Invariant NKT (iNKT) cells can be activated to stimulate a broad inflammatory response. In murine models of sickle cell disease (SCD), interruption of iNKT cell activity prevents tissue injury from vaso-occlusion. NKTT120 is an anti-iNKT cell monoclonal antibody that has the potential to rapidly and specifically deplete iNKT cells and, potentially, prevent vaso-occlusion. We conducted an open-label, multi-center, single-ascending-dose study of NKTT120 to determine its pharmacokinetics, pharmacodynamics and safety in steady-state patients with SCD. Doses were escalated in a 3+3 study design over a range from 0.001 mg/kg to 1.0 mg/kg. Twenty-one adults with SCD were administered NKTT120 as part of 7 dose cohorts. Plasma levels of NKTT120 predictably increased with higher doses. Median half-life of NKTT120 was 263 hours. All subjects in the higher dose cohorts (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) demonstrated decreased iNKT cells below the lower limit of quantification within 6 hours after infusion, the earliest time point at which they were measured. In those subjects who received the two highest doses of NKTT120 (0.3, 1 mg/kg), iNKT cells were not detectable in the peripheral blood for a range of 2 to 5 months. There were no serious adverse events in the study deemed to be related to NKTT120. In adults with SCD, NKTT120 produced rapid, specific and sustained iNKT cell depletion without any infusional toxicity or attributed serious adverse events. The next step is a trial to determine NKTT120's ability to decrease rate of vaso-occlusive pain episodes. Trial Registration: clinicaltrials.gov NCT01783691.

The iNKT cell (invariant Natural Killer T cell) is a unique immune cell that has been shown to be associated with chronic inflammation in sickle cell disease. One approach to reducing iNKT mediated inflammation would be to reduce the number of iNKT cells in the tissue of SCD patients. NKTT120 is a humanized monoclonal antibody directed to the unique T cell receptor of invariant NKT cells that can deplete iNKT cells by antibody dependent cellular cytotoxicity. It recently completed a first in man Phase 1 study that demonstrated that intravenous single doses in adults with SCD specifically reduced iNKT cells without dose limiting toxicity in the therapeutic range of 0.3 and 1.0 mg/kg.

Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.

Background: Painful vaso-occlusive crises (pVOC) are caused by the polymerization of sickle deoxyhemoglobin, red cell sickling and vaso-occlusion. Vaso-occlusion is exacerbated by an inflammatory cascade that is initiated by iNKT cell activation. Townes sickle cell mice have mouse globins replaced by human globins, including the mutant human sickle b-globin gene (SS). NKT-14 is a monoclonal antibody directed specifically to the mouse invariant T cell receptor (iTCR). The antibody is a chimeric mouse IgG2a that, when bound to the mouse iNKT cell, promotes a rapid, specific and long lasting (> 14 days) depletion.

Background: Invariant NKT (iNKT) cells are a lymphocyte subset that can be rapidly activated to produce cytokines and fuel tissue inflammatory responses that are implicated in sickle cell disease (SCD) pathogenesis. In mouse models of SCD, interrupting the activation of iNKT cells decreases tissue injury. Patients with stable SCD, defined as no recent vaso-occlusive crisis (VOC), illness or transfusion, were enrolled into a phase 1 study utilizing an anti-iNKT cell monoclonal antibody, NKTT120, aimed at establishing a safe and effective dose to reduce or deplete their blood and tissue iNKT cells. Since monitoring iNKT cell response to our depleting antibody is a critical measure of both efficacy (iNKT cell depletion) and recovery (iNKT cell return to peripheral circulation) we assessed stability of the fraction of activated iNKT cells as well as their percentage as a fraction of total CD3+ lymphocytes measured over two time points.

