Adaptive NK Cell Therapy Modulated by Anti-PD-1 Antibody in Gastric Cancer Model

Recently, adaptive NK cell therapy has become a promising treatment but has limited efficacy as a monotherapy. The identification of immune checkpoint inhibitor (ICI) molecules has opened a new horizon of immunotherapy. Herein, we aimed to demonstrate the cytotoxic effects of a polytherapy consisting of ex vivo expanded IL-2-activated NK cells combined with human anti-PD-1 antibody as an important checkpoint molecule in a xenograft gastric cancer mouse model. EBV-LCL cell is used as a feeder to promote NK cell proliferation with a purity of 93.4%. Mice (NOG, female, 6–8 weeks old) with xenograft gastric tumors were treated with PBS, ex vivo IL-2-activated NK cells, IL-2-activated NK cell along with human anti-PD-1 (Nivolumab), and IL-2-activated pretreated NK cells with anti-PD-1 antibody. The cytotoxicity of ex vivo expanded NK cells against MKN-45 cells was assessed by a lactate dehydrogenase (LDH) assay. Tumor volume was evaluated for morphometric properties, and tumor-infiltrating NK cells were assessed by immunohistochemistry (IHC) and quantified by flow cytometry. Pathologic responses were considered by H and E staining. Ex vivo LDH evaluation showed the cytotoxic potential of treated NK cells against gastric cancer cell line. We indicated that the adoptive transfer of ex vivo IL-2-activated NK cells combined with anti-PD-1 resulted in tumor growth inhibition in a xenograft gastric cancer model. Mitotic count was significantly decreased (*p < 0.05), and the tumor was associated with improved infiltration of NK cells in the NK-anti-PD-1 pretreated group (*p < 0.05). In conclusion, the combination approach of activated NK cells and anti-PD-1 therapy results in tumor growth inhibition, accompanied by tumor immune cell infiltration in the gastric tumor model.

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Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽

10.1182/blood-2019-131947 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3607-3607

Author(s):

Grace Lee ◽

Sheela Karunanithi ◽

Zachary Jackson ◽

David Wald

Keyword(s):

Cell Therapy ◽

Cytotoxic Activity ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Akt Activation ◽

Nk Cell Cytotoxicity ◽

Rock Inhibition ◽

Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'>0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

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Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽

10.1182/blood.v112.11.3666.3666 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3666-3666

Author(s):

Tarun K. Garg ◽

Susann Szmania ◽

Jumei Shi ◽

Katie Stone ◽

Amberly Moreno-Bost ◽

...

Keyword(s):

Bone Marrow ◽

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

Scid Mice ◽

Target Cells ◽

Recent Trial ◽

Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

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

Cancers ◽

10.3390/cancers11101534 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1534 ◽

Cited By ~ 10

Author(s):

Sooyeon Oh ◽

Joo-Ho Lee ◽

KyuBum Kwack ◽

Sang-Woon Choi

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Genetic Defects ◽

New Paradigm ◽

Nk Cell Therapy

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.

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Haploidentical Mbil-21 Ex Vivo Expanded NK Cells (FC21-NK) for Patients with Multiple Relapsed and Refractory Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-134858 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-12

Author(s):

Stefan O. Ciurea ◽

Jolie Schafer ◽

Piyanuch Kongtim ◽

Julianne Chen ◽

Doris Soebbing ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Median Number ◽

