722 INBRX-121 is an NKp46-targeted detuned IL-2 with antitumor activity as a monotherapy or in combination with multiple cancer immunotherapy modalities

BackgroundNatural Killer (NK) cells play a pivotal role in cancer immunosurveillance due to their potent cytolytic activity and NK cell-centric therapies have emerged as safer alternatives to targeting T cells.1 2 Interleukin 2 (IL-2) drives NK cell expansion and activity, but its therapeutic utility is limited by rapid clearance, expansion of immunosuppressive regulatory T cells, and by severe dose-limiting toxicities.3 INBRX-121 overcomes these liabilities through specific targeting of an affinity-detuned IL-2 variant to cells expressing NKp46.MethodsAn IL-2 variant was engineered to eliminate binding to CD25 and to have attenuated affinity for CD122. This detuned cytokine was fused to a high-affinity single-domain antibody targeting NKp46 to generate INBRX-121. The ability of INBRX-121 to target IL-2-like signaling specifically to NKp46-expressing cells was evaluated in vitro using human lymphocytes by measuring STAT5 signaling and cytotoxic activity in tumor cell co-cultures. Characterization of the pharmacokinetic/pharmacodynamic relationship of INBRX-121 was completed in non-human primates across escalating dose levels, while anti-tumor activity as a monotherapy and in combination with Rituximab or PD-1 checkpoint blockade was tested in Raji xenografts and syngeneic CT-26 mouse models, respectively.ResultsINBRX-121 induces a STAT5 signal equal to that of wild-type IL-2 in human lymphocytes but shows an NK cell-centric activity profile. Cells targeted by INBRX-121 have increased proliferative capacity and improved cytotoxicity in antibody-dependent and -independent tumor cell killing assays. INBRX-121 shows prolonged pharmacokinetic exposure in vivo and is well-tolerated in mice and cynomolgus monkeys. The NKp46-specific IL-2 stimulus in these models results in a robust, dose-dependent NK cell expansion. As predicted by its in vitro activity, INBRX-121 also enhances the cytotoxic capacity of NK cells in vivo measured via elevated intracellular levels of Granzyme B. In a Raji xenograft model, INBRX-121 slows tumor growth as a single agent and synergizes with Rituximab to induce complete tumor regression. Similarly, co-treatment with INBRX-121 improves the incomplete suppression of CT-26 tumor growth by a PD-1 blocking antibody to yield complete responses that show immunological memory upon re-challenge.ConclusionsINBRX-121 offers a unique approach to overcoming the limitations of current IL-2 therapeutics. NKp46-targeting of a detuned IL-2 variant helps to avoid IL-2-mediated toxicity while enhancing the antitumor activities of NK cells. Through its novel therapeutic concept INBRX-121 provides a promising treatment option for multiple cancer indications both as a monotherapy and in combination with a variety of frontline agents.ReferencesShimasaki N, Jain A, Campana D. NK cells for cancer immunotherapy. Nat Rev Drug Discov 2020;19:200–218.Liu S, Galat V, Galat Y, Lee Y, Wainwright D, Wu J. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol 2021;14:7.Overwijk W, Tagliaferri M, Zalevsky J. Engineering IL-2 to give new life to T Cell immunotherapy. Annu Rev Med 2021;72:281–311.Ethics ApprovalAll animal studies were conducted in accordance with AAALAC regulations and were approved by the IACUC for Explora BioLabs (#SP17-010-013) and BTS Research (20-015 Enrollment 05).

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Optimal NK Cell Expansion Depends on Accessory Cells, Synergy between Physiologic Concentrations of IL-2 and IL-15, and Umbilical Cord Blood (UCB) NK Cell Precursors Expand Better Than Adult NK Cells.

Blood ◽

10.1182/blood.v108.11.3642.3642 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3642-3642 ◽

Cited By ~ 1

Author(s):

