-HSCT) and other therapies are limited. Natural killer (NK) cells apply cytotoxicity against tumor cells independently of antigen-presenting cells and the adaptive immune system. The primary endpoint of this trial was to assess the safety of the injection of allogenic, ex vivo-expanded and primed NK cells in relapsed/refractory neuroblastoma patients after auto-HSCT. The secondary endpoint included the efficacy of this intervention in controlling tumors. NK cells were isolated and primed ex vivo (by adding interleukin [IL]-2, IL-15, and IL-21) in a GMP-compliant CliniMACS system and administered to four patients with relapsed/refractory MYCN-positive neuroblastoma. NK cell injections (1 and 5 × 107 cells/kg in the first and second injections, respectively) were safe, and no acute or sub-acute adverse events were observed. During the follow-up period, one complete response (CR) and one partial response (PR) were observed, while two cases exhibited progressive disease (PD). In follow-up evaluations, two died due to disease progression, including the case with a PR. The patient with CR had regular growth at the 31-month follow-up, and another patient with PD is still alive and receiving chemotherapies 20 months after therapy. This therapy is an appealing and feasible approach for managing refractory neuroblastomas post-HSCT. Further studies are needed to explore its efficacy with higher doses and more frequent administrations for high-risk neuroblastomas and other immunologically cold tumors.Trial registration number: irct.behdasht.gov.ir (Iranian Registry of Clinical Trials, No. IRCT20201202049568N1).

TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant.

High-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in HR-NBL patients; however, graft-versus-host-disease (GVHD) and disease progression have limited clinical application. Ex-vivo stimulated allogeneic natural killer (NK) cells represent a potential approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL. Ex-vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble IL-15/IL-15Rα alone or with irradiated CD137L/CD54+ AgN2a-4P (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-TIM-3. Lethally irradiated B6AJF1 Mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish their contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and overall survival. Co-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Ki-67 and NKG2D, NKp46, TRAIL and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared to IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared to NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomes, TRAIL and FasL expression observed. Analysis of RNA from 15-4P NK cells exposed to TIM-3 blockade showed gene expression of chemokines, NKG2D/DAP12 signaling, non-canonical NF-κb pathway and TRAIL signaling. Blockade of NKG2D, TRAIL or FasL on 15-4P NK cells abrogated cytotoxicity. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared to NK cells or anti-TIM-3 alone, without inducing GVHD. Depletion of NK cells, but not T cells, abrogated the GVT effect. Allo-HCT can be a platform for treating NBL using combination ex-vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD.

Boosting cytotoxicity of adoptive allogeneic NK cell therapy with an oncolytic adenovirus encoding a human vIL-2 cytokine for the treatment of human ovarian cancer

Despite good results in the treatment of hematological malignancies, Natural killer (NK) cells have shown limited effectiveness in solid tumors, such as ovarian cancer (OvCa). Here, we assessed the potential of an oncolytic adenovirus expressing a variant interleukin-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, to enhance NK cell therapy efficacy in human OvCa ex vivo. Human OvCa surgical specimens were processed into single-cell suspensions and NK cells were expanded from healthy blood donors. OvCa sample digests were co-cultured ex vivo with NK cells and vIL-2 virus and cancer cell killing potential assessed in real time through cell impedance measurement. Proposed therapeutic combination was evaluated in vivo with an OvCa patient-derived xenograft (PDX) in mice. Addition of vIL-2 virus significantly enhanced NK cell therapy killing potential in treated OvCa co-cultures. Similarly, vIL-2 virus in combination with NK cell therapy promoted the best in vivo OvCa tumor control. Mechanistically, vIL-2 virus induced higher percentages of granzyme B in NK cells, and CD8+ T cells, while T regulatory cell proportions remained comparable to NK cell monotherapy in vivo. Ad5/3-E2F-d24-vIL2 virus treatment represents a promising strategy to boost adoptive NK cell therapeutic effect in human OvCa.

Clinical-grade natural killer cells expanded from donor peripheral blood and anti-leukemia effect against myeloid leukemia.

