scholarly journals Engineered Memory-like NK Cars Targeting a Neoepitope Derived from Intracellular NPM1c Exhibit Potent Activity and Specificity Against Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Han Dong ◽  
Guozhu Xie ◽  
Yong Liang ◽  
James Dongjoo Ham ◽  
Juliana Vergara ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Nk Cell ◽  
Leukemia Cells ◽  
Tumor Resistance ◽  
Cell Therapies ◽  
Car T

Introduction: Acute myeloid leukemia (AML) continues to be a major therapeutic challenge. There is an emerging need to develop less toxic and more effective targeted therapies. Natural Killer (NK) cells possess many of the key attributes critical for effective cancer therapies- "born to kill" but without apparent risk of graft versus host disease, cytokine release syndrome, or neurotoxicity. Furthermore, their intrinsic propensity to target myeloid blasts makes them particularly attractive for AML. Despite promising clinical results in blood cancer, the development of NK cell-based therapy remains challenging mostly due to NK cells' short lifespan, inadequate proliferation and lack of specific tumor targeting. Here, we utilized a new approach to arm NK cells for adoptive immunotherapy based on innate cell memory. Chimeric antigen receptors (CARs) significantly enhance anti-tumor specificity and activity of immune effector cells. Our innovative CAR-NK cells target a tumor-specific neoepitope in AML and harness potent function pathways in their design to enhance efficacy and minimize toxicity. Methods: 1. Mutated NPM1c as a CAR Target in AML. Most CAR-T cell therapies target tumor-associated antigens (TAAs), which could lead to on-target/off-tumor toxicity as well as tumor resistance. One way to overcome these drawbacks is to target tumor-specific oncogenic driver mutations. The four-nucleotide duplication in nucleophosmin, referred to as NPM1c, is a driver oncogene mutation in about 35% of AML. The mutation creates a neoepitope that is presented by the most common HLA-A2 allele. Using yeast surface display, we have isolated a human single-chain variable fragment (scFv) that specifically binds to the NPM1c epitope-HLA-A2 complex, but not HLA-A2 alone or HLA-A2 loaded with control peptides. 2. Cytokine-Induced Memory-Like (CIML) NK Cells as a CAR Platform. CIML NK cells can provide a unique platform for development of NK cell CARs based on the favorable safety profile, increased proliferation, prolonged persistence and enhanced anti-leukemia function that we have observed in pre-clinical models (Romee et al, Blood 2012) and in patients (Romee et al, Science Trans Med 2016) treated with un-modified CIML NK cells. 3. Efficient Gene Editing in Primary NK Cells. We have overcome the transduction block in primary human and mouse NK cells by utilizing an unconventional pseudotyped lentivirus based on a unique protein with high expression on CIML NK cells. Results: 1. Engineered CAR-T cells with the isolated scFv exhibit potent cytotoxicity both in vitro and in vivo against NPM1c+HLA-A2+ leukemia cells (OCI-AML3) and primary AML blasts, but not NPM1c-HLA-A2+ leukemia cells (OCI-AML2) or HLA-A2- tumor cells (PC-3). 2. The in vivo anti-leukemia efficacy of anti-NPM1c CAR-T cells was however transient (overall survival extended from 28 to 42 days, median survival extended from 21 to 37 days, compared with the control mice adoptively transferred with untraduced T cells), with unneglectable toxicity. 3. Utilizing an unconventional pseudotyped lentivirus to transduce CIML NK cells from healthy donor blood (n = 5 donors), we have successfully generated anti-NPM1c CAR-NK cells with high transduction efficiency (using MOI = 10: transduction rate mean 48%, range 32% to 65%; compared with 2%, range 0.8% to 4.5% for the conventional approach with VSVG pseudotyped lentivirus). 4. Harnessing key cytokine pathways in the CAR design substantially promoted CAR-NK cell survival (indicated by the enhanced cell viability from 29.7% to 75.2%) and proliferation (marked by the increased levels of ki-67 from 60.2% to 94.5%). 5. Anti-NPM1c CAR significantly promoted anti-tumor function (represented by CD107a, IFN-gamma) and tumor-specific killing (measured by annexin V and 7-AAD) of CIML NK cells against AML with NPM1c oncogene (OCI-AML3). 6. Dual-armed CIML NK cells with CAR and cytokine signaling exhibited optimal specificity and sustainability against AML targets. Conclusion: These results demonstrate that the innovative CAR-CIML NK cells could be developed as an efficient cellular immunotherapy for treating NPM1c+HLA-A2+ AML with potentially reduced on-target/off-tumor toxicity and tumor resistance. Our study should drive novel conception and design of CAR-NK cell therapies against myeloid malignancies in the clinic. Figure Disclosures Ritz: Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy.