Purvi Gada ◽

Michelle Gleason ◽

Valarie McCullar ◽

Philip B. McGlave ◽

Jeffrey S. Miller

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Cell Expansion ◽

Nk Cell ◽

High Dose ◽

Unexpected Result ◽

Accessory Cells

Abstract Allogeneic NK cells may play a therapeutic role in treating patients with AML. We have previously shown that high dose cyclophosphamide (120 mg/kg × 1 day) and fludarabine (125 mg/m2 × 5 days) can clear lymphoid space and induce a surge of endogenous IL-15 to expand haploidentical NK cells obtained from CD3-depleted lymphapheresis products from adult donors. In this initial study, 5 of 19 patients achieved remissions and in vivo NK cell expansion. Limitations of this therapy includeinability of NK cells to expand in most patients,development of PTLD (in one patient) andinadequate disease control.We hypothesized that contaminating T cells could compete for NK cell expansion, that B-cells may contribute to PTLD, and that a 2-step NK cell purification method using CD3 depletion followed by CD56 selection (CliniMacs) may overcome these problems. We tested this in 9 patients with advanced AML. The purified NK cells, activated with 1000 U/ml IL-2 (16–20 hours), were infused 48 hours after the last fludarabine dose. Patients then received subcutaneous IL-2 (10 MU) every other day × 6 doses to expand NK cells in vivo. None of the 9 pts treated on this protocol achieved remission or exhibited evidence of in vivo expansion. Several studies were designed to investigate this unexpected result. First, we found that the more extensive processing resulted in approximately 1/3 the NK cell recovery compared to CD3 depletion alone (38±% viable NK cells vs. 91±2% respectively). In addition, we questioned whether the contaminating B cells and monocytes that were removed in the 2-step depletion strategy had served a critical role in NK cell activation or expansion. Cytotoxicity assays performed against K562 targets showed that the killing was about 3-fold higher with the purified (CD3-CD56+) product compared the CD3-depleted product alone (P=0.001 at E:T of 6.6:1). Proliferation, measured by a 6-day thymidine assay, was higher in proportion to the higher NK cell content. The only difference between the two NK products was their expansion after 14 days of culture, where the CD3-depleted product, with contaminating B-cells and monocytes, gave rise to greater NK cell expansion (14 ±3-fold) compared to the 2-step purified product (4.5±0.9, n=6, P=0.005). If this finding holds true in vivo, the co-infusion of accessory cells may be required for NK cell expansion. We next developed in vitro assays using very low concentrations (0.5 ng/ml) of IL-2 and IL-15 to understand their role in expansion. IL-2 or IL-15 alone induced low proliferation and the combination was synergistic. Lastly, UCB, a rich source of NK cell precursors, was compared to adult NK cells. In a short term proliferation assay, CD56+ NK cells stimulated with IL-2 + IL-15 expanded better from adult donors (61274±12999, n=6) than from UCB (20827± 6959, n=5, P=0.026) but there was no difference after 14 days in expansion culture suggesting that the only difference is in kinetics. However, UCB depleted of T-cells (enriching for NK cell precursors) exhibited higher fold expansion over 14 days under different culture conditions conducive to NK cell progenitors. In conclusion, NK cell expansion in vitro depends on cell source, IL-2 and IL-15 (increased in vivo after lymphoid depleting chemotherapy) as well as accessory cells. The role of these factors to enhance in vivo expansion is under clinical investigation to further exploit the NK cell alloreactivity against AML targets.

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114 Preclinical development of a novel iPSC-derived CAR-MICA/B NK cell immunotherapy to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0114 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A126-A126

Author(s):

John Goulding ◽

Mochtar Pribadi ◽

Robert Blum ◽

Wen-I Yeh ◽

Yijia Pan ◽

...

Keyword(s):