The iNKT cell represents a novel therapeutic target for important hematologic diseases such as sickle cell disease (SCD) and myeloma. While an antibody specifically targeting human iNKT cells is now in a clinical trial, no surrogate reagent that specifically recognizes murine iNKT cells has been previously reported. This abstract defines work on a unique, recently developed antibody specifically directed to the T cell receptor of the mouse iNKT cell. These cells are a small subset of T lymphocytes that share characteristics with adaptive as well as innate immune cells. In contrast to conventional T cells they recognize glycolipid antigens presented on the MHC-I like molecule CD1d. Upon activation they can rapidly release either pro-inflammatory or anti-inflammatory cytokines, depending on stimulus and microenvironment. This enables them to direct downstream immune functions into inflammatory or tolerizing modes. iNKT cell activation has been implicated as a mediator of the chronic inflammation that is found in patients with SCD (Field et al. Blood 121:3321, 2013) suggesting that reduction of activity or iNKT cell depletion may be an effective therapy. The activation of iNKT cells has been shown to have therapeutics effects in multiple hematologic tumors including myeloma, lymphoma, and leukemia (Dhodapkar and Richter Clin.Immunol.140:160, 2011). Until now, the role of iNKT cells in immune regulation has been studied using iNKT cell deficient inbred mouse strains like CD1d and Ja18 knockout mice or with the iNKT cell activating agent alpha-Galactosyl-Ceramide (aGalCer). These tools have weaknesses and limitations. CD1d deficient mice are not only deficient in invariant NKT cells but also other CD1d restricted cells, such as Type 2 NKT cells. Ja18 knockout mice have recently been shown to have a substantial decrease in TCR diversity in addition to their iNKT cell deficiency (Bedel et al.,Nat Immunol. 2012 Jul 19;13(8):705-6.). Furthermore, these mouse strains lack iNKT cells from birth and little is known about pharmacologic suppression in iNKT cell competent mouse strains. Although aGalCer can be used to activate iNKT cells in vivo, it induces a persistent iNKT cell anergy after activation. NKT Therapeutics has developed human iNKT cell specific humanized monoclonal antibodies, one of which is currently being evaluated in a Phase I study in patients with sickle cell disease. The human iNKT cell specific antibodies are not cross-reactive to murine iNKT cells. In order to better understand the potential of pharmacologic modulation of iNKT cell function in pre-clinical disease models, we developed a mouse iNKT specific monoclonal antibody. We have a generated both a depleting version (NKT-14) and by manipulating the FC-function through mutations we have also generated a non-depleting, activating version (NKT-14m). Both are highly specific for mouse iNKT cells and recognize all aGalCer -loaded CD1d tetramer binding cells (Fig. 1A) in multiple inbred mouse strains tested (C57BL/6, BALB/c, NOD, DBA, C3H,NZW, NZW/NZB F1, AKR, SJL and A/J). NKT-14 rapidly and very specifically depletes iNKT cells in vivo (Fig. 1B). NKT-14m can activate iNKT cells in vivo and induces release if IFn-Gamma (Fig. 1C). These novel mouse invariant TCR specific monoclonal antibodies will allow us to better understand the role of iNKT cells in health and disease in order to inform clinical trials of therapeutics which manipulate these unique immune regulatory cells for the treatment of disease.

Abstract Background Multiple lines of evidence suggest that invariant NKT (iNKT) cells generate an inflammatory cascade that promotes and sustains sickle cell vaso-occlusion. In prior studies of mouse models and patients with sickle cell disease (SCD), iNKT cells are increased in number and more likely to be activated compared to controls. Depleting iNKT cells in a mouse model of SCD decreases inflammation and prevents end-organ injury. NKTT120 is a humanized monoclonal antibody that specifically depletes iNKT cells. Preclinical studies show that NKTT120 has high affinity and specificity for iNKT cells. NKTT120 depletes iNKT cells in a dose-dependent manner. Return of iNKT cells to the peripheral circulation following NKTT120 administration occurs in a dose- and time-dependent manner. Our global hypothesis is that NKTT120 will deplete iNKT cells, reduce inflammation and prevent painful vaso-occlusive crises. In this phase 1 dose-escalation study, we will examine the safety of NKTT120 in steady state adults with SCD. Objective To determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of NKTT120 in steady state adults with SCD. The optimal dose for a phase 2 study of NKTT120 will deplete iNKT for approximately 3 months allowing for periodic dosing. Methods Phase 1 study utilizing a 3+3 design to evaluate single doses escalated over a range from 0.001 mg/kg to 0.1 mg/kg (0.001, 0.003, 0.01, 0.03, and 0.10 mg/kg). Primary outcome measure is safety. Secondary outcomes include pain, analgesic use, quality of life (QoL), and pulmonary function. During a screening run-in period and after dosing of NKTT120, subjects will maintain a daily smartphone eDiary (eSCaPe) to report pain, respiratory symptoms and analgesic use. ASCQ-Me and PROMIS QoL questionnaires will be administered at clinic visits. The screening run-in outcomes will be used as baseline comparison for values obtained post-dosing. Results One month of follow-up data on iNKT cell numbers is available for the first four patients in the study (Figure 1). Three subjects received the lowest dose of 0.001 mg/kg and 1 subject received 0.003 mg/kg. All 4 subjects showed a reduction in the iNKT cell percent of CD3+ T cells 6 hours after NKTT120 administration. Three subjects have completed the study with iNKT cells returning to pre-dosing levels at day 7 while the remaining subject is awaiting iNKT cell recovery five weeks after dosing. The effects of NKTT120 were specific to iNKT cells as T cell, B cell and NK cell percent of lymphocytes was not affected. NKTT120 has been well tolerated with no adverse events reported. Conclusions In steady state adults with SCD, NKTT120 administered at the lowest dose of 0.001 mg/kg specifically reduces iNKT cells without toxicity. The dose of 0.003 mg/kg is currently being evaluated in this ongoing trial and higher doses of NKTT120 are anticipated to further deplete iNKT cells in the blood and tissue with longer times to recovery. This reduction of iNKT cells should result in a suppression of the inflammatory stimuli that promote many of the pathophysiologic sequelae seen in SCD. Disclosures: Field: NKT Therapeutics: Consultancy. Eaton:NKT Therapeutics: Employment, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Nathan:NKT Therapeutics: Consultancy.