e19018 Background: Natural killer (NK) cells play a crucial role in immune surveillance and tumor clearance. It has been reported that allogeneic NK cell therapy has good safety and curative effect in patients with refractory and relapsed acute myeloid leukemia (AML). However, the clinical application of adoptive NK infusion is limited due to the lower number of human NK cells. We established a feeder-free NK cell expansion system from peripheral blood (PB) and explore the anti-leukemia potential in preclinical and clinical studies. Methods: The killing capacity of ex vivo-expanded NK cells were detected by Flow cytometry. The in vivo expansion, survival and anti-leukemia activity of the expanded NK cells were detected in K562-bearing NSG mice. Six AML patients with hematological/extra medullary relapsed (n = 3) or persistent positive minimal residual disease (n = 3) after consolidation and maintenance therapy were enrolled in Peking University Institute of Hematology from 2021 to 2022. The patients were treated with high-dose cyclophosphamide and fludarabine, then combined with adoptive infusion of donor-derived expanded NK cells. The chimerism, the number and function of NK cells were analyzed, and the safety and clinical efficacy were evaluated. Results: The ex vivo-expanded NK cells exhibit highly anti-leukemia activity. NK cells proliferate in mice and the CD56+ cells to reach the peak time in each organ is 3-7 days and drop to the level before infusion after 14 days. NK cell infusion significantly improve the survival of leukemia mice, and the anti-leukemia effect is NK cell dose-dependent. In the clinical trial, six patients received a total of 7 cases of NK cell infusion, including 3 cases in low-dose group and 4 cases in middle-dose group, among which 1 patient received 2 dose (1 low-dose and 1 middle-dose). In the low-dose group, 1 patient got response (1/3), with the minimal residual disease turned negative. In the middle-dose group, 3 patients responded (3/4), of which 1 patient had the fusion gene CBFB /ABL decreased from 0.99% to 0.13%, 1 case of extramedullary recurrence of AML turned negative, and NK cells could be detected in cerebrospinal fluid, 1 case of hematological recurrence got hematological remission after treatment. The survival time of NK cells in vivo was 7d-30d, with longer time in middle dose group than those in low dose group. The proportion and number of NK cells in responding cases were significantly higher than those in non-responsive group. Conclusions: Clinical grade expanded NK cells has strong anti-leukemia activity in patients with relapse or minimal residual positive AML patients. NK cells can survive for 2 weeks and can enter the central nervous system without obvious adverse reactions. The response rate in middle-dose group is higher than that in low-dose group. However, the efficacy of NK cells still needs to be further confirmed in a large sample size. Clinical trial information: NCT04221971 .

Abstract 2898: CAT-248, an allogeneic CD70-directed CAR-NK cell therapy effectively controls CD70-positive tumor xenografts

Abstract Engineered, off-the-shelf, allogeneic natural killer (NK) cell therapy is an attractive approach for targeting solid tumors, given their emerging clinical safety, efficacy, and intrinsic anti-tumor recognition and activity. However, improvements to support persistence and maintain durable anti-tumor activity within the tumor microenvironment may be necessary to achieve meaningful clinical efficacy. Here we describe the preclinical activity of CAT-248, a CD70-directed CAR-NK cell therapy, engineered using the TcBuster™ Transposon System (Bio-Techne) multiplexed with CRISPR/Cas9 editing. CD70 is highly expressed in many tumor types while normal tissue expression is restricted to a subset of activated immune cells. CAT-248 is an allogeneic, healthy donor peripheral blood-derived NK cell product designed for durable efficacy against CD70 expressing tumors. CAT-248 is engineered to express CD70 CAR, interleukin 15 (IL-15), and transforming growth factor β (TGFβ) dominant negative receptor (DNR). In addition, CAT-248 includes CRISPR/Cas9 knockout of CD70 to mitigate fratricide due to endogenous CD70 expression in activated NK cells. IL-15 enhances persistence of CAT-248 to enable durable efficacy, and TGFβ DNR enables CAT-248 to maintain high activity in TGFβ-enriched and immunosuppressive solid tumor microenvironments. CAT-248 is manufactured using transposon-based engineering which enables stable integration of the three transgenes and CRISPR/Cas9 knockout of CD70 in a single electroporation step, resulting in 40-80% CAR expression and 80-90% knockout of CD70 in CAT-248 NK cells. CAT-248 activity was characterized across a panel of in vitro assays to evaluate the function of CD70 CAR, TGFβ DNR, and IL-15 transgenes. CD70-directed cytotoxicity was assessed against a panel of tumor cell lines with a broad range of CD70 expression. In vitro, CAT-248 cells demonstrated both CAR-dependent cytotoxicity and over 2-fold greater secretion of effector cytokines IFNγ and TNFα than control NK cells. TGFβ DNR effectively prevented TGFβ-induced SMAD phosphorylation and TGFβ-induced downregulation of DNAM-1, an NK cell activating receptor. IL-15 secretion enabled in vitro NK cell expansion over a 9-day time course without exogenous cytokine support. To confirm cytolytic activity in vivo, CAT-248 cells were administered therapeutically in a 786-O CD70+ renal cell carcinoma xenograft model. CAT-248 cells effectively controlled tumor, demonstrating >99% reduction in tumor burden relative to control NK cells (p<0.01). Further, CAT-248 cells demonstrated significant in vivo persistence beyond 4 weeks post-dosing in peripheral blood. Overall, the results demonstrate the potential for CAT-248 as a novel off-the-shelf, cryopreserved, allogeneic NK cell therapy for CD70-positive renal cell carcinoma and other solid tumor malignancies. Citation Format: Bashar Hamza, Eugene Choi, Dominic Picarella, Finola Moore, Angela Nunez, Henry Moreno, Marilyn Marques, Alexia Barandiaran, Meghan Walsh, Kisha Pradhan, Krista Daniel, Kelly Becker, Jennifer Johnson, Karl Malakian, Keith H. Wong, Mark A. Omobono, Charlotte Franco, Andres Alvarez, Jenna Nguyen, Billy Blanco, Priya Khurana, Beatriz Marques, Adriana Rivera, Michele McAuliffe, Jae-Woong Chang, Joshua Krueger, Beau R. Webber, Branden S. Moriarity, Joseph Gold, Cherry Thomas, Vipin Suri. CAT-248, an allogeneic CD70-directed CAR-NK cell therapy effectively controls CD70-positive tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2898.