T Cells ◽

Cancer Immunotherapy ◽

Tumor Immunity ◽

Immune Cell ◽

Nk Cell ◽

Tumor Escape ◽

Car T Cells ◽

Car T

BackgroundMHC class I related proteins A (MICA) and B (MICB) are induced by cellular stress and transformation, and their expression has been reported for many cancer types. NKG2D, an activating receptor expressed on natural killer (NK) and T cells, targets the membrane-distal domains of MICA/B, activating a potent cytotoxic response. However, advanced cancer cells frequently evade immune cell recognition by proteolytic shedding of the α1 and α2 domains of MICA/B, which can significantly reduce NKG2D function and the cytolytic activity.MethodsRecent publications have shown that therapeutic antibodies targeting the membrane-proximal α3 domain inhibited MICA/B shedding, resulting in a substantial increase in the cell surface density of MICA/B and restoration of immune cell-mediated tumor immunity.1 We have developed a novel chimeric antigen receptor (CAR) targeting the conserved α3 domain of MICA/B (CAR-MICA/B). Additionally, utilizing our proprietary induced pluripotent stem cell (iPSC) product platform, we have developed multiplexed engineered, iPSC-derived CAR-MICA/B NK (iNK) cells for off-the-shelf cancer immunotherapy.ResultsA screen of CAR spacer and ScFv orientations in primary T cells delineated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control against MICA+ cancer cells. The novel CAR-MICA/B design was used to compare efficacy against NKG2D CAR T cells, an alternative MICA/B targeting strategy. CAR-MICA/B T cells showed superior cytotoxicity against melanoma, breast cancer, renal cell carcinoma, and lung cancer lines in vitro compared to primary NKG2D CAR T cells (p<0.01). Additionally, using an in vivo xenograft metastasis model, CAR-MICA/B T cells eliminated A2058 human melanoma metastases in the majority of the mice treated. In contrast, NKG2D CAR T cells were unable to control tumor growth or metastases. To translate CAR-MICA/B functionality into an off-the-shelf cancer immunotherapy, CAR-MICA/B was introduced into a clonal master engineered iPSC line to derive a multiplexed engineered, CAR-MICA/B iNK cell product candidate. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iNK cells displayed MICA specificity, resulting in enhanced cytokine production, degranulation, and cytotoxicity. Furthermore, in vivo NK cell cytotoxicity was evaluated using the B16-F10 melanoma cell line, engineered to express MICA. In this model, CAR-MICA/B iNK cells significantly reduced liver and lung metastases, compared to untreated controls, by 93% and 87% respectively.ConclusionsOngoing work is focused on extending these preclinical studies to further support the clinical translation of an off-the-shelf, CAR-MICA/B iNK cell cancer immunotherapy with the potential to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing.ReferenceFerrari de Andrade L, Tay RE, Pan D, Luoma AM, Ito Y, Badrinath S, Tsoucas D, Franz B, May KF Jr, Harvey CJ, Kobold S, Pyrdol JW, Yoon C, Yuan GC, Hodi FS, Dranoff G, Wucherpfennig KW. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018 Mar 30;359(6383):1537–1542.

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DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

Journal of Experimental Medicine ◽

10.1084/jem.20081752 ◽

2008 ◽

Vol 205 (13) ◽

pp. 2965-2973 ◽

Cited By ~ 195

Author(s):

Susan Gilfillan ◽

Christopher J. Chan ◽

Marina Cella ◽

Nicole M. Haynes ◽

Aaron S. Rapaport ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Adhesion Molecules ◽

Cd8 T Cells ◽

Nk Cell ◽

Cd8 T Cell ◽

Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

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Stem Cell Factor Enhances Interleukin-2–Mediated Expansion of Murine Natural Killer Cells In Vivo

Blood ◽

10.1182/blood.v90.9.3647 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3647-3653 ◽

Cited By ~ 26

Author(s):

Todd A. Fehniger ◽

William E. Carson ◽

Ewa Mrózek ◽

Michael A. Caligiuri

Keyword(s):

Nk Cells ◽

Cell Expansion ◽

Stem Cell Factor ◽

Low Dose ◽

Nk Cell ◽

Interleukin 2 ◽

Innate Immune ◽

Ifn Γ

Abstract The administration of low dose interleukin-2 (IL-2) results in a selective expansion of natural killer (NK) cells in vivo, and promotes the differentiation of NK cells from hematopoietic precursor cells in vitro. We have previously shown that stem cell factor (SCF ), the ligand to the c-kit tyrosine kinase receptor, enhances IL-2–induced NK cell proliferation and differentiation in vitro. Here, we investigated the effects of SCF plus IL-2 delivered to mice in vivo. Eight-week-old C57BL/6 mice were treated with a continuous subcutaneous infusion of IL-2 (1 × 104 IU/d) plus a daily intraperitoneal dose of SCF (100 μg/kg/d), IL-2 alone, SCF alone, or vehicle alone for 8 weeks. The in vivo serum concentration of IL-2 ranged between 352 ± 12.0 pg/mL and 606 ± 9.0 pg/mL, achieving selective saturation of the high affinity IL-2 receptor, while the peak SCF serum concentration was 296 ± 13.09 ng/mL. Alone, the daily administration of SCF had no effect on the expansion of NK cells. The continuous infusion of IL-2 alone did result in a significant expansion of NK1.1+CD3− cells compared to mice treated with placebo or SCF. However, mice treated with both SCF and IL-2 showed an increase in the absolute number of NK cells that was more than twofold that seen with IL-2 alone, in the spleen (P ≤ .005), bone marrow (P ≤ .025), and blood (P < .05). NK cytotoxic activity against YAC-1 target cells was significantly higher for mice treated with SCF plus IL-2, compared to mice treated with IL-2 alone (P ≤ .0005). Interferon-γ (IFN-γ) production in cytokine-activated splenocytes was also greater for the SCF plus IL-2 group, over IL-2 treatment alone (P ≤ .01). The effect of SCF plus IL-2 on NK cell expansion was likely mediated via NK cell precursors, rather than mature NK cells. In summary, we provide the first evidence that SCF can significantly enhance expansion of functional NK cells induced by the prolonged administration of low dose IL-2 in vivo. Since the NK cell is a cytotoxic innate immune effector and a potent source of IFN-γ, this therapeutic strategy for NK cell expansion may serve to further enhance innate immune surveillance against malignant transformation and infection in the setting of cancer and/or immunodeficiency.