scholarly journals Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 407-407
Author(s):  
Frank Cichocki ◽  
Jode P Goodridge ◽  
Ryan Bjordahl ◽  
Svetlana Gaidarova ◽  
Sajid Mahmood ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Abstract Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape. Accordingly, we generated triple gene-modified iPSC-derived NK (iNK) cells, termed "iDuo" NK cells, tailored to facilitate multi-antigen targeting. The iPSC line was clonally engineered to express high-affinity, non-cleavable CD16a (hnCD16), an anti-CD19 CAR optimized for NK cell signaling, and a membrane-bound IL-15/IL-15R fusion (IL-15RF) molecule to enhance NK cell persistence (Fig. 1A). To model antigen escape, we generated CD19 knockout AHR77 lymphoma cells alongside wild type AHR77 cells (both CD20 +) as targets in cytotoxicity assays. Activated peripheral blood NK (PBNK) cells, non-transduced iNK cells, and iDuo NK cells were tested as effectors. Unlike PBNK cells or non-transduced iNK cells, iDuo NK cells efficiently eliminated wild type AHR77 cells with or without the addition of rituximab at all tested E:T ratios. Similarly, iDuo NK cells in combination with rituximab were uniquely able to efficiently eliminate CD19 KO AHR77 cells due to enhanced antibody-dependent cellular cytotoxicity (ADCC) driven by hnCD16 (Fig. 1B-E). Cytotoxicity mediated by iDuo NK cells was also evaluated using primary chronic lymphocytic leukemia (CLL) cells. Compared to expanded PBNK cells and non-transduced iNK cells, only iDuo NK cells (in the absence of rituximab) were able to kill primary CLL cells (Fig. 1F). Expression of IL-15RF by iDuo NK cells uniquely supports in vitro expansion without the need for cytokine supplementation. To determine whether IL-15RF supports in vivo persistence of iDuo NK cells, CD19 CAR iNK cells (lacking IL-15RF) and iDuo NK cells were injected into NSG mice without the addition of cytokines or CD19 antigen availability. iDuo NK cell numbers peaked within a week after injection and persisted at measurable levels for ~5 weeks, in marked contrast to CD19 CAR iNK cell numbers that were undetectable throughout (Fig. 1G). To evaluate the in vivo function of iDuo NK cells, NALM6 leukemia cells were engrafted into NSG mice. Groups of mice received tumor alone or were treated with 3 doses of thawed iDuo NK cells. iDuo NK cells alone were highly effective in this model as evidenced by complete survival of mice in the treatment group (Fig. 1H). To assess iDuo NK cells in a more aggressive model, Raji lymphoma cells were engrafted, and groups of mice received rituximab alone, iDuo NK cells alone, or iDuo NK cells plus rituximab. Mice given the combination of iDuo NK cells and rituximab provided extended survival compared to all other arms in the aggressive disseminated Raji lymphoma xenograft model (Fig. 1I). One disadvantage of anti-CD19 CAR T cells is their inability to discriminate between healthy and malignant B cells. Because NK cells express inhibitory receptors that enable "self" versus "non-self" discrimination, we reasoned that iDuo NK cells could have higher cytotoxicity against tumor cells relative to healthy B cells. To address this, we labeled Raji cells, CD19 + B cells from healthy donor peripheral blood mononuclear cells (PBMCs) and CD19 - PBMCs. Labeled populations of cells were co-cultured with iDuo NK cells, and specific killing was analyzed. As expected, iDuo NK cells did not target CD19 - PBMCs. Intriguingly, iDuo NK cells had much higher cytotoxic activity against Raji cells compared to primary CD19 + B cells, suggesting a preferential targeting of malignant B cells compared to healthy B cells. Together, these results demonstrate the potent multi-antigen targeting capability and in vivo antitumor function of iDuo NK cells. Further, these data suggest that iDuo NK cells may have an additional advantage over anti-CD19 CAR T cells by discriminating between healthy and malignant B cells. The first iDuo NK cell, FT596, is currently being tested in a Phase I clinical trial (NCT04245722) for the treatment of B-cell lymphoma. Figure 1 Figure 1. Disclosures Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Bjordahl: Fate Therapeutics: Current Employment. Gaidarova: Fate Therapeutics, Inc: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Rogers: Fate Therapeutics, Inc: Current Employment. Huffman: Fate Therapeutics, Inc: Current Employment. Lee: Fate Therapeutics, Inc: Current Employment. Szabo: Fate Therapeutics, Inc: Current Employment. Wong: BMS: Current equity holder in publicly-traded company; Fate Therapeutics, Inc: Current Employment. Cooley: Fate Therapeutics, Inc: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment. Miller: Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3090-3090 ◽  
Author(s):  
Folashade Otegbeye ◽  
Nathan Mackowski ◽  
Evelyn Ojo ◽  
Marcos De Lima ◽  
David N. Wald
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