Publicly Traded ◽

Advisory Committees ◽

Nk Cell Therapy

Background: Allogeneic stem-cell transplantation (alloSCT) remains the only curative treatment for patients with advanced AML. However, only a minority of these patients achieve disease control prior to transplantation. Natural Killer (NK) cells have potent anti-leukemic activity but are functionally deficient in AML. Adoptive NK-cell therapy using high-doses of functionally active NK-cells could overcome these limitations. We previously developed an ex vivo NK-cell expansion method based on K562 feeder cells modified to express membrane bound IL-21 (mbIL-21) and 4-1BB ligand, (FC21), which resulted in high numbers of hyperfunctional FC21-NK cells with enhanced cytotoxicity and cytokine production. Here we report outcomes of a phase I clinical trial designed to assess the safety, feasibility and maximum tolerated dose (MTD) of haploidentical FC21-NK cells for patients with relapse/refractory (R/R) AML at MD Anderson Cancer Center. Methods: Eligible patients were ≥18 years, KPS ≥70 with good organ function. Patients with relapsed AML after alloSCT were eligible if they had no active GVHD and did not require immunosuppression. Haploidentical donors were selected based on KIR characteristics, when multiple donors were available. Donor NK cells were expanded over 3 weeks and cryopreserved. Three dose levels between 106-108 cells/kg were planned. Patients received cytoreductive chemotherapy with fludarabine 30 mg/m2/day and cytarabine 2 g/m2/day for 5 days (4 days for age >60) and G-CSF (subsequently eliminated). 3-7 days after chemotherapy, patients received FC21-NK cell infusions 3 times per week, up to 6 infusions. Results: As of 4/14/2020, 15 patients were screened, 12 of whom were eligible and received the FC21-NK cells. Median age was 60 years (range 25-70); 6 (50%) had adverse cytogenetics, 8 (66.7%) had adverse ELN genetic risk, 6 (50%) had primary induction failure, 2 (16.7%) had CNS disease and 4 (33.3%) had secondary AML. Median number of prior treatment regimens was 5 (range 2-8), median blast count at enrollment was 47% (range 7-88). Median time from diagnosis to enrollment and to first NK-cell infusion was 16.6 (range 2.5-98.1) and 17.2 (range 3.1-98.6) months, respectively. Donor-recipient NK-cell alloreactivity was seen in 5 patients (41.7%). Median number of NK-cell infusion was 6 (range 3-6); 8 (66.7%) and 4 (33.3%) patients received NK-cell dose of 1 X106 and 1 X107 cells/kg, respectively. MTD was not reached. Seven patients had ANC recovery post-NK cell infusion with cumulative incidence (CI) of ANC recovery to 500/mm3 at 60 days of 58.3%. Eight patients (66.7%) achieved complete remission (CR) (N=4, 33.3%) or CR with incomplete hematologic recovery (CRi) (N=4, 33.3%) at 30 days post-NK cell infusion. One patient with CR had negative minimal residual disease (MRD). Five patients (41.7%) proceeded to haploidentical alloSCT from the same donor and were transplanted in CR/CRi, all but one with persistent MRD. With a median follow-up of 13 months (range 4.1-42.7), median OS and DFS were 17.6 and 3.3 months, and 28 and 20 months for patients receiving alloSCT, respectively. Other outcomes including 2-year OS, DFS, relapse and TRM are shown in Figure 1 and Table 1. No infusion related toxicity or cytokine release syndrome was observed. Two patients were evaluable for FC21-NK cell persistence with haplotype-specific anti-HLA antibodies. FC21-NK cells were detected 5 and 6 weeks after the last FC21-NK cell infusion, respectively. A progressive decrease of the blast population with progressive expansion of the FC21-NK cell population after repeated NK-cell infusions was noted in samples collected from one pt (Figure 2). Persistence is also being evaluated by STR chimerism. Conclusions: Multiple infusions of FC21-NK cells yielded unprecedented outcomes with 66.7% of patients responding and approximately half proceeding to alloSCT in a heavily pre-treated, ultra-refractory, high-risk patient population. Responses were observed irrespective of dose. FC21-NK cell therapy was very well tolerated with no attributable AEs and were shown to persist for at least 5 weeks after infusion. These encouraging results warrant further clinical evaluation of FC21-NK cells in R/R AML patients. Disclosures Ciurea: Kiadis Pharma: Current equity holder in publicly-traded company, Research Funding. Schafer:Kiadis Pharma: Current Employment. Shpall:Zelluna: Membership on an entity's Board of Directors or advisory committees; Adaptimmune: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Licensing Agreement. Konopleva:Calithera: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Forty-Seven: Consultancy, Research Funding; Sanofi: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Ablynx: Research Funding; AbbVie: Consultancy, Research Funding; Ascentage: Research Funding; Rafael Pharmaceutical: Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Omeros: Consultancy; DKMS America: Membership on an entity's Board of Directors or advisory committees; Cytonus: Consultancy; Genzyme: Speakers Bureau; Takeda: Patents & Royalties.

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Donor-Derived Ex-Vivo Activated NK Cells in Management of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation in High-Risk Acute Leukemia

Blood ◽

10.1182/blood.v124.21.312.312 ◽

2014 ◽

Vol 124 (21) ◽

pp. 312-312

Author(s):

Xing-Yu Cao ◽

Tong Wu ◽

Bi-Ping Deng ◽

Rui-Juan Sun ◽

Yue Lu ◽

...

Keyword(s):

High Risk ◽

Cell Therapy ◽

Acute Leukemia ◽

Nk Cells ◽

Culture System ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Original Culture ◽

Nk Cell Therapy

Abstract Introduction: Relapse remains the main cause of failure of hematopoietic stem cell transplantation (HSCT) in acute leukemia. NK cells have the property of killing leukemia cells without GVHD aggravation theoretically. Moreover, in some cases, leukemia cells may lost HLA-I and/HLA-II antigens which would result in poor response to the immunotherapy except NK-based adoptive effectors. Objective: In present study, the safety and efficacy of donor-derived ex-vivo activated NK cells in management of relapse after allogeneic HSCT in high-risk acute leukemia were examined. Patients and methods: Between July 2012 and July 2014, 29 patients with acute leukemia who received NK cell infusion after HSCT were analyzed retrospectively. Some cases failed to chemotherapy combined with donor lymphocyte infusion (DLI) before NK cell therapy. The diagnosis were ALL (10 cases), AML (18 cases) and mixed acute leukemia (1 case). All patients were high-risk leukemia. The disease status before transplant was CR1 in 8 cases, CR2 in 7, CR3 in 1 and non-remission in 13. The types of donor included identical sibling (5 cases), haploientical family member (21 cases) and unrelated donor (3 cases). The conditioning and GVHD prophylactic regimens were reported previously (Lu DP et al., Blood 2006; 107:3065). Minimal residual disease (MRD) was detected by either quantitative RT-PCR for fusion genes or flow cytometry or both. The expression of HLA-I and HLA-II antigens in leukemia cells was evaluated by flow cytometry. Donor-derived either peripheral blood stem cells or lymphocytes were cultured for 6 days using original culture system (AIM-V medium with IL-2, IL-12, IL-15 and IL-21) or modified culture system (SCGM medium with IL-2, IL-12, IL-15, IL-18 and IL-21). Escalated dosage of NK cells were infused starting with 1×105 cells/kg (recipient’s body weight) with or without IL-2 injection. Nine patients were in prevention group and 20 cases were in treatment group. The patients with hematologic relapse received NK cells 3 days later after chemotherapy. Results: Compared with our original culture system, the modified culture system enhanced approximately 10% to 20% of the purity and 4 to 8 fold in number of NK cells by day 6. Furthermore, our modified culture system elevated the expression of function phenotype including TRAIL, NKG2D and CD62L on NK cells in approximately 8 to 10 folds at day 6 and simultaneously stimulated higher level of IFN-γ. One to 4 NK cell infusions were given in each case with two week interval. Two of 29 cases developed mild skin GVHD. No transfusion-related side effects were noted. In prevention group, four of 9 cases remain complete remission, and the other 5 patients became MRD positive or relapse. In treatment group, seven of 20 cases have response to NK cell therapy, and two out of 7 cases who response to NK cells had failed to chemotherapy plus DLI before. Among 11 patients who had response to NK cells, eight of them are AML, and the remaining 3 patients are ALL. Higher response rate (10/23 cases) was seen with NK cell therapy by our modified culture system compared with the one (1/6 cases) by our original culture system. Conclusions: Our preliminary results have demonstrated that donor-derived ex-vivo activated NK cells are safe and effective modality in the management of relapse after allogeneic HSCT in high-risk acute leukemia even failed to chemotherapy combined with DLI. Optimal culture system has improved not only NK cell’s purity, number and function phenotype but also clinical efficacy. Disclosures No relevant conflicts of interest to declare.

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Preclinical assessment of PM21-particle expanded natural killer cells for ovarian cancer treatment.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.127 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 127-127

Author(s):

Jeremiah Oyer ◽

Sarah B. Gitto ◽

Deborah Altomare ◽

Dean A. Lee ◽

Alicja Copik

Keyword(s):

Ovarian Cancer ◽

Cell Therapy ◽

Nk Cells ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

Feeder Cell ◽

Nsg Mice ◽

Nk Cell Therapy

127 Background: Ovarian cancer has high recurrence rate and could benefit from immunotherapy with NK cells. A necessity for NK cell therapy is an efficient way to generate high doses of NK cells. The best method currently used in clinical trials is ex vivo NK cell expansion by co-culture with K562 CML cells, modified to express 41-BBL and membrane bound IL21 (K562.mb21). However feeder cell based methods are limited to ex vivo co-culture, difficult to disseminate, and not allowed in many jurisdictions. To overcome these limitations and to further improve NK cell therapy, we developed a feeder cell free particle based method for NK cell stimulation. These particles (PM21) are nano-scale, made from cell membranes of K562.mb21 cells, and efficiently stimulate NK cell expansion (mean 825 fold in 14 days, range 163–2216, n = 13). Methods: PM21 particles were prepared from K562.mb21 cells with a procedure developed by our group. NK cells were expanded by culturing CD3 depleted PBMCs with PM21 particles or by co-culture with K562.mb21 cells for 14 days as previously described. NSG mice ( ≥ 8 per group) were implanted ip with 1 x 106 SKOV3 ovarian tumor cells, seeded 8 days, and then treated with vehicle or NK cells expanded with PM21 or K562.mb21 cells (two doses of 10 x 106, injected 6 days apart), with or without in vivo administration of PM21 particles (600 µg, 3x weekly), and IL2 (25 KU, 3x weekly), all delivered ip. Survival analysis was performed with log rank (Mantel-Cox) test. Results: Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with K562.mb21 cells or with PM21 particles, allowed significant ( < 0.0001) 10 day increase in survival compared to untreated animals that succumbed on average 21 days after start of treatment. Administration of ip PM21 particles enhanced survival by 5 days (p = 0.056) over no in vivo PM21 groups. Conclusions: NK cells prepared with PM21 particles or with K562.mb21 cells are equivalent in anti-SKOV3 efficacy and in vivo application of PM21 particles provides further benefit. Clinical translation is underway and clinical trials are being planned. PM21 particles can be the next step in development of NK cell therapy for enhancing both efficacy and dissemination of NK cell therapeutics for ovarian cancer.

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799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.799 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A834-A834

Author(s):

Xue Yao ◽

Sandro Matosevic

Keyword(s):

Tumor Microenvironment ◽

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Tumor Cells ◽

Nk Cell ◽

Killer Cell ◽

Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

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Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

Frontiers in Immunology ◽

10.3389/fimmu.2021.668307 ◽

2021 ◽

Vol 12 ◽

Author(s):

Paul D. Bates ◽

Alexander L. Rakhmilevich ◽

Monica M. Cho ◽

Myriam N. Bouchlaka ◽

Seema L. Rao ◽

...

Keyword(s):

Tumor Growth ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

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Biological treatment of pediatric sarcomas by combined virotherapy and NK cell therapy

BMC Cancer ◽

10.1186/s12885-019-6387-5 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Chihab Klose ◽

Susanne Berchtold ◽

Marina Schmidt ◽

Julia Beil ◽

Irina Smirnow ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Real Time ◽

Nk Cell ◽

Sarcoma Cell ◽

Pediatric Sarcoma ◽

Pediatric Sarcomas ◽

Sarcoma Cells ◽

Human Sarcoma ◽

Nk Cell Therapy

Abstract Background In pediatric sarcomas, outcomes of established therapies still remain poor, especially due to high-grade resistances to chemotherapeutic compounds. Taking novel biological approaches into account, virotherapy was found to be efficient in many pediatric sarcoma types. Also NK cell therapy was denoted to represent a promising upcoming strategy for pediatric sarcoma patients. We here investigated a combinatorial approach employing oncolytic measles vaccine virotherapeutics (MeV) together with activated human NK cells (or PBMCs). Methods The human sarcoma cell lines A673 and HT1080 were used to evaluate the efficacy of this combinatorial treatment modality. Oncolysis was determined by measuring real-time cell proliferation using the xCELLigence RTCA SP system. Furthermore, expression of receptors on NK cells and the respective ligands on A673 cells was analyzed by flow cytometry. To measure the protein release of activated NK cells a LEGENDplex™ assay was performed. Results Monotherapy with MeV led to a time- and dose-dependent oncolytic reduction of A673 and HT1080 sarcoma tumor cell masses. Concurrently, such MeV infections did not change the expression of NK cell ligands MICA/B, ULBP1, 2, and 3, CD112, and CD155. As shown by real-time proliferation assays, infections of A673 and HT1080 sarcoma cells with MeV followed by co-culture with activated NK cells or PBMCs led to enhanced sarcoma cell destruction when compared to the respective monotherapies. In parallel, this dual therapy resulted in an increased release of granzymes, perforin, and granulysin from NK cells. In contrast, expression of activation and ontogenesis receptors on NK cells was not found to be altered after co-culture with MeV-infected A673 sarcoma cells. Conclusions Taken together, the combined treatment strategy comprising oncolytic MeV and activated NK cells resulted in enhanced oncolysis of A673 and HT1080 cells when compared to the respective monotherapies. In parallel, we observed an increased release of NK cell activation markers upon co-culture with MeV-infected A673 human sarcoma cells. These results support the onset of clinical trials combining oncolytic virotherapy with NK cell based immunotherapies.