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Mechanisms of IL-21 Enhancement of Rituximab Efficacy in a Lymphoma Xenograft Model.

Blood ◽

10.1182/blood.v104.11.1404.1404 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1404-1404

Author(s):

Steve D. Hughes ◽

Ken Bannink ◽

Cecile Krejsa ◽

Mark Heipel ◽

Becky Johnson ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Antibody Therapy ◽

Xenograft Model ◽

Tumor Model ◽

Cell Types ◽

Current Evidence

Abstract Interleukin 21 (IL-21) is an IL-2 family cytokine produced by activated CD4+ T cells. Potent effects of IL-21 have been observed on the growth, survival, and functional activation of T cells, B cells, and natural killer (NK) cells. A Phase I clinical trial of IL-21 in metastatic melanoma and renal cell carcinoma is currently in progress. We recently reported that IL-21 significantly enhanced rituximab mediated clearance of CD20+ lymphoma cell lines both in vitro and in vivo, and that these effects were potentially mediated through IL-21 enhancement of NK cell capacity to effect antibody dependent cellular cytotoxicity (ADCC). Specifically, NK cells treated with IL-21 showed increased cytotoxicity, granzyme B and IFNg production. Current studies aim to further evaluate the mechanisms by which IL-21 enhances ADCC. A number of observations suggest a multi-factorial basis for IL-21 synergy with rituximab. In a xenograft tumor model, SCID mice were injected IV with HS Sultan cells on day 0. Treatment with recombinant murine IL-21 (mIL-21; starting day 1) combined with rituximab (starting day 3) resulted in significantly increased survival (70% vs. 20% on day 100), compared to rituximab alone. In separate studies, the spleens of mice treated with mIL-21 showed increased numbers of activated macrophages and granulocytes. As macrophages and granulocytes can participate in ADCC, IL-21 synergy with rituximab in vivo may be partly dependent on its activation of these cell types. We have also evaluated whether direct effects of IL-21 on lymphoma cells contribute to enhancement of rituximab efficacy. The xenogeneic B lymphoma models in which IL-21 plus rituximab exhibited enhanced survival are highly aggressive and these models were not shown to respond to treatment with mIL-21 alone. In vitro studies were performed to determine if IL-21 could potentiate the growth inhibitory and pro-apoptotic effects of rituximab. In the absence of effector cells synergistic interaction was not observed. In addition, we tested the ability of IL-21 to enhance cytotoxicity when combined with antibodies targeting non-hematopoietic tumor cells (e.g. trastuzumab). Human NK cells treated with IL-21 displayed significantly increased cytotoxicity in ADCC assays using trastuzumab to target breast cancer cells expressing varying levels of HER-2 antigen. In summary, the current evidence suggests that IL-21 can enhance antibody-mediated tumor cell lysis through activation of multiple effectors of ADCC. Thus IL-21 may prove to be broadly applicable to monoclonal antibody therapy of cancer.

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The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T-, B-, and NK-cell development

Blood ◽

10.1182/blood-2002-02-0438 ◽

2003 ◽

Vol 101 (3) ◽

pp. 1015-1023 ◽

Cited By ~ 83

Author(s):

Remko Schotte ◽

Marie-Clotilde Rissoan ◽

Nathalie Bendriss-Vermare ◽

Jean-Michel Bridon ◽

Thomas Duhen ◽

...

