Receptor Alpha Chain Research Articles

ALLO-715, an Allogeneic BCMA CAR T Therapy Possessing an Off-Switch for the Treatment of Multiple Myeloma

Autologous chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) have demonstrated promising clinical activity, inducing durable responses in patients with relapsed/refractory multiple myeloma (MM). Development of autologous CAR T therapies is however limