IntroductionThe serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy.MethodsTo assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo.ResultsIn a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo, STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti–PD-L1 antibody in the MCA205 model.ConclusionsIn summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo. This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

Abstract Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a serine/threonine kinase that has been demonstrated to have suppressive effects across a range of immune cells, including T cells and dendritic cells. Loss of MAP4K1 kinase activity is sufficient to enhance T cell receptor (TCR) signaling resulting in robust anti-tumor immunity alone and in combination with checkpoint inhibition. These data support that MAP4K1 is a novel and attractive target for cancer immunotherapy. We have designed a series of potent, selective, and orally bioavailable inhibitors of MAP4K1. Treatment of primary human T cells or peripheral blood with either BLU2069 or BLU6348 was able to inhibit phosphorylation of pSLP76, a scaffolding protein that regulates MAPK downstream of the TCR. In addition, we show that compound treatment can enhance cytokine secretion and proliferation in human T cells in response to TCR crosslinking. The therapeutic benefit of MAP4K1 inhibition alone and in combination with anti-PD-L1 was evaluated in multiple syngeneic mouse tumor models including MCA205, MC38 and EMT-6. Treatment with either compound alone led to a reduction in tumor growth that was further enhanced when combined with anti-PD-L1 therapy. When tumors were grown in immunocompromised mice (MCA-205) or in the setting of CD8+ T cell depletion (MC-38), the anti-tumor effect of BLU2069 and BLU6348 respectively was lost, confirming the importance of immune cells in compound mediated antitumor effects. We further show that MCA205 tumors harvested from mice treated with BLU2069 had increased intratumoral CD8+ T cell infiltration, resulting in enhanced CD8/Treg ratios. In addition, transcriptional analysis of tumor lysates showed that BLU2069 significantly increased genes associated with an effector phenotype. These data support that pharmacological inhibition of MAP4K1 reduced tumor burden and enhanced antitumor immunity in preclinical tumor models. Finally, we show that MAP4K1 inhibition can enhance CD3/CD28-induced IL2 and IFNγ in human tumor infiltrating lymphocytes (TILs) generated from melanoma or non-small cell lung cancer (NSCLC) primary tumors. This work describes the identification of potent small molecule inhibitors of MAP4K1 which could be novel therapeutic agents and induce an effective immune response either alone or in combination with approved checkpoint inhibitors. Citation Format: Kerrie Faia, Alberto Toso, Kristina Fetalvero, Marly Roche, Steven Bench, Erin O'Hearn, Qiongfang Cao, Kerry-Ann Bright, Debora Paduraru, Andrea Romagnani, Weifan Weng, Tina Zimmermann, Michael Burke, Joshua Close, Luke Green, Joseph Kim, Chandra Miduturu, Alison Ribeiro, Marina Bacac, Sylvia Herter, Emanuele Perola, Michael Sheets, Jan Eckmann, Gordon Heidkamp, Tary Traore, Erik Gerson, Rich Woessner, Carsten Wolter, Felix Scheuplein, Nisha Perez, Timothy LaBranche, Grace Silva, Chaoyang Ye, Caitlin Utt, Stefan Gross, James R. Bischoff, Marion Dorsch, Tim Guzi, Klaus Hoeflich, Jason Brubaker. MAP4K1 inhibition enhances immune cell activation and anti-tumor immunity in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1717.

Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK) cells, and T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigens. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. To this end, we have characterized the iNKT cell stimulatory properties of NKTT320, a novel, recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iTCRs. Strikingly, immobilized NKTT320 mediated robust iNKT cell activation (upregulation of CD25 and CD69) and proliferation (carboxyfluorescein succinimidyl ester (CFSE) dilution), as well as Th1 and Th2 cytokine production. Additionally, iNKTs stimulated by plate-bound NKTT320 exhibited increased intracellular levels of granzyme B and degranulation (exposure of CD107 on the cell surface). Furthermore, both soluble and immobilized NKTT320 induced iNKT cell-mediated activation of bystander immune cells, suggesting that this novel anti-iTCR antibody facilitates both direct and indirect iNKT cell cytotoxicity. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes.

iNKT cells and mast cells have both been implicated in the syndrome of allergic asthma through their activation-induced release of Th2 type cytokines and secretion of histamine and other mediators, respectively, which can promote airways hyperresponsiveness (AHR) to agents such as methacholine. However, a mechanistic link between iNKT cells and mast cell recruitment or activation has never been explored. Our objective was to determine whether iNKT cells are necessary for the recruitment of mast cells and if iNKT cells can influence the acute allergen induced bronchoconstriction (AIB) caused by mast cell mediator release. To do so, we pharmacologically eliminated iNKT cells using a specific antibody (NKT-14) and examined its impact on airway inflammation and physiological phenotype. In mice treated with NKT-14, the elimination of iNKT cells was sufficient to prevent AHR and pulmonary eosinophilic inflammation elicited by administration of the iNKT cell agonist αGalCer. In mice treated with NKT-14 and then sensitized and challenged with house dust mite extract (HDM), eliminating the iNKT cells significantly reduced both AHR and AIB but did not affect pulmonary inflammation, the mast cell population, nor the release of the mast cell mediators mast cell protease-1 and prostaglandin D2. We conclude that while iNKT cells contribute to the phenotype of allergic airways disease through the manifestation of AIB and AHR, their presence is not required for mast cell recruitment and activation, or to generate the characteristic inflammatory response subsequent to allergen challenge.