Keyword(s):

T Cells ◽

Transcription Factor ◽

Nk Cells ◽

Progenitor Cells ◽

Nk Cell ◽

Cdna Subtraction ◽

Common Precursor ◽

Functional Features

Abstract Human plasmacytoid dendritic cells (pDCs), also called type 2 dendritic cell precursors or natural interferon (IFN)–producing cells, represent a cell type with distinctive phenotypic and functional features. They are present in the thymus and probably share a common precursor with T and natural killer (NK) cells. In an effort to identify genes that control pDC development we searched for genes of which the expression is restricted to human pDC using a cDNA subtraction technique with activated monocyte-derived DCs (Mo-DCs) as competitor. We identified the transcription factor Spi-B to be expressed in pDCs but not in Mo-DCs. Spi-B expression in pDCs was maintained on in vitro maturation of pDCs. Spi-B was expressed in early CD34+CD38− hematopoietic progenitors and in CD34+CD1a− thymic precursors. Spi-B expression is down-regulated when uncommitted CD34+CD1a− thymic precursors differentiate into committed CD34+CD1a+ pre-T cells. Overexpression of Spi-B in hematopoietic progenitor cells resulted in inhibition of development of T cells both in vitro and in vivo. In addition, development of progenitor cells into B and NK cells in vitro was also inhibited by Spi-B overexpression. Our results indicate that Spi-B is involved in the control of pDC development by limiting the capacity of progenitor cells to develop into other lymphoid lineages.

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127 Preclinical evaluation of NKX019, a CD19-targeting CAR NK Cell

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0127 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A140-A140

Author(s):

Nadege Morisot ◽

Sarah Wadsworth ◽

Tina Davis ◽

Nicole Dailey ◽

Kyle Hansen ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Half Life ◽

Nk Cell ◽

Cytolytic Activity ◽

Nsg Mice ◽

Animal Care

BackgroundNatural killer (NK) cells are highly effective and fast-acting cytolytic cells capable of eradicating target cells with limited adverse effects such as cytokine release syndrome (CRS) or graft-versus-host disease. Chimeric antigen receptors (CARs)-engineered NK cells have been recently used against leukemia with encouraging clinical outcomes.1 The surface antigen CD19, expressed by B-lymphoblasts, represents an ideal CAR target against B cell acute lymphoblastic leukemia (B-ALL). We developed a highly potent CD19 -directed CAR NK cell therapy, NKX019, with an extended in vivo half-life aimed at killing CD19-expressing target.MethodsNK cells isolated from healthy PBMCs were expanded in the presence of NKSTIM cells, IL-2, IL-12, IL-18 and transduced with both a CD19-targeted CAR construct and a membrane-bound form of IL-15 (mbIL-15). Control (non-engineered) NK cells were produced in parallel. Cytotoxic activity of NKX019 against CD19+ B-ALL cell line (REH), pre-B ALL cell line (Nalm-6), allogeneic PBMCs was assessed using Incucyte® or flow cytometry. NSG mice bearing either Nalm-6.fluc (Nalm6) or REH.fluc (REH) tumor received different concentrations of NKX019 or control NK cells. In-life analysis of tumor-bearing and naïve NSG mice include: 1) bioluminescence imaging, 2) clinical observations, 3) serum cytokines and 4) CAR+ NK cell persistency.ResultsNKX019 showed enhanced cytolytic activity against REH and Nalm-6 tumor cells compared to control NK cells and CAR19+ T cells. The superiority of NKX019 over CAR19+ T cells was more pronounced at the earlier time point (24 hours) with near identical calculated EC50 observed at 72 hours for both cell types. Increased cytolytic activity of NKX019 was limited to CD19+ cells in bulk PBMCs. Consistent with our in vitro observations, NKX019 controlled Nalm-6 and REH tumor growth in doses as low as 2 × 106 cells/kg for up to 30 days with no apparent increase in cytokines commonly associated with CRS. Increased Nalm-6 tumor growth coincided with an apparent decrease in measurable NKX019 in the periphery. In tumor-naïve NSG mice, NKX019 was detectable in the blood for up to 9 weeks post-infusion consistent with its extended half-life.ConclusionsNKX019 expresses mbIL-15 and is produced in the presence of IL-12 and IL-18, resulting in enhanced in vitro expansion and longer in vivo half-life than non-engineered NK cells. NKX019 also exhibited advantages compared to CAR19+ T cells including faster cytotoxic kinetics and limited production of cytokines associated with CRS. A first-in-human trial of NKX019 in B cell malignancies is planned for 2021.Ethics ApprovalThe animal procedures described in this abstract were conducted in accordance with Explora BioLabs Animal Care and Use Protocol approved by Explora BioLabs Institutional Animal Care and Use Committee.ReferenceLiu, et al. 2020 NEJM

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Development of a Novel MICA/B-Specific CAR As a Pan-Tumor Targeting Strategy for Off-the-Shelf, Cell-Based Cancer Immunotherapy

Blood ◽

10.1182/blood-2020-141095 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 5-6

Author(s):