Current state of NK cell-mediated immunotherapy in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has become one of the most common hematological diseases in western countries, with an annual incidence of 42/100,000. Conventional chemotherapy and targeted therapeutic drugs showed limitations in prognosis or in efficiency in high-risk patients. Immunotherapy represented is one of the most effective therapeutic approaches with the potential of better effect and prognosis. Natural killer (NK) cells are good options for immunotherapy as they can effectively mediate anti-tumor activity of immune system by expressing activating and inhibiting receptors and recognizing specific ligands on various tumor cells. NK cells are critical in the immunotherapy of CLL by enhancing self-mediated antibody-dependent cytotoxicity (ADCC), allogeneic NK cell therapy and chimeric antigen receptor-natural killer (CAR-NK) cell therapy. In this article, we reviewed the features, working mechanisms, and receptors of NK cells, and the available evidence of the advantages and disadvantages of NK cell-based immunotherapies, and put forward future study directions in this field.

Phase I Study: Safety and Efficacy of an Ex Vivo-Expanded Allogeneic Natural Killer Cell (MG4101) with Rituximab for Relapsed/Refractory B Cell Non-Hodgkin Lymphoma

The prognosis of non-Hodgkin lymphoma (NHL) remains poor, with an unmet need for novel therapies. MG4101, an ex vivo-expanded allogeneic natural killer (NK) cell, can enhance rituximab antibody-dependent cytotoxicity in relapsed/refractory (r/r) B cell non-Hodgkin lymphoma. This study assessed the safety and efficacy of MG4101 plus rituximab for patients with r/r NHL. Patients received escalating doses of i.v. MG4101 plus rituximab every 2 weeks. IL-2 was administered s.c. after MG4101 treatment. Fludarabine plus cyclophosphamide was administered i.v. before rituximab treatment in cycles 1, 3, and 5. A 3+3 design was used to determine the maximum tolerated dose (MTD) and maximum feasible dose. Assessments were performed over a 6-cycle period, with an extended maintenance period of up to 8 cycles. Nine patients received 3 different doses of MG4101 and rituximab. MTD could not be determined because of the absence of dose-limiting toxicity. Treatment-related adverse events, mostly grade 1 or 2, occurred in 89% of patients. Only 1 patient experienced grade 1 cytokine release syndrome. MG4101 persisted for at least 7 days in 7 patients. Four patients achieved a partial response and 1 patient attained a complete response, for an overall response rate of 55.6%. Two patients showed prolonged responses and low exhaustion marker levels in T cells. For allogeneic NK cell therapy, strategies including the use of the high-affinity hFcγRIIIaV158 variant of the KIR B/x haplotype with lymphodepleting chemotherapy may be promising options for improving clinical efficacy in the antibody combination therapeutic setting as an off-the-shelf product. MG4101 plus rituximab presented a favorable safety profile and overall response rate in patients with r/r NHL.

Abstract 10 Interim Results of a Phase I Study Investigating a Cord Blood-Derived Natural Killer Cell Therapy for Patients Hospitalized with COVID-19

IntroductionIn the setting of viral infections, including infection with SARS-CoV-2, lymphocyte exhaustion and lymphopenia are common. Patients with COVID-19 who develop lymphopenia, particularly low numbers of circulating natural killer (NK) cells, are at high risk for disease progression. Thus, strategies that overcome profound lymphopenia and restore innate immune function may reduce the severity of COVID-19. One approach is the direct infusion of an exogenous source of healthy, functional NK cells intended to boost the patient’s immune system during the viral infection, enabling elimination of infected cells. “DVX201” is a cord blood-derived allogeneic NK cell therapy that is cryopreserved and intended for “off-the-shelf use” without HLA-matching.ObjectiveThe primary objective of this study is to investigate the safety and to identify the recommended phase II dose and/or the maximum tolerated dose (MTD) of DVX201 in patients hospitalized with COVID-19.MethodsWe are conducting an open-label, nonrandomized phase I safety and dose-finding (modified 3+3) study of DVX201 at three dose levels in patients hospitalized with COVID-19. Adult patients with documented SARS-CoV-2 infection were eligible if they were admitted to the hospital, were receiving ≤6 L supplemental oxygen by low flow delivery, and did not have other evidence of cytokine storm based on levels of serum IL-6, C-reactive protein, and ferritin.ResultsTo date, 9 patients have been enrolled: 3 subjects at each dose level. All dose levels have been well tolerated, with no dose-limiting toxicities, infusion toxicities, or cytokine release syndrome observed. All patients were discharged from the hospital at an average of 5 days post infusion. Blood samples were drawn for correlative studies including persistence of DVX201, pre- and post-infusion immune system characterization, and cytokine panels. Accrual is continuing at the MTD.DiscussionThis trial will serve as the proof of concept for the use of DVX201 as an anti-viral treatment for COVID-19. These results will inform the potential role of DVX201 for treatment of other life-threatening viral infections by harnessing NK cells’ innate function to kill virally infected cells, regardless of the etiology of the infection.