scholarly journals NKG2D-CAR Transduced Primary Natural Killer Cells Efficiently Target Multiple Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 590-590 ◽  
Author(s):  
Alejandra Leivas ◽  
Paula Rio ◽  
Rebeca Mateos ◽  
Mari Liz Paciello ◽  
Almudena Garcia-Ortiz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Car T ◽  
Equity Ownership

Abstract Introduction Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity. Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer. The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients. Methods The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells. Results In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target). In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1). Conclusions It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. Disclosures Rio: Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

scholarly journals Importance of T, NK, CAR T and CAR NK Cell Metabolic Fitness for Effective Anti-Cancer Therapy: A Continuous Learning Process Allowing the Optimization of T, NK and CAR-Based Anti-Cancer Therapies

Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 183
Author(s):  
Adrien Krug ◽  
Adriana Martinez-Turtos ◽  
Els Verhoeyen
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Building Blocks ◽  
Cancer Therapies ◽  
Cell Therapies ◽  
Anti Cancer ◽  
Car T ◽  
Metabolic Fitness

Chimeric antigen receptor (CAR) T and CAR NK cell therapies opened new avenues for cancer treatment. Although original successes of CAR T and CAR NK cells for the treatment of hematological malignancies were extraordinary, several obstacles have since been revealed, in particular their use for the treatment of solid cancers. The tumor microenvironment (TME) is competing for nutrients with T and NK cells and their CAR-expressing counterparts, paralyzing their metabolic effective and active states. Consequently, this can lead to alterations in their anti-tumoral capacity and persistence in vivo. High glucose uptake and the depletion of key amino acids by the TME can deprive T and NK cells of energy and building blocks, which turns them into a state of anergy, where they are unable to exert cytotoxic activity against cancer cells. This is especially true in the context of an immune-suppressive TME. In order to re-invigorate the T, NK, CAR T and CAR NK cell-mediated antitumor response, the field is now attempting to understand how metabolic pathways might change T and NK responses and functions, as well as those from their CAR-expressing partners. This revealed ways to metabolically rewire these cells by using metabolic enhancers or optimizing pre-infusion in vitro cultures of these cells. Importantly, next-generation CAR T and CAR NK products might include in the future the necessary metabolic requirements by improving their design, manufacturing process and other parameters. This will allow the overcoming of current limitations due to their interaction with the suppressive TME. In a clinical setting, this might improve their anti-cancer effector activity in synergy with immunotherapies. In this review, we discuss how the tumor cells and TME interfere with T and NK cell metabolic requirements. This may potentially lead to therapeutic approaches that enhance the metabolic fitness of CAR T and CAR NK cells, with the objective to improve their anti-cancer capacity.