Ryan Bjordahl ◽

John Goulding ◽

Mochtar Pribadi ◽

Robert Blum ◽

Chiawei Chang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cell ◽

Cell Lines ◽

Nk Cell ◽

Tumor Cell Lines ◽

Publicly Traded ◽

Cell Immunotherapy

Surface expression of the HLA-I related molecules MICA and MICB (MICA/B) in response to oncogenic and cellular stress acts as a natural anti-cancer immunosurveillance mechanism. The recognition of MICA/B by the activating immunoreceptor NKG2D, which is expressed by natural killer (NK) and T cell subsets, is responsible for the removal of many transformed and virally infected cells. However, tumors frequently evade NKG2D-mediated immunosurveillance by proteolytic shedding of MICA/B, which can inhibit NKG2D function and promote tumor immune escape. Recently, we demonstrated that monoclonal antibodies targeting the conserved, membrane-proximal α3 domain of MICA/B can prevent MICA/B shedding and enhance NK cell anti-tumor efficacy. With the goal of leveraging the ubiquity of MICA/B expression on malignant cells, we have developed a novel chimeric antigen receptor targeting the α3 domain of MICA/B (CAR-MICA/B) and are currently evaluating application of CAR-MICA/B in an off-the-shelf NK cell immunotherapy platform for both solid and hematopoietic tumor indications. Optimization of CAR-MICA/B design was performed by primary T cell transduction using a matrix of CAR spacers and ScFv heavy and light chain orientations. Six candidate CAR-MICA/B designs were screened in vitro against a panel of tumor cell lines and in vivo against the Nalm6 leukemia cell line engineered to express MICA (Nalm6-MICA). All tested constructs demonstrated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control (Figure 1A). Additional studies utilizing the optimal CAR-MICA/B configuration demonstrated MICA/B-specific reactivity against a panel of solid and hematopoietic tumor cell lines in vitro, including melanoma, renal cell carcinoma, and lung cancer lines (Figure 1B). Further, CAR-MICA/B T cells were superior to NKG2D-CAR T cells in clearing A2058 melanoma cells in an in vivo xenograft metastasis model (Figure 1C). Although MICA/B expression has primarily been studied in the context of solid tumors, moderate MICA/B mRNA expression was identified in a number of hematopoietic tumor cell lines, including acute myeloid leukemia (AML) and multiple myeloma (MM) lines. Following the confirmation of surface MICA/B protein expression on a selection of MM and AML cell lines, we utilized MICA/B CAR primary T cells to further demonstrate MICA/B-specific activation and cytotoxicity and to confirm CAR-MICA/B targeting of hematological malignancies (Figure 1D). To further advance CAR-MICA/B development, we introduced the CAR-MICA/B construct into an induced pluripotent stem cell (iPSC) line designed for production of off-the-shelf natural killer (NK) cell immunotherapies. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iPSC-derived NK (iNK) cells displayed specific MICA reactivity, resulting in enhanced cytokine production, degranulation, and CAR-mediated cytotoxicity compared to CAR-negative iNK control cells (Figure 1E). In addition to MICA/B-specific cytotoxicity mediated by CAR, iNK cells also mediated innate cytotoxicity against cancer cells through endogenous NKG2D and other NK cell activating receptors, highlighting the multifaceted targeting capacity of CAR iNK cells. In order to isolate CAR-directed cytotoxicity from the iNK cells' innate anti-tumor capacity, an in vivo proof of concept study was performed using mouse B16-F10 melanoma cells engineered to express human MICA. In this model, iNK expressing CAR-MICA/B significantly reduced B16-F10-MICA liver and lung metastases from CAR-MICA/B iNK cells compared to CAR negative control cells, with reductions of the number of metastases by 87% in the lung (p<0.0001) and 93% in the liver (p<0.006) for CAR-MICA/B iNK cells vs non-CAR controls (Figure 1F). Additionally, CAR-MICA/B iNK cells were effective at controlling Nalm6-MICA progression in a disseminated leukemia model, suggesting potential application against both hematopoietic and solid tumors. Ongoing work is focused on extending these studies into disease-specific models of endogenous MICA/B expression to further advance CAR-MICA/B iNK cells in both solid and hematologic cancers. In summary, these preclinical data support the development and translation of an off-the-shelf NK cell immunotherapy targeting the conserved α3 domain of MICA/B with potential therapeutic application to multiple hematopoietic and solid tumor types. Figure 1 Disclosures Bjordahl: Fate Therapeutics: Current Employment. Goulding:Fate Therapeutics: Current Employment. Blum:Fate Therapeutics: Current Employment. Chang:Fate Therapeutics: Current Employment. Wucherpfennig:Fate Therapeutics: Research Funding. Chu:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company; Roche Holding AG: Current equity holder in publicly-traded company. Chu:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Liu:Fate Therapeutics: Current Employment. Sikaroodi:Fate Therapeutics: Current Employment. Fong:Fate Therapeutics: Current Employment. Huffman:Fate Therapeutics: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