P482: NOVEL BISPECIFIC INNATE CELL ENGAGER AFM28 FOR THE TREATMENT OF CD123 POSITIVE ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME

Background: Novel treatments for patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS) with relapsed or refractory (R/R), or measurable residual disease are urgently needed. Targeting of the surface antigen CD123 expressed on both leukemic blasts and stem cells (LSCs) of most patients allows for depletion of both compartments, which is considered a prerequisite for deep anti-tumor responses and induction of prolonged remission. AFM28 is a novel bispecific Innate Cell Engager (ICE®) designed for bivalent high-affinity binding of CD16A on natural killer (NK) cells, redirecting effector cell cytotoxicity to CD123+ tumor cells. Allogeneic NK cell therapy is emerging as a next-generation treatment with demonstrated activity in R/R AML and a very benign safety profile. Retargeting of NK cells with AFM28 may be a particularly effective treatment strategy in patients with CD123+ AML and HR-MDS. Aims: 1. Preclinical characterization of AFM28 pharmacology and toxicology in vitro and in vivo to support clinical development. 2. Assessment of binding and activation of NK cells by AFM28 to test suitability for combination development with allogeneic NK cell therapy. Methods: Flow cytometry (FC) was used to assess binding and retention of AFM28 on NK cells, NK cell activation (degranulation, IFNγ expression) and depletion of leukemic cells from AML and MDS patient specimens. Quantification of secreted cytokines in leukocyte cultures employed bead-based luminex methodology. In vivo efficacy data were acquired by BLI measurement from a murine AML model. Cynomolgus data were acquired from non-GLP dose range finding and a GLP toxicology study using standard methods (ELISA, IHC, FC). Results: AFM28 induced NK cell activation at low picomolar concentrations and mediated efficacious depletion of CD123+ tumor cell lines and primary leukemic cells, including cells with low CD123 expression. NK cell activation was induced more potently than by an effector-enhanced IgG1 and was accompanied by more pronounced degranulation and IFNγ production. Similarly, AFM28 induced depletion of leukemic cells in patient bone marrow samples, without lysis of CD34+/CD123− cells, suggesting sparing of healthy hematopoietic progenitors. Potent anti-tumor activity was confirmed in vivo in a murine tumor model. In preclinical toxicology models, AFM28-induced target cell depletion was associated with minimal release of inflammatory cytokines, including IL-6, TNFα and IFNγ from leukocytes in vitro, and only low-level release of IL-6 in cynomolgus monkeys, despite efficacious depletion of CD123+ basophils and plasmacytoid dendritic cells (pDCs). Characterization of NK cell binding revealed high-avidity interaction, long cell surface retention, and potent induction of NK cell-mediated tumor cell lysis with no impact on NK cell viability. Summary/Conclusion: These data demonstrate that AFM28 exhibits potent pharmacological activity in vitro and in vivo and efficaciously induces NK cell-mediated depletion of primary leukemic cells. Further, toxicology data suggest good tolerability with a low risk of cytokine release syndrome. Together, these findings support the clinical investigation of AFM28 as a potential novel treatment of CD123+ AML and HR-MDS. A first-in-human trial investigating safety and activity of single agent AFM28 in patients with R/R AML is currently in preparation. In addition, these data support the combination of AFM28 with allogeneic NK cell therapy, which holds potential as an effective, novel treatment strategy.

CD155 axis modulation promotes natural killer cell-mediated graft-versus-tumor effects against osteosarcoma