scholarly journals Human Cytokine-Induced Memory-like NK Cells Exhibit in Vivo Anti-Leukemia Activity in Xenografted NSG Mice and in Patients with Acute Myeloid Leukemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 101-101
Author(s):  
Rizwan Romee ◽  
Rosario Maximillian ◽  
Melissa M Berrien-Elliott ◽  
Julia A Wagner ◽  
Brea A Jewell ◽  
...  
Keyword(s):  
Nk Cells ◽  
Myeloid Leukemia ◽  
Low Dose ◽  
Nk Cell ◽  
Leukemia Cells ◽  
Cell Frequency ◽  
Nsg Mice ◽  
Post Infusion

Abstract Natural killer (NK) cells mediate anti-AML responses and previously published clinical trials of adoptive allogeneic NK cell therapy provide proof-of-principle that NK cells may eliminate leukemia cells in patients. However, complete remissions occur in 30-50% of patients with active AML and are typically of limited duration. Thus, improvements are needed for this promising cellular immunotherapy strategy. Following paradigm-shifting studies in mice, it was established that human NK cells exhibit an innate 'memory-like' responses following a brief, combined pre-activation with IL-12, -15, and -18 (Romee R et. al., Blood, 2012). These long-lived memory-like NK cells have an enhanced ability to produce IFN-g in response to restimulation with cytokines or activating receptor ligation, even following extensive proliferation. We hypothesized that memory-like NK cells exhibit enhanced responses to myeloid leukemia. Compared to control NK cells from the same donor, IL-12/15/18-induced memory-like NK cells produced significantly increased IFN-g upon co-culture with primary AML blasts in vitro (P<0.001), following 7 days of rest in low dose IL-15 vitro. In addition, memory-like NK cells had increased granzyme B expression (P<0.01), and enhanced killing of K562 leukemia targets in vitro (P<0.05). Utilizing an in vivo xenograft model of human NK cells in NSG mice (Leong J et. al., BBMT, 2014), IL-12/15/18-induced memory-like NK cells that differentiated in NSG mice for 7 days exhibited increased IFN-g responses after ex vivo re-stimulation with K562 leukemia, confirming their memory-like functionality (P<0.05). To test in vivo responses to human leukemia in this model, luciferase-expressing K562 cells were engrafted into NSG mice (1x106/mouse, IV), and on day 3, groups of mice were injected with IL-12/15/18-pre-activated or control NK cells from the same donor (4x106/mouse). Mice treated with a single dose of memory-like NK cells exhibited significantly improved in vivo leukemia control measured by whole mouse bioluminescent imaging (P=0.03), as well as overall survival (P<0.05), compared to mice treated with control or no NK cells. Based on these pre-clinical findings, we initiated a first-in-human clinical trial of HLA-haploidentical IL-12/15/18-induced memory-like NK cells in patients with AML (NCT01898793). Relapsed/refractory (rel/ref) AML patients receive lymphodepleting non-myeloablative flu/cy conditioning, infusion of a single dose of CD56+CD3- memory-like donor NK cells, followed by two weeks of low dose rhIL-2. Three patients were treated at dose level 1 (0.5x106 cells/kg) and two patients treated at dose level 2 (1.0x106/kg) with no DLTs observed, and accrual continues. Correlative analyses utilizing donor-specific HLA mAbs allow tracking of donor memory-like NK cell frequency and function following adoptive transfer. Donor memory-like NK cells were detectable in the PB and BM of all tested patients with informative HLA (4/5), peak in frequency at 7-8 days post-infusion, and contract after 14-21 days as expected following recipient T cell recovery (Figure). Memory-like NK cells exhibit significantly increased Ki67%+ as a marker of proliferation at day 7 [97.8+1.0% (donor) vs. 21.6+5.5% (recipient), mean+SEM, P<0.001]. Moreover, functional analyses of NK cells at days 7-8 post-infusion reveal increased numbers of donor IFN-g+ NK cells following restimulation with K562 leukemia cells in the same blood [1009+590 (donor) vs. 8+3 (recipient) IFN-g+ NK cells] or BM [686+423 (donor) vs. 4+2 (recipient) IFN-g+ NK cells] samples. Two of four evaluable patients treated with memory-like NK cells had leukemia free BM and PB at days 14 post-therapy, which correlated with BM NK cell frequency and IFN-g production (Figure). CIML007 had rel/ref AML with 48% BM blasts pre-therapy, and had no evidence of leukemia on day 14, 28, and 100 BM biopsies, and has an ongoing complete remission more than 100 days after this therapy. CIML009 had 80% BM blasts pre-therapy, and had no evidence of leukemia on day 14 BM biopsy post-infusion. Thus, human IL-12/15/18-induced memory-like NK cells expand and have enhanced anti-AML function following adoptive transfer in patients, thereby constituting a promising translational innovation for immunotherapy of AML. Figure 1. Figure 1. Disclosures Fehniger: Celgene: Research Funding.