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Enhancing Human NK Cell Function and Specificity for Cancer Immunotherapy

Blood ◽

10.1182/blood-2018-99-120173 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2044-2044

Author(s):

Pomeroy Emily ◽

Hunzeker John ◽

Kluesner Mitchell ◽

Crosby Margaret ◽

Laura Bendzick ◽

...

Keyword(s):

Nk Cells ◽

Cancer Immunotherapy ◽

Peripheral Blood ◽

High Efficiency ◽

Cell Function ◽

Nk Cell ◽

Negative Regulator ◽

Car T

Abstract Natural Killer (NK) cells are cytotoxic lymphocytes capable of immune surveillance and represent an excellent source of cells for cancer immunotherapy for numerous reasons: 1) they mediate direct killing of transformed cells with reduced or absent MHC expression, 2) they can carryout antibody-dependent cell-mediated cytotoxicity (ADCC) on cells bound by appropriate antibodies via CD16, 3) they are readily available and easy to isolate from peripheral blood, 4) they can be expanded to clinically relevant numbers in vitro. Moreover, as NK cells do not cause graft versus host disease, they are inherently an off-the-shelf cellular product, precluding the need to use a patient's own NK cells to treat their cancer. In light of these attributes, NK cells have been used in many clinical trials to treat a number of cancer types; however, the results have not been as successful as other cellular based immunotherapies, such as CAR-T. In light of this, many groups have taken approaches to augment NK cell function, such as high dose IL15, CARs and Bi- or Tri-specific killer engagers. A synergistic or even alternative approach to these technologies is the use of CRISPR/Cas9-based genome editing to disrupt or manipulate the function of NK genes to improve their utility as an immunotherapeutic agent. In order to enhance the immunotherapeutic efficacy of NK cells we have implemented the CRISPR/Cas9 system to edit genes and deliver CARs. To this end, we have developed methods for high efficiency nucleic acid delivery to NK cells using electroporation. First, primary human NK cells are immunomagnetically isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Purified NK cells are then activated and expanded using artificial antigen presenting cells (aAPCs) expressing membrane bound IL21 and 41BB for 7 days and subsequently electroporated (Figure 1A). Using this approach with EGFP encoding mRNA, we achieve high rates of transfection (>90%) and high viability (>90%) (Figure 1B). We next developed gRNAs targeting PD1, CISH, and ADAM17. PD1 is a negative regulator of NK cell function and its cognate receptor, PD-L1, is upregulated in a number of cancers. ADAM17 mediates CD16 cleavage on NK cells to negatively regulate their ability to perform ADCC. CISH is a recently described negative regulator of NK cell activation and integrates cytokine signals, including IL-15. We consistently achieved high rates (up to 90%) of gene inactivation in primary human NK cells across multiple donors (Figure 1C). Importantly, these gene edits do not affect expansion potential and are stable over several rounds of expansion (Figure 1D, E). Moreover, ADAM17 KO NK cells are highly resistant to CD16 cleavage upon activation (Figure 2A-E) and PD1 KO NK cells demonstrate significantly enhanced function against PD-L1 expressing cancer cell lines in vitro and in vivo (Figure 2F-J). These data demonstrate that high efficiency gene editing of NK cells can significantly enhance their function while maintaining in vitro expansion. In an effort to engineer NK cell specificity for cancer immunotherapy, we recently developed CAR molecules designed for use in NK cells (Li et al., 2018, Cell Stem Cell 23, 1-12). To this end, we engineered and tested 10 mesothelin CAR molecules with NK specific transmembrane domains (CD16, NKp44, NKp46, or NKG2D) and intracellular signaling domains (2B4, DAP10, DAP12, CD3ζ, and/or CD137). Utilizing several cancer models, we identified an architecture that significantly enhanced NK activation compared to T-CAR architectures (CAR4: scFv-NKG2D-2B4-CD3ζ). Moreover, NK-CAR4 cells demonstrated increased in vivo expansion, improved activity, and reduced toxicity compared to CAR-T cell therapy. In our studies to develop novel NK CARs, CARs were delivered to iPSC derived test NK cells (iNKs) using the PiggyBac transposon system. In order to deliver NK-CAR4 to peripheral blood NK cells we developed methods for high frequency, site specific integration. To this end, we utilized CRISPR/Cas9 combined with non-integrating recombinant Adeno-Associated Virus (rAAV) DNA donor for homologous recombination. Using an EGFP reporter we were able to optimize this process and deliver EGFP reporter to the AAVS1 safe harbor site with efficiencies >80% in NK cells. We are now utilizing our optimized gene editing approaches to generate multiplex edited CAR-NK cells and results from these studies will be presented. Disclosures Webber: BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership. Felices:GT Biopharma: Research Funding. Moriarity:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership.

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IL-15 Administration to In-Vivo Expand Adoptively Transferred NK Cells In Rhesus Macaques

Blood ◽

10.1182/blood.v116.21.960.960 ◽

2010 ◽

Vol 116 (21) ◽

pp. 960-960

Author(s):

Rebecca Lopez ◽

Stephanie Sellers ◽

Cynthia E. Dunbar ◽

Richard Childs

Keyword(s):

Bone Marrow ◽

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Rhesus Macaques ◽

Cell Transfer ◽

Regulatory Phenotype

Abstract Abstract 960 Immunotherapy using natural killer (NK) cells is currently being explored as a treatment option for patients with advanced malignant diseases. Although pilot clinical trials have shown adoptive NK cell transfer can result in tumor regression in humans with cancer, additional insight from animal models is needed to optimize methods to enhance the function and in vivo persistence of these adoptively infused lymphocytes. In contrast to mice, rhesus macaques have orthologues to most of the human MHC class I and II genes and possess NK cells expressing KIRs that are phenotypically and functionally similar to human NK cells, thus providing an excellent model to evaluate adoptive NK cell therapy. To characterize their in vivo longevity and tissue trafficking following adoptive infusion, we developed a method to expand large numbers of rhesus NK cells in vitro. NK cells enriched from peripheral blood mononuclear cells by depleting CD3+ cells using immunomagnetic beads were expanded in vitro with autologous plasma and a human EBV-LCL feeder cell line using culture conditions identical to those used for human NK cell expansion. Expanded rhesus NK cells were both phenotypically and genotypically similar to their human counterparts; NK cell cultures expanded up to 1000 fold within 2–3 weeks, were greater than 99% CD3 negative, and had a large proportion of CD16/CD56 double positive cells. In addition, expanded NK cells up-regulated receptors involved in tumor killing, including NKG2D, Granzyme B, TRAIL and Fas-ligand and were highly cytotoxic to K562 cells. Adoptive transfer of (3.2×107 – 1×108) CFSE-labeled ex vivo expanded rhesus NK cells has been well tolerated without any overt toxicities noted to date. Remarkably, despite the infusion of large cell numbers, CFSE labeled NK cells were detectable in the peripheral blood, lymph nodes, and bone marrow compartments at very low levels for only a few hours following infusion. Combining adoptive transfer of ex vivo expanded NK cells with IL-15 administration (rhesus recombinant IL-15 10 ug/kg s.c. × 5 days) resulted in only a minimal and transient 24 hour increase in the number of detectable CFSE labeled NK cells in the circulation and bone marrow. Although IL-15 administration did not substantially expand the number of circulating CFSE labeled NK cells that were adoptively transferred, it did result in a substantial increase in circulating numbers of endogenous NK and T-cells (4.74 fold and 5.2 fold increase in CD3-/CD56+ NK cells and CD3+ T-cells respectively). Surprisingly, IL-15 administration also resulted in a significant expansion of circulating T-regs (CD4-/CD25+/CD127Dim/FOXP3 +) which have previously been shown to suppress NK cell effector function in vitro and vivo; T-cells with a regulatory phenotype expanded 4.54 fold. Expansion of circulating T-regs occurred both when IL-15 was administered alone or in conjunction with adoptive NK cell transfer. Conclusions: IL-15 administration in macaques at the doses used in this study did not expand circulating numbers of adoptively transferred ex-vivo expanded NK cells, although it did significantly expand the numbers of circulating endogeneous NK cells. Remarkably, IL-15 administration was also associated with a significant expansion of T-cells with a regulatory phenotype. We are currently evaluating whether lympho-depletion followed by adoptive NK cell transfer can be used as a method to prevent the expansion of T-regs associated with IL-15 administration. Disclosures: No relevant conflicts of interest to declare.

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