Abstract Patients with relapsed/refractory osteosarcoma (OSA) have a poor prognosis with few treatment options. Immunotherapy with allogeneic natural killer (NK) cells after allogeneic bone marrow transplant (BMT) is an attractive approach to employ a graft-versus-tumor (GVT) effect against OSA. However, NK cells have had limited success against solid tumors in vivo. CD155 is overexpressed on OSA and interacts with DNAM-1, an activating ligand, and TIGIT, an inhibitory ligand, on NK cells. The impact of blocking CD155 after allogeneic BMT is unknown. IL-15-expanded C57BL/6 (B6, H-2b) and BALB/c (H-2d) murine NK cells were co-cultured with K7M2 murine OSA (H-2d) after CD155 and CD155 ligand blockade, and analyzed by flow cytometry and cytotoxicity assays. BALB/c mice were also transplanted with T cell depleted allogeneic B6 or syngeneic BALB/c bone marrow, challenged with K7M2 and treated with IL-15-expanded NK cells and CD155 blockade. Allogeneic NK cells showed increased degranulation, interferon-gamma production and cytotoxicity against K7M2 OSA in vitro compared to syngeneic NK cells. NK cell activation and cytotoxicity were further enhanced by CD155 and TIGIT blockade, but decreased with DNAM-1 blockade. In vivo, allogeneic NK cell infusion and anti-CD155 treatment decreased tumor growth and increased survival compared to syngeneic NK cell treatment, without induction of graft-versus-host-disease. CD155 blockade augments NK cell activation, cytotoxicity and GVT effects against OSA without toxicity, suggesting TIGIT is the dominant CD155 checkpoint during allogeneic BMT. CD155 blockade with allogeneic NK cell therapy after BMT may be an effective combination immunotherapy platform for treating relapsed/refractory OSA. Supported by NIH grants (T32 CA009135, R01 CA215461)

Tafasitamab mediates killing of B-cell non-Hodgkin’s lymphoma in combination with γδ T cell or allogeneic NK cell therapy

Tafasitamab is an Fc-modified monoclonal antibody that binds to CD19, a cell-surface antigen that is broadly expressed on various types of B-cell non-Hodgkin’s lymphoma (NHL). Antibody-dependent cellular cytotoxicity (ADCC), a key mode of action of tafasitamab, is mediated through the binding of tafasitamab’s Fc region to FcγRIIIa receptors on immune effector cells and results in antitumor activity. Despite the proven clinical activity of tafasitamab in combination with lenalidomide in the treatment of diffuse large B-cell lymphoma (DLBCL), a higher number of immune cells in cancer patients may improve the activity of tafasitamab. Here, we characterized two ex vivo-expanded FcγRIIIa receptor—expressing cell types—γδ T and MG4101 natural killer (NK) cells—as effector cells for tafasitamab in vitro, and found that in the presence of these cells tafasitamab was able to induce ADCC against a range of NHL cell lines and patient-derived cells. We also explored the concept of effector cell supplementation during tafasitamab treatment in vivo by coadministering MG4101 NK cells in Raji and Ramos xenograft models of NHL. Combination treatment of tafasitamab and allogeneic MG4101 NK cells in these models demonstrated a survival benefit compared with tafasitamab or MG4101 monotherapy (Raji: 1.7- to 1.9-fold increase in lifespan; Ramos: 2.0- to 4.1-fold increase in lifespan). In conclusion, adoptive cell transfer of ex vivo-expanded allogeneic NK or autologous γδ T cells in combination with tafasitamab treatment may potentially be a promising novel approach to increase the number of immune effector cells and enhance the antitumor effect of tafasitamab.

AFM28, a Novel Bispecific Innate Cell Engager (ICE ®), Designed to Selectively Re-Direct NK Cell Lysis to CD123+ Leukemic Cells in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are common forms of adult hematologic malignancies with incidences of approximately 1-6/100.000/year. Progress in the management of AML and high-risk MDS has lagged behind other hematological malignancies, and, in particular, treatment for relapsed or refractory (R/R) disease and minimal residual disease (MRD) are needed to induce long-term remission.Here we describe the preclinical development of AFM28, a novel bispecific Innate Cell Engager (ICE ®) developed using the Redirected Optimized Cell Killing (ROCK ®) platform, designed to specifically activate NK cells and induce depletion of leukemic blasts and LSCs. Natural killer (NK) cell-based innate immunotherapy is emerging as a promising treatment option in AML and MDS based on the demonstrated susceptibility of leukemic blasts for NK cell killing and clinical activity of allogeneic NK cell therapy in R/R disease. Depletion of leukemic stem cells (LSCs) alongside leukemic blasts is considered a critical goal of treatment in order to eradicate MRD and prevent relapse. Accordingly, treatments that effectively target both cell types hold the potential to induce long-term remission in patients with AML or MDS.AFM28 binds the surface antigen CD123, which is almost universally expressed on blasts and LSCs in AML and MDS, and selectively targets CD16A expressed on NK cells, with no detectable binding to CD16B expressed on neutrophils. Through high affinity, bivalent binding to CD16A in a region distinct from the Fc binding site of IgGs, AFM28 remains stably bound to NK cells for extended periods and potently induces antibody-dependent cell-mediated cytotoxicity (ADCC) towards CD123+ AML and MDS cells. When compared with conventional anti-CD123 antibodies, including Fc-enhanced IgG1, AFM28 mediates more potent lysis of tumor cells even with low levels of CD123 expression. Furthermore, in contrast to conventional monoclonal CD123 antibodies, AFM28-induced lysis is not affected by the addition of competing IgG, which otherwise could impact efficacy. Similarly, ex vivo analysis of NK cell activation and inflammatory cytokine release in the ID.Flow human whole-blood loop system demonstrates that AFM28, compared with Fc-enhanced IgG1, produces 100-fold more potent NK cell activation associated with dose-dependent release of pro-inflammatory IL-6, TNFα, and IFNγ, concomitant with depletion of CD123+ plasmacytoid dendritic cells (pDCs) and basophils. In contrast, cytokine release was substantially lower than observed with a CD123-directed T cell-engaging bispecific antibody suggesting a low risk of cytokine release syndrome with AFM28. In vivo, AFM28 demonstrates a monoclonal antibody-like pharmacokinetic profile in mice and cynomolgus monkeys. A pre-clinical toxicology model in cynomolgus monkey suggested that AFM28 is safe and well tolerated and demonstrated pharmacodynamic activity.In summary, these data demonstrate AFM28 could be a novel treatment modality for AML and MDS that has good tolerability and the potential to effectively target MRD by depleting leukemic blasts and LSCs. AFM28 is currently being prepared for first-in-human clinical investigation. High affinity binding, potent induction of NK cell activation, and extended cell surface retention suggest AFM28 endows NK cells with CAR-like properties and may hold particular promise when combined with allogeneic NK cell therapy, for example within a pre-complexed NK cell product. DisclosuresGötz: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Pahl: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Schmitt: Affimed GmbH: Research Funding. Müller: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Haneke: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Kozlowska: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Sarlang: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Knackmuss: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Peters: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Reusch: Affimed: Current Employment, Current holder of stock options in a privately-held company. Ross: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Nowak: AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; Takeda: Honoraria. Hofmann: Affimed GmbH: Research Funding. Merz: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company.

Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Immune Monitoring of CD34+ Placental Cell Derived Natural Killer Cell Therapy (PNK-007) in Phase I Study of Multiple Myeloma

Background: Natural Killer (NK) cells are innate immune lymphocytes with cytotoxic function and are critical for immune surveillance. Unlike T cells which rely on antigen-specific responses, NK cells recognize transformed cells through a variety of NK cell-specific receptor-ligand interactions. Clinical studies have highlighted the therapeutic potential of NK cell-based therapies and Celularity has developed a GMP procedure for generating PNK-007: an off-the-shelf and allogeneic NK cell product culture-expanded and differentiated from human placental CD34+ stem cells. PNK-007 was evaluated as a single-dose allogeneic cell therapy in a phase I dose-escalation study for multiple myeloma (MM) patients receiving autologous stem cell transplant (ASCT) (NCT02955550). 12 of 15 patients received subcutaneous IL-2 every 48 hours for 10 days following PNK-007 cell infusion while 3 patients in a separate cohort did not receive IL-2. Here, we report translational studies evaluating post-treatment immune reconstitution, minimal residual disease (MRD) detection at 10-5 threshold, and serum profiling of cytokines and chemokines. Methods: Patient bone marrow aspirate was collected for EuroFlow MRD assessment and immune phenotyping by flow cytometry at baseline, 90-100 days, 6 months and 1 year post-ASCT. Peripheral blood was collected at baseline, then weekly for six weeks following PNK-007 infusion, at 6 months and at 1 year post-ASCT. PNK-007 persistence, leukocyte populations, and HLA antibody panels were determined by flow cytometry. Serum was analyzed by Luminex for panel cytokines, chemokines and soluble cytokine receptors. All patients and PNK-007 drug product were typed for HLA and killer-cell immunoglobulin-like (KIR) genotype. Results: PNK-007 infusion did not interfere with immune reconstitution kinetics post-ASCT. Cohorts receiving PNK infusion 14 days post-ASCT had already recovered white blood cell counts to normal levels. One cohort receiving 3x107 PNK cells/kg 7 days post-ASCT showed an immune deficient environment (0.05x103 ± 0.004x103 leukocytes/ml, n=3), but recovered their white blood cell counts by day 21 (6.8x103 ± 1.8x103 leukocytes/ml, n=3). Using a validated Euro-flow MRD assay, 4/15 patients were MRD(-) at pre-ASCT baseline, and by day 90, 10/15 pts were MRD(-). PNK-007 persistence was not detected in patients by flow cytometry with the earliest timepoint tested being 7 days post-infusion. Panel HLA serum antibodies were not detected at any timepoint indicating the absence of alloantibodies. Withholding IL-2 administration in one cohort allowed us to evaluate its potential effects on NK cell recovery and immune reconstitution. We found that subcutaneous IL-2 q.o.d. for 10 days following PNK-007 infusion did not affect recovery kinetics and concentration of endogenous NK cells. However, these patients showed a 5-10 fold increase in serum soluble IL-2RA from baseline and elevated regulatory T cell (Treg) count in the blood versus baseline (167 ± 107 Treg/mL vs. 61 ± 33 Treg/mL, p=0.0075). Patients not receiving IL-2 saw no change in serum soluble IL-2RA or blood Treg level from baseline. CD4 and CD8 T cells from all patients retained their ability to become activated in response to ex vivo stimulation and favored release of IL-2 and IFNg. T cell effector function was maintained in all patients post-ASCT but was lost in a subset of patients at 1 year. Serum analysis showed low levels of free IL-15 at the time of PNK dosing despite the transient lymphodepleted state associated with ASCT. Cytokines associated with myeloid inflammation and T cell immunity in the month post dosing were within normal homeostatic level. Serum TGFβ was significantly lower at the day of PNK infusion compared to normal homeostatic levels. Conclusion: Translational data from a Phase I study of PNK-007 in post-ASCT MM established that dosing up to 3x107 cells/kg at 14 or 7 days post-transplant did not impair engraftment or immune reconstitution. EuroFlow MRD assessment of the bone marrow showed conversion of 6/11 patients from MRD(+) to MRD(-) by day 90 post transplant. The administration of IL-2 in this clinical study did not appear to benefit NK cell reconstitution but instead favored soluble IL2RA antagonism and increased systemic levels of Treg. These results demonstrate the feasibility of allogeneic NK cell therapy in the MM + ASCT setting and will help inform the design of further clinical studies. Disclosures Van Der Touw: Celularity, Inc.: Employment, Patents & Royalties. Kang:Celularity, Inc.: Employment, Patents & Royalties. Stout:Celularity, Inc.: Employment. Voskinarian-Berse:Celularity, Inc.: Employment. Rousseva:Celularity, Inc.: Employment. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