Invariant Natural Killer T (iNKT) cells are a subset of T cells recognizing glycolipid antigens presented by CD1d. Human iNKT cells express a conserved T cell receptor (TCR)-α chain (Vα24-Jα18) paired with a specific beta chain, Vβ11. The cells are both innate-like, with rapid cytokine release, and adaptive-like, including thymic positive selection. Over activation of iNKT cells can mediate tissue injury and inflammation in multiple organ systems and play a role in mediating the pathology associated with clinically important inflammatory diseases. At the same time, iNKT cell activation can play a role in protecting against infectious disease and cancer or modulate certain autoimmune diseases through its impact on both the innate and adaptive immune system. This suggests that approaches to cause iNKT cell reduction and/or depletion could treat inflammatory diseases while approaches to promote activation may have therapeutic potential in certain infections, cancer or autoimmune disease. This report summarizes the characterization of a humanized monoclonal depleting antibody (NKTT120) in the cynomolgus macaque. NKTT120 is being developed to treat iNKT mediated inflammation that is associated with chronic inflammatory conditions like sickle cell disease and asthma. NKTT120 binds to human iTCRs and to FCγRI and FCγRIII and has been shown to kill target cells in an ADCC assay at low concentrations consistent with the FCγR binding. iNKT cells were depleted within 24 hours in cynomolgus macaques, but T cell, B cell, and NK cell frequencies were unchanged. iNKT cell recovery was dose and time dependent. T cell dependent antigen responses were not impaired by NKTT120 mediated iNKT depletion as measured by response to KLH challenge. NKTT120 administration did not induce an inflammatory cytokine release at doses up to 10 mg/kg. These data support the use of NKTT120 as an intervention in inflammatory diseases where iNKT reduction or depletion could be beneficial.