scholarly journals A Phase I Study of FT538, a First-of-Kind, Off-the-Shelf, Multiplexed Engineered, iPSC-Derived NK Cell Therapy As Monotherapy in Relapsed/Refractory Acute Myelogenous Leukemia and in Combination with Daratumumab or Elotuzumab in Relapsed/Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Murali Janakiram ◽  
Ravi Vij ◽  
David S. Siegel ◽  
Ted Shih ◽  
Sara Weymer ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Myelogenous Leukemia ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Cell Therapies ◽  
Acute Myelogenous

Background: Allogeneic natural killer (NK) cell therapies have been well-tolerated with documented anti-tumor activity in patients with relapsed/refractory (r/r) hematologic malignancies including acute myelogenous leukemia (AML) and multiple myeloma (MM) (Lupo et al. 2019). Allogeneic NK cell therapies may offer an improved safety profile characterized by the absence of cytokine release syndrome and neurologic toxicity compared with T-cell therapies (Liu et al. 2020). However, in comparison to T cells, NK cells have limited in vivo expansion and a short half-life, and the potential to generate deeper and more durable anti-tumor responses through multi-dose administration is limited by the inability to consistently manufacture and administer more than one dose of allogeneic NK cells. The monoclonal antibodies (mAbs) daratumumab and elotuzumab have demonstrated clinical benefit and are approved for the treatment of MM (Darzalex® USPI, Empliciti® USPI). However, durable responses and disease-free survival remain limited. Engagement of the Fc portion of the mAb with CD16 on NK cells, which promotes antibody-dependent cellular cytotoxicity (ADCC), is a major contributor to the efficacy of daratumumab and elotuzumab. It is hypothesized that more clinically meaningful outcomes may be achieved by combining therapeutic mAbs with allogeneic NK cells engineered to enhance ADCC. FT538 is an investigational, first-of-kind, multiplexed engineered NK cell therapy generated from a clonal master engineered induced pluripotent stem cell (iPSC) line, which can be used as a renewable source for the mass production of off-the-shelf NK cells for broad patient access. FT538 is engineered with three modalities for enhanced innate immunity: (1) high-affinity 158V, non-cleavable CD16 Fc receptor for augmented ADCC; (2) interleukin (IL)-15/IL-15 receptor fusion that promotes cytokine-autonomous persistence; and (3) CD38 knockout to mitigate NK cell fratricide by CD38-directed mAbs. In preclinical studies, FT538 combined with daratumumab against MM targets demonstrated avoidance of daratumumab-mediated fratricide and significantly enhanced ADCC in vitro in a serial stimulation cytotoxicity assay compared with peripheral blood NK cells, and the combination of FT538 with daratumumab led to highly effective tumor control compared with daratumumab alone in an in vivo MM xenograft model (Bjordahl et al. 2019). Study Design and Methods: This study is a multicenter, multi-dose, Phase I clinical trial of FT538 in patients with r/r AML or r/r MM. The primary objectives are to determine the recommended Phase II dose of FT538 as monotherapy in r/r AML and in combination with daratumumab or elotuzumab in r/r MM. Key secondary objectives include evaluation of FT538 safety and tolerability, anti-tumor activity, and pharmacokinetics (PK) as monotherapy in r/r AML and combined with mAbs in r/r MM. Exploratory objectives include characterization of FT538 pharmacodynamics as assessed by peripheral blood biomarkers, assessment of minimal residual disease, and characterization of the tumor microenvironment in pre- and post-treatment tumor biopsies. The dose-escalation part of the trial utilizes a 3+3 design to identify the maximum tolerated dose of up to three doses of FT538 on Days 1, 8, and 15 as a monotherapy in r/r AML (Regimen A) and in combination with daratumumab (Regimen B) or elotuzumab (Regimen C) in r/r MM. The dose-expansion part of the trial will further characterize the safety, efficacy, and PK of FT538 in all regimens. The trial will test up to five FT538 dose levels ranging from 50 million to 1.5 billion cells. Up to 105 patients will be enrolled. The mAbs in Regimens B and C will be administered based on dosing schedules per their respective prescribing information. Lympho-conditioning consisting of three consecutive days of fludarabine and cyclophosphamide will be administered prior to the first dose of FT538. Key inclusion criteria include r/r disease after standard approved therapies for r/r AML or r/r MM, as applicable, measurable disease for r/r MM, and adequate organ function. Key exclusion criteria include active central nervous system disease, need for systemic immunosuppressive therapy, and prior allograft organ transplant. This trial is expected to begin patient enrollment in 2020. Disclosures Janakiram: Takeda, Fate, Nektar: Research Funding. Siegel:Karyopharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; Merck: Consultancy, Honoraria, Speakers Bureau; Celulatiry: Consultancy; BMS: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Shih:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Weymer:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Chu:Roche Holding AG: Current equity holder in publicly-traded company; Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as lympho-conditioning therapy prior to FT538 administration.