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Development of Multi-Engineered iPSC-Derived CAR-NK Cells for the Treatment of B-Cell Malignancies

Blood ◽

10.1182/blood-2021-148438 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1729-1729

Author(s):

Luis Borges ◽

Mark A Wallet ◽

Chiamin-Liao Bullaughey ◽

Michael F Naso ◽

Buddha Gurung ◽

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Keyword(s):

Tumor Growth ◽

Growth Inhibition ◽

B Cell ◽

Nk Cells ◽

Stock Options ◽

Tumor Growth Inhibition ◽

B Cell Malignancies ◽

Privately Held

Abstract Induced-pluripotent stem cells (iPSCs) can be differentiated into various somatic cells, including different immune cell types. We have engineered iPSC-derived NK cells with multiple features to generate therapeutic candidates designed to eliminate cancer cells while avoiding recognition by the host immune system. The unlimited replication capacity of iPSCs facilitates the engineering of several genetic modifications without the risk of driving cells to exhaustion as in the case of cell products derived from fully differentiated immune cells. Once all edits are completed, our cells are single-cell cloned and each clone is genetically characterized to select clones without off-target insertions or deletions. Following the genetic characterization, selected clones are differentiated and tested in vitro and in vivo to identify the final clinical candidate. The use of a single-cell iPSC clone enables the generation of a master cell bank producing a highly uniform cell product that can be made available off-the-shelf at any clinical site. CNTY-101 is an iPSC-derived CAR-NK clinical candidate for the treatment of B-cell malignancies. It incorporates six gene edits designed to improve persistence and functionality as well as safety. These modifications include edits to reduce graft rejection due to alloreactivity, the expression of a homeostatic cytokine to improve functionality and persistence, the introduction of a chimeric antigen receptor (CAR) targeting CD19 to mediate tumor cell engagement and killing, as well a safety switch to eliminate the cells, if ever necessary. To prevent rejection by the patient's CD8 T cells, the beta-2-microbulin (ß2M) gene was disrupted with simultaneous insertion of a transgene encoding the HLA-E protein tethered with ß2M and a peptide. HLA-E was introduced to prevent NK cell cytotoxicity against the engineered cells, which lack HLA-I. For resistance to CD4 T cell-mediated allogenic immune rejection, the class II major histocompatibility complex transactivator (CIITA) gene was disrupted with simultaneous insertion of a transgene encoding the extra-cellular and transmembrane domains of EGFR, and the NK cell growth factor IL-15. EGFR provides an elimination tag that can be engaged by clinically approved anti-EGFR antibodies, such as cetuximab. Finally, the CAR transgene targeting the CD19 antigen was inserted into the AAVS1 safe harbor locus. Our data indicates that CNTY-101 iNK cells have strong antitumor activity against lymphoma cell lines both in vitro and in vivo. In vitro, CNTY-101 eliminates lymphoma cell lines through multiple rounds of killing without reaching exhaustion. Clones expressing higher levels of IL-15 tend to have better persistence and functionality, with some clones showing robust cytotoxicity for over fifteen rounds of serial killing. In vivo, the clones that demonstrated better in vitro serial killing tend to mediate the best anti-tumor activity in lymphoma xenograft models. Upon 3 weekly doses, the most active candidate clone demonstrated significant tumor growth inhibition after administration of fresh (91 % tumor growth inhibition) or cryopreserved cells (76 % tumor growth inhibition). The efficacy of the EGFR-safety switch was also investigated both in vitro and in vivo. In vitro, addition of cetuximab to co-cultures of IL-2-activated PBMC and cells mediated antibody-dependent cellular cytotoxicity (ADCC) in a concentration-dependent fashion, with an EC50 of 2 ng/ml. In vivo, there was a 96% reduction in the number of iPSC-derived CAR-NK cells in the lungs and a 95% reduction in the number of CAR-NK cells in the blood of mice that received cetuximab versus PBS-treated mice. In summary, CNTY-101 is a novel, multi-engineered, allogeneic CAR-iNK product candidate for the treatment of B-cell malignancies. It includes multiple immune evasion features to prevent recognition by the patient's immune system and expression of IL-15 to facilitate persistence and functionality. We have initiated GMP manufacturing of CNTY-101 and plan to enter clinical trials in 2022. Disclosures Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bullaughey: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Naso: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Gurung: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Keating: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Carton: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Wheeler: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Campion: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Jessup: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Chin: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

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