Abstract Given recent approvals of anti-PD-1 inhibitors Keytruda (pembrolizumab) and Opdivo (nivolumab) for treatment of metastatic melanoma and non-small cell lung cancer NSCLC, we asked whether additional anti-PD-1 inhibitors with different epitopes or potentially differentiated mechanisms of action can provide clinical benefit beyond the two marketed therapies. Using Enumeral's proprietary single cell technology, we generated more than 300 anti-PD-1 monoclonal antibodies from primary B cells. Bioinformatics analysis of sequences show these antibodies comprise 26 distinct clades, or families, that bind to PD-1. Results from binding studies further indicate we have discovered a family of novel antibodies that do not appear to compete with currently marketed antibodies for binding to PD-1, nor do they appear to compete with PD-L1, suggesting a differentiated mechanism of action. We have humanized two lead antibodies for preclinical testing and in preparation for clinical studies. Here we describe functional characterization of the two lead antibodies using cell-based ex vivo assays, patient-derived tumor profiling assays using our single-cell platform, and in vivo studies in humanized NSG mice. Results show that the two lead anti-PD-1 antibodies exhibit a higher level of T cell activation in mixed lymphocyte reaction (MLR) assays using primary human immune cells. Second, these antibodies also demonstrated dose-dependent increases in T cell CD25 expression. Next, to test the effect of the two lead anti-PD-1 antibodies on tumor growth, we are conducting a study using a patient-derived tumor (PDX) model on humanized NSG mice. NSG mice reconstitute a full human immune system including a functional T, B cell repertoire, enabling the direct, in vivo evaluation of our human anti-PD-1 antibodies. Effects of drug treatment on tumor growth, transcriptome (RNA seq) and protein expression (IHC) will be described. Finally, results from ex vivo tumor profiling comparing the immmunomodulatory effects of our anti-PD-1 antibodies on tumor-infiltrating lymphocyte function will also be presented. Citation Format: Felix Scheuplein, Sheila Ranganath, Thomas McQuade, Lei Wang, Vikki Spaulding, Sri Vadde, Jennifer Watkins-Yoon, Bin Feng, Shanu Mehta, Maria Isabel Chiu, Cokey Nguyen. Discovery and functional characterization of novel anti-PD-1 antibodies using ex vivo cell-based assays, single-cell immunoprofiling, and in vivo studies in humanized mice. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B30.

Abstract We have previously described the identification of more than 300 anti-PD-1 monoclonal antibody candidates using Enumeral's proprietary single cell immune-profiling technology, on which our antibody discovery platform is based. Bioinformatics analysis of heavy chain CDR3 sequences show they comprise diverse families, encompassing 26 phylogenetic clades. Functional studies from cell-based, ex vivo human assays led to the discovery of two distinct lead candidates, 244C8 and 388D4. The former represents a novel class of anti-PD-1 antibodies with a potentially differentiated mechanism of action, as they appear not to compete with currently marketed antibodies for PD-1 nor do they compete with PD-L1 for PD-1 binding. Further, antibodies from the 244C8 family elicit both a higher T cell activation and an increased expression of the high affinity IL-2 receptor CD25 than currently marketed anti-PD-1 antibodies in ex vivo human cell based assays. Lead antibodies from both anti-PD-1 classes, 244C8 and 388D4, have been humanized for preclinical testing in preparation for Phase 1 clinical studies. Here we describe the in vivo efficacy testing of these two antibodies in a human PDX tumorgraft model derived from a core needle biopsy of a patient with metastatic non-small cell lung carcinoma (LG1306). Direct testing of the anti-human PD-1 antibodies was made possible by the use of immune-humanized NSG mice. Pembrolizumab, a currently marketed anti-PD-1 antibody, served as control, along with vehicle alone. Treatment with each of the humanized lead PD-1 antibodies, 388D4 and 244C8, was well tolerated at 5 mg/kg and led to significant tumor growth inhibition over a 28-day study period. Both showed equivalent efficacy to pembrolizumab with tumor growth inhibition (%TGI) at 40% and 38% respectively compared to 37% for pembrolizumab. All three treatment agents showed significant tumor growth inhibition relative to vehicle, with Student T-test p values < 0.003 at end of study assessment. These results show that, contrary to expectation, competition for binding to PD-L1 ligand by an anti-PD-1 antibody is not a pre-requisite for functional efficacy in vivo, as we observed with 244C8. In addition to the in vivo efficacy studies, we will report on post-treatment analyses of all treatment cohorts by immunohistochemistry and RNAseq of the tumor samples, immunoprofiling analysis of tumor infiltrating lymphocytes (TILs) from all treatment cohorts, as well as donor-specific differences that influence response to treatment. Citation Format: Felix Scheuplein, Sheila Ranganath, Bin Feng, Thomas McQuade, Lei Wang, Vikki Spaulding, Sri Vadde, Shanu Mehta, Maria Isabel Chiu, Cokey Nguyen. Two novel anti-PD-1 antibodies, 244C8 and 388D4, elicit in vivo antitumor efficacy in a lung PDX tumorgraft in immuno-humanized NSG mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4871.