scholarly journals Cytokine-Induced Memory-like NK Cells Have a Distinct Single Cell Transcriptional Profile and Persist for Months in Adult and Pediatric Leukemia Patients after Adoptive Transfer

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3825-3825
Author(s):  
Jennifer A. Foltz ◽  
Melissa M. Berrien-Elliott ◽  
David A. Russler-Germain ◽  
Carly C. Neal ◽  
Jennifer Tran ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
Transcription Factors ◽  
Nk Cells ◽  
Single Cell ◽  
Adoptive Transfer ◽  
Research Funding ◽  
Nk Cell ◽  
Mass Cytometry

Abstract Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies. To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2). We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design. Figure 1 Figure 1. Disclosures Foltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.

Expression of Glucocorticoid-Induced TNF Receptor Ligand on Acute Myeloid Leukemia Cells Mediates the Release of Immunosuppressive Cytokines and Impairs NK Cell-Mediated Immune Surveillance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1941-1941
Author(s):  
Matthias Krusch ◽  
Katrin M. Baltz ◽  
Tina Baessler ◽  
Mercedes Kloss ◽  
Ingrid Kumbier ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Cellular Proliferation ◽  
Nk Cell ◽  
Leukemia Cells ◽  
Tnf Receptor ◽  
Ifn Γ ◽  
Acute Myeloid

Abstract NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance of hematological malignancies including acute myeloid leukemia (AML). Among the molecules influencing host-tumor interaction are many members of the TNF superfamily, which mediate multiple cellular functions including cellular proliferation, differentiation and cell death. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and nuclear factor kappa B and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while solid tumors express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell cytotoxicity and IFN-γ production. Here we analyzed the role of GITR and its ligand in AML. We report for the first time that GITRL is expressed on primary AML cells in 18 of 30 patients as determined by FACS and RT-PCR analysis. Reverse signaling through GITRL using a recombinant GITR-Ig fusion protein induces the release of the immunoregulatory cytokines IL-10 and TNF as determined by ELISA. GITRL-mediated cytokine production of AML cells is abrogated by inhibition of mitogen activated protein kinase (MAPK) pathways as demonstrated by addition of the specific p38 MAPK inhibitor SB202190, the specific JNK inhibitor SP600125 and the specific ERK Inhibitor II. Furthermore, binding of AML-expressed GITRL to GITR on NK cells downregulates cellular cytotoxicity and IFN-γ production in AML-NK cell cocultures, which can be overcome by addition of GITR-blocking antibodies as determined by cytotoxicity assays and ELISA. Thus, our data indicate that GITRL expression in AML substantially influences tumor immunoediting and enables the escape of leukemia cells from NK cell-mediated immunosurveillance.

scholarly journals A Bicistronic Vector Expressing CD16 and a Membrane Bound IL-15 Construct in iPSC Derived NK Cells Increased Cytotoxicity and Persistence

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4809-4809
Author(s):  
Alexander G Allen ◽  
Rithu Pattali ◽  
Kaitlyn M Izzo ◽  
Jared A Getgano ◽  
Kevin M Wasko ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Constitutive Expression ◽  
Publicly Traded ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T ◽  
Membrane Bound

Abstract Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability. Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence. CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity. The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time. In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. Disclosures Allen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Mutated GM-CSF-Based CAR T-Cells Targeting CD116/CD131 Complexes Exhibit Enhanced Anti-Tumor Effects Against Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-37
Author(s):  
Shoji Saito ◽  
Aiko Hasegawa ◽  
Mika Nagai ◽  
Yoichi Inada ◽  
Hirokazu Morokawa ◽  
...  
Keyword(s):  
T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Antigen Recognition ◽  
Gm Csf ◽  
Car T Cells ◽  
Car T ◽  
Myelomonocytic Leukemia

Background: The prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor; therefore, novel treatment strategies are required urgently. Meanwhile, recent clinical trials have demonstrated that CAR-T cells for AML have been less successful than those targeting CD19 for B cell malignancies. Recently, we developed piggyBac-modified ligand-based CAR-T cells that target CD116, also called granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) α chain, for treating juvenile myelomonocytic leukemia (Nakazawa, et al. J Hematol Oncol. 2016). Since CD116 is overexpressed in 60%-80% of AML cases, the present study aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. Methods: CD116 expression in AML cell lines or primary leukemia cells were examined using flow cytometry. The original piggyBac transposon plasmid for GMR CAR comprises GM-CSF as an antigen recognition site, IgG1 CH2CH3 hinge region, CD28 costimulatory domain, and CD3ζ chain. To improve the in vivo persistency and anti-tumor effects, two types of spacer (∆CH2H3 and G4S) that lack CH2CH3 lesion were newly constructed. In order to modulate the antigen recognition ability, mutated ligand-based GMR CAR vectors were constructed with a mutation at residue 21 of GM-CSF that is reported to play a critical role in its biological activity (Lopez, et al. Embo j. 1992). All the GMR CAR-T cells were generated with piggyBac gene modification. To investigate the in vitro anti-tumor activity, GMR CAR-T cells were co-cultured with AML cell lines. In order to evaluate the in vivo anti-tumor effects, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were intravenously injected with THP-1, THP1-ffLuc, or MV4-11 and then treated with GMR CAR-T cells. To characterize the safety profile of GMR CAR-T cells, peripheral blood mononuclear cells or polymorphonuclear cells were co-cultured with GMR CAR-T cells at an effector:target ratio of 1:1 for 3 days. Thereafter, B cells, NK cells, neutrophils, and monocytes were quantified using flow cytometry using counting beads. Results: Approximately 80% of the AML cells predominant in myelomonocytic leukemia expressed CD116. PiggyBac-modified GMR CAR-T cells displayed a favorable CD45RA+CCR7+-dominant phenotype, consistent with our previous findings. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. GMR CAR-T cells incorporating a G4S spacer significantly improved the long-term in vitro and in vivo anti-tumor effects as compared to those incorporating a ∆CH2CH3 spacer. Furthermore, by employing a mutated GM-CSF at residue 21 (E21K and E21R) as an antigen recognition site, the in vivo anti-tumor effects were also substantially improved along with prolonged survival (Figure 1) over controls (PBS or CD19.CAR-T cells) (all, p &lt; 0.01) as well as over GMR CAR-T cells with a wild-type GM-CSF ligand (E21R: p &lt; 0.01; E21K: p = 0.02), with 4 out of 5 mice surviving for &gt; 150 days. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. It is noteworthy that the cytotoxic effects of GMR CAR-T cells on normal neutrophils, T cells, B cells, and NK cells were minimal. Conclusions: GMR CAR-T cell therapy appears to be a potentially useful strategy for CD116+ R/R AML. Based on the promising results, we plan to perform the first-in-human clinical trial of GMR CAR-T cells. Disclosures Saito: Toshiba Corporation: Research Funding. Hasegawa:Toshiba Corporation: Research Funding. Inada:Kissei Pharmaceuticals: Ended employment in the past 24 months. Nakashima:Toshiba Corporation: Research Funding. Yagyu:Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

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