Natural Killer Cell Therapy: A New Treatment Paradigm for Solid Tumors.

In treatments of solid tumors, adoptive transfer of ex vivo expanded natural killer (NK) cells has dawned as a new paradigm. Compared with cytotoxic T lymphocytes, NK cells take a unique position targeting tumor cells that evade the host immune surveillance by down-regulating self-antigen presentation. Recent findings highlighted that NK cells can even target cancer stem cells. The efficacy of allogeneic NK cells has been widely investigated in the treatment of hematologic malignancies. In solid tumors, both autologous and allogeneic NK cells have demonstrated potential efficacy. In allogeneic NK cell therapy, the mismatch between the killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) can be harnessed to increase the antitumor activity. However, the allogeneic NK cells cause more adverse events and can be rejected by the host immune system after repeated injections. In this regard, the autologous NK cell therapy is safer. This article reviews the published results of clinical trials and discusses strategies to enhance the efficacy of the NK cell therapy. The difference in immunophenotype of the ex vivo expanded NK cells resulted from different culture methods may affect the final efficacy. Furthermore, currently available standard anticancer therapy, molecularly targeted agents, and checkpoint inhibitors may directly or indirectly enhance the efficacy of NK cell therapy. A recent study discovered that NK cell specific genetic defects are closely associated with the tumor immune microenvironment that determines clinical outcomes. This finding warrants future investigations to find the implication of NK cell specific genetic defects in cancer development and treatment, and NK cell deficiency syndrome should be revisited to enhance our understanding. Overall, it is clear that NK cell therapy is safe and promises a new paradigm for the treatment of solid tumors.

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.

An important discovery on combination of irreversible electroporation and allogeneic natural killer cell immunotherapy for unresectable pancreatic cancer.

PurposeTo study the safety and clinical efficacy on combination of irreversible electroporation and allogeneic natural killer cell therapy for treating Stage III/IV pancreatic cancer, evaluating median progression free survival (PFS), and overall survival (OS).ResultsAdverse events of all patients were limited to grades 1 and 2, including local (mainly tussis 13.4%, nausea and emesis 7.1%, pain of puncture point 29.6% and duodenum and gastric retention 4.3%) and systemic (mainly fatigue 22.3%, fever 31.6%, and transient reduction of intraoperative blood pressure 25.1% and white cell count reduction 18.3%) reactions, fever was the most frequent. The serum amylase level at 24 h and 7 d after IRE was not significantly changed compared to those before IRE (P > 0.05). CA19–9 value was lower in IRE-NK group than in IRE at 1 month after treatment (P < 0.05). After a median follow-up of 7.4 months (3.6–11.2 months): in stage III group, median PFS was higher in IRE-NK group (9.3 months) than in IRE group (8.1 months, P = 0.0465), median OS was higher in IRE-NK (13.2 months) than in IRE (11.4 months, P = 0.0411), and median PFS was higher in who received multiple NK than single NK (9.8 months vs.8.1 months, P = 0.0423, respectively), median OS who received multiple NK was higher than single NK (13.9 months vs.12.3 months, P = 0.0524, respectively), the RR in IRE-NK (63.2%) was higher than in IRE (50.0%, P < 0.05); in stage IV group, median OS was higher in IRE-NK (9.8 months) than in IRE (8.7 months, P = 0.0397), the DCR in IRE-NK (66.7%) was higher than in IRE (42.9%, P < 0.05).Materials and MethodsBetween July 2016 and May 2017, we enrolled 71 patients who met the enrollment criteria. The patients were divided into stage III (32 patients, 17 patients received only IRE and 15 patients received IRE-NK (Irreversible electroporation- natural killer): 8 patients underwent a course of NK and 7 patients underwent ≥ 3 courses) and stage IV (39 patients, 22 patients received only IRE and 17 patients received IRE-NK: 9 patients underwent a course of NK and 8 patients underwent ≥ 3 courses). The safety and short-term effects were evaluated firstly, then the median PFS, median OS, response rate (RR) and disease control rate (DCR) were assessed.ConclusionsCombination of irreversible electroporation and allogeneic natural killer cell immunotherapy significantly increased median PFS and median OS in stage III pancreatic cancer and extended the median OS of stage IV pancreatic cancer. Multiple allogeneic natural killer cells infusion was associated with better prognosis to stage III pancreatic cancer.

Short-term clinical efficacy of percutaneous irreversible electroporation combined with allogeneic natural killer cell for treating metastatic pancreatic cancer

This study was to determine how the short-term clinical efficacy of irreversible electroporation (IRE) combined with allogeneic natural killer (NK) cell therapy for treating metastatic pancreatic cancer. Between March and December 2016, we enrolled 40 patients who met the enrollment criteria and assigned them to two groups: simple IRE (IRE group, n=20) and IRE plus allogeneic NK cell therapy (IRE-NK, n=20). We evaluated immune function changes, quality of life, clinical response, and other related indicators. Combining allogeneic NK cells with IRE had a synergistic effect, not only enhancing the immune function of the patients, but also reducing the expression of carbohydrate antigen 19-9 (CA19-9) and CA242 and significantly exhibiting good short-term outcome and improving the quality of life of the patients. This is the first clinical trial to combine allogeneic NK cells with IRE for treating metastatic pancreatic cancer, and proves the safety and efficacy of the treatment.

SAT-107 - Phase I study of HLA-half matched allogeneic natural killer cell therapy in patients with hepatocellular carcinoma

SAT-107 - Phase I study of HLA-half matched allogeneic natural killer cell therapy in patients with hepatocellular carcinoma

Abstract A52: Efficacy of specified natural killer (NK) cells in ovarian cancer xenograft models.

Abstract Objective: Natural killer (NK) cells are one of the most essential cell types, and they rank with cytotoxic T lymphocytes (CTLs) as regards strong cytotoxic activity against malignant tumor cells in the cellular immune response. Recent advances have led to the hope that NK cells could be harnessed as a therapy for cancers. Expanded specified NK cells show potent cytotoxic activity against tumor cells in vitro and in vivo. Recently, some evidence suggests that NK cells can help to control ovarian tumor growth has been provided by the analysis of tumor prevalence in immunodeficient mice. Therefore, we investigated whether expanded NK cells have potent cytotoxic activity against human ovarian cancer xenografts Methods: We have established a novel method for the expansion and activation of NK cells from healthy volunteers for allogeneic NK cell therapy. A highly pure population of CD16+CD56+ NK cells can be obtained with minimal contamination by T cells, B cells, or monocytes. The specified NK cells which were selected after in vitro experiment screening of candidates by Facscan analysis and OVCAR3-Luc cells were selected. For NK cells, its anti-tumor efficacy was confirmed in mice through experiments of autopsies. For in vivo screening of anti-cancer cell therapy, an experimental xenograft model using OVCAR3-Luc cells was established and optimized by optical imaging method considering time and degree of tumorigenesis. The injection of 5x106 OVCAR3-luc cells were turned out suitable for screening model, because of rapid saturation time of optical signal and survival period of mouse. Tumor luminescence was static until 4 weeks postimplantation, after which a steady increase was noted. In addition the mice were weighed and examined for abdominal distension during each imaging session. Results: In this experimental animal model, tumor was predominantly generated in peritoneal area and in about 10% mice a small number of tumor-mass was spread under the peritoneal area. Despite the detectable increase in tumor burden by bioluminescence, measurable changes in girth and weight were not seen until later in the study. At Day 60, specified NK cell treatment displayed effective inhibition of ovarian cancer growth, while negative control showed negligible anticancer effect. Total flux of NK cell was much lower than that of negative control, but significance was low (T-Test, p=0.092). Paclitaxel acts in different mechanism to NK cell, was used as a positive control and showed gentle inhibition of ovarian cancer growth. Notable change of body weights in the mice was not observed in any group, indicating severe toxicity did not occur in those mice. Conclusions: As the results, specified NK cells showed potent inhibition of ovarian cancer proliferation in the OVCAR3-Luc cancer model. Therefore, specified NK cell therapy, has been shown to be an important role in the control and cure of at least some ovarian cancer diseases. Citation Format: Young Jae Lee, Ha Young Lee, Yu Kyung Hwang, Kyung Jin Lee, Shin Wha Lee, Yong Man Kim. Efficacy of specified natural killer (NK) cells in ovarian cancer xenograft models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A52.