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.

Abstract Invariant natural killer T cells (iNKTs) are innate-type lipid-reactive T lymphocytes that directly kill tumor cells as well as exhibit robust capacity to trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK), T and B cells. Based on their dual functions as potent cytokine producers and tumor killers, iNKTs serve as a powerful tool for use in cell-based immunotherapies for cancer. In most cases, iNKT cell functions are T cell receptor (TCR)-dependent and require engagement of the invariant T cell receptor (iTCR) by lipids such as the potent iNKT agonist α-galactosyl ceramide (αGC). However, transformed cells often down-regulate CD1d expression and as a result, they become invisible to iNKT cell attack. To circumvent this critical barrier, which limits our ability to capitalize on the therapeutic potential of iNKTs, we recently characterized the iNKT stimulatory properties of a novel monoclonal antibody (NKTT320), which is specific for the human iTCR. NKTT320 is a recombinant DNA derived humanized monoclonal antibody that binds selectively and with high affinity to the CDR3 loop of the alpha chain within the α-β hetero-dimer of the extracellular domain of the human iTCR. For these studies, we have developed methods to successfully expand human iNKTs up to 1000-2000 fold and purify them from human peripheral blood mononuclear cells. To assess whether the NKTT320 antibody induces human iNKT cell functions, highly purified human iNKTs were cultured with or without varying concentrations (0.01-10.0 μg/ml) of plate-bound NKTT320 antibody. At varying times, culture supernatants were collected and cells were harvested, counted and viability assessed. iNKT cell activation (by examining for upregulation of activation markers [CD25, CD69], degranulation (measured by exposure of CD107 on the cell surface) and proliferation (by measuring CFSE dilution) was assessed by flow cytometry. Cell culture supernatants were evaluated for the secretion of a wide array of cytokines and chemokines using Luminex. Strikingly, immobilized NKTT320 antibody induced a robust dose-dependent iNKT cell activation, proliferation and degranulation. Additionally, iNKTs stimulated by the plate-bound antibody secreted increased levels of Th1 (IL-1β, IFN-γ, TNF-α, IL-2, IL-6) and Th2 (IL-4, IL-5, IL-10) cytokines as well as chemokines in a dose-dependent manner. Our in vitro studies are consistent with in vivo data in Vα24 transgenic mice, which express the human iTCR alpha chain. Dosing of NKTT320 in these animals led to iNKT cell activation (upregulation of activation markers and intracellular IFN-γ production), as well as incorporation of BRDU indicating in vivo iNKT cell proliferation. These studies provide a framework by which human iNKT cell functions could be enhanced for therapeutic purposes in cancer. Citation Format: Rupali Das, Felix Scheuplein, Nishant P. Patel, Peng Guan, Robert G. Schaub, Kim E. Nichols. NKTT320, a novel monoclonal antibody activates the immuno-stimulatory functions of human invariant natural killer T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2014-653

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.

T cells constitutively express low amounts of a toxin-related ADP-ribosylating ecto-enzyme, ART2.2. In inflammatory settings, cells release NAD, the substrate for ART2.2. The ART2.2 catalyzed ADP-ribosylation of cell surface proteins induces cell death. However, the low expression levels of ART2.2 have hampered analysis of ART2.2 in physiological settings. Here we report the generation of transgenic mice over-expressing ART2.2 under the control of the H2K promoter and Igμ enhancer. ART2.2 transgenic mice were healthy and fertile and exhibited normal development of the major lymphocyte subsets. Most T cells and a small subpopulation of B cells from transgenic mice showed more than 10-fold higher levels of ART2.2 expression than their wild-type counterparts. Exposure of ART2.2-transgenic T cells to low, submicromolar concentrations of NAD caused cell membrane alterations including uptake of propidium iodide, externalization of phosphatidylserine, and shedding of CD62L, while ART2.2-transgenic B cells were resistant to NAD. The ART2.2-overexpressing animals described here confirm that ART2.2 is an essential component for the regulation of T-cell functions by extracellular NAD and provide a useful tool to further elucidate the function of ART2.2 in vivo.