Abstract Abstract 4836 Invariant Natural Killer T (iNKT) cells are a small subset of T lymphocytes (ranging from 0.01 – 0.1% of CD3+ T cells) that share surface markers and functional characteristics with T cells and natural killer (NK) cells. Unlike other T cells, they recognize glycolipid antigens presented by the MHC class-I-like protein CD1d rather than peptide antigens. In contrast to most T cells which express diverse T cell receptor (TCR) sequences, iNKT cells express a unique, highly conserved, semi-invariant TCR-α chain (Vα24-Jα18 in humans), which preferentially pairs with specific TCR-β chains (Vβ11 in humans). Like cells of the innate immune system, iNKT cells are rapid-onset cells with a universal receptor. They also share properties of T cells like requiring thymic positive selection and recognition of antigen presented on the MHC-I like molecule CD1d. As such, they serve as a bridge between the innate and adaptive immune systems. They can play either a pro-inflammatory role to enhance or an immuno-regulatory role to attenuate a developing immune response. iNKT cells have been shown to be involved in mediating tissue injury and inflammation in multiple organ systems. There is a growing recognition that chronic inflammation is associated with the pathophysiology of Sickle Cell Disease (SCD). Wallace et al. (Blood 114:667, 2009) found an increased ratio of activated iNKT cells in peripheral blood of patients with SCD compared to normal volunteers. Our study of peripheral blood iNKT cells from 5 SCD patients and 10 unaffected African American volunteers found that an average of 50.5% of the iNKT cells of the SCD patients were activated as indicated by up regulation of the lymphocyte activation marker CD69. In the healthy controls an average of only 8.9% of iNKT cells expressed CD69. A key role of iNKT cell activation in the pathology of SCD is supported by studies in a mouse model of SCD (Blood 114:667, 2009) that suggest iNKT cell depletion could reduce inflammation in the SCD patient. We have developed a humanized monoclonal antibody (NKTT120) that binds the CDR3 loop of the human and the old-world non-human primate invariant TCR with high specificity. NKTT120 depletes iNKT cells in human iTCR transgenic mice and cynomolgus monkeys (Macaca fascicularis). In the transgenic mice, iNKT cells reappear in the peripheral circulation within weeks after complete depletion. We conducted a study in cynomolgus monkeys to explore the relationship between dose and duration of iNKT cell depletion. Each group of animals (n=3) received a single dose of NKTT120. The dosing groups were 10, 30, 100 and 300 ug/kg, respectively. iNKT cells and other lymphocytes were monitored 24, 48, 72, 96 and 168 hours following dosing and weekly thereafter using flow cytometry. In all dose groups, iNKT cells were depleted within 24 hours with no significant changes in the other cells of the lymphocytic series. No adverse events have been noted to date. iNKT cells in the 10 ug/kg and the 30 ug/kg dose group were recovered by week 5 and week 7 respectively. As of week 12 post-dosing, iNKT cells in the 100 and the 300 ug/kg dose groups have not recovered. The kinetics of recovery in these higher dose animals will shed light on the impact of what is expected to be full tissue depletion on iNKT cell recovery. Overall, our study has shown that we can safely deplete iNKT cells in non-human primates and that iNKT cells can recover after depletion. Percent of Cells (Mean±SD) Estimated by FACS Analysis Before and Following NKTT120 IV Administration Shows Recovery Following Depletion Lymphocyte Profiles Pre Sample Day 1 Post Dose Week 5 Post Dose Week 7 Post Dose 10 ug/kg 30 ug/kg 100 ug/kg 300 ug/kg 10 ug/kg 30 ug/kg 100 ug/kg 300 ug/kg 30 ug/kg 100 ug/kg 300 ug/kg iNKT Cells* 0.29±0.17 0.02±0.03 0 0 0 0.14±0.12 0.01±0.005 0 0 0.05±0.01 0 0.01±0.01 NK Cells** 11.6±3.9 8.1±1.8 8.5±2.1 7.8±4 11.1±2.5 11.4±2.6 16.2±4.3 8.9±3.1 14.3±4.3 13.2±3.1 9.2±4.1 13.2±2.5 Mature T cells** 73.1±5.5 69.6±4 70±7.5 75.9±4.3 65.1±3.6 70.6±4.5 67.4±5.1 78.2±5.7 66.5±4.4 72.3±4.9 80.8±6.3 69.3±3.2 CD8 T Cells** 26.0±4.7 22.1±4.2 20.7±2.8 27.8±3.5 22.4±4.1 26.2±5.5 24.1±6.3 31.8±3.7 26.5±6.1 25.4±5.8 32.7±5.1 27.2±6.9 CD4 T Cells** 42.0±4 44.3±7.9 45.2±5.2 43.2±1.5 38.3±4.1 41.1±6.9 39.5±1.7 40.9±2.6 35.4±3.3 43.2±7.7 42.2±0.7 37.4±5.4 B Cells** 3.4±2.1 8.9±2.4 6.8±1.6 5.1±1 8.5±5.1 7±1.1 7.4±2.9 4.3±0.6 7.8±4.9 5.5±1.5 2.4±0.2 6.1±3.5 * % of CD3 ** % of Lymphocytes Disclosures: Scheuplein: NKT Therapeutics: Employment, Equity Ownership. Macdonald:NKT Therapeutics: Employment, Equity Ownership. Zeigler:MPI Research: Employment; NKT Therapeutics: Research Funding. LeBel:MPI Research: Employment; NKT Therapeutics: Research Funding. Thariath:NKT Therapeutics: Employment, Equity Ownership. Truneh:NKT Therapeutics: Consultancy, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Nathan:NKT Therapeutics: Consultancy. Schaub:NKT Therapeutics: Employment, Equity Ownership.

Abstract Abstract 2150 Invariant Natural Killer T (iNKT) cells are a small subset of T lymphocytes (ranging from 0.01 – 0.1% of CD3+ T cells). iNKT cells recognize glycolipid antigens presented by the MHC class-I-like protein CD1d rather than peptide antigens. In contrast to most T cell subpopulations, which have diverse sequences for their T Cell Receptors (TCRs), iNKT cells express a uniquely rearranged, highly conserved, semi-invariant TCR-α chain (Vα24-Jα18 in humans), which preferentially pairs with specific TCR-β chains (Vβ11 in humans). iNKT cells are similar to innate cells in their rapid release of cytokines following iTCR antigen binding. They are also adaptive-like, with T cell properties including thymic positive selection and antigen recognition by CD1d presentation. iNKT cells are involved in mediating tissue injury and inflammation in multiple organ systems. Chronic inflammation is associated with the pathophysiology of Sickle Cell Disease (SCD) and our studies and those of others have found an increased ratio of activated iNKT cells in peripheral blood of patients with SCD. The role of iNKT cell activation in the pathology of SCD is supported by studies in a mouse model of SCD (Wallace et al. Blood 114:667, 2009). These data suggest that iNKT cell reduction and/or depletion would be effective in reducing the inflammatory state in SCD. To this end, we have developed a humanized monoclonal antibody (NKTT120) that exclusively binds to the CDR3 loop of the human and non-human primate (NHP) invariant T cell receptor and depletes iNKT cells. This antibody could provide an effective therapeutic intervention to modulate iNKT cell numbers, and thus their ability to mediate inflammation in SCD. The current study was designed to assess the overall safety of NKTT120 in the cynomolgus monkey (Macaca fascicularis). The cynomolgus monkey was selected for testing because NKTT120 is only active in human and old world NHP species. Thirty-two cynomolgus monkeys (3–5 kg) equally divided between males and females were studied. The groups consisted of a vehicle control and treatment groups that received 0.3 mg/kg, 3 mg/kg, and 10 mg/kg NKTT120 IV weekly for a total of 5 doses. Two animals per sex in the vehicle and the 10 mg/kg groups were recovered for an additional 2 months following the last dose. Animals were evaluated for food intake, body weight and general health. Standard hematology, coagulation testing and clinical chemistry testing were also performed. In addition, iNKT cell number and other lymphocytes were monitored during the study by FACS analysis. The repeat dose injections were well tolerated by all dose groups. No deaths or serious adverse events were reported during dosing or in the recovery period. Body weight, food intake and clinical evaluation, hematology, coagulation assays, and clinical chemistry were similar for vehicle control and all dosing groups. As expected, iNKT cells were depleted within 24 hours to below level of detection and remained depleted throughout the dosing period at all doses tested. The iNKT cell numbers of the 10 mg/kg recovery animals remained depleted throughout the 2 month recovery. However, there was no change in other cells of the lymphocytic series at any dose or at any time point evaluated. Overall our study showed that we can safely and specifically deplete iNKT cells in non-human primates following administration of NKTT120. These data support the use of NKTT120 as a therapeutic intervention in conditions such as sickle cell disease where iNKT modulation could be beneficial. Percent of Gated Cells (Mean ± SD) Estimated by FACS Analysis Before and Following NKTT120 IV Administration to Cynomolgus Macaques Shows Specific iNKT Cell Depletion at all Doses and Time points Lymphocyte Profiles Post Dose 1 Post Dose 5 2 Months Post Dose 5 Vehicle 0.3 mg/kg 3 mg/kg 10 mg/kg Vehicle 0.3 mg/kg 3 mg/kg 10 mg/kg Vehicle 10 mg/kg iNKT Cells* 0.05±.03 0.00 0.00 0.00 0.06±0.05 0.00 0.00 0.00 0.06±.01 0.00 NK Cells** 11±6 10±3 11±5 12±7 10±5 9±5 6±4 7±6 7±3 5±4 Mature T cells** 69±7 61±7 63±6 57±7 64±8 61±10 59±11 59±8 78±6 70±7 CD8 T Cells** 26±4 25±6 23±3 19±6 22±4 22±8 20±7 19±5 28±2 20±2 CD4 T Cells** 40±6 35±6 37±6 35±7 37±6 35±7 35±5 37±8 46±4 47±5 B Cells** 5±3 8±2 7±4 9±3 5±3 5±2 2±2 6±3 6±3 8±3 * %CD3 Cells. ** % Lymphocytes. Disclosures: Macdonald: NKT Therapeutics: Employment, Equity Ownership. Scheuplein:NKT Therapeutics: Employment, Equity Ownership. Thariath:NKT Therapeutics: Employment, Equity Ownership. LeBel:MPI Research: Employment; NKT Therapeutics: Research Funding. Zeigler:MPI Research: Employment; NKT Therapeutics: Research Funding. Truneh:NKT Therapeutics: Consultancy, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Schaub:NKT Therapeutics: Employment, Equity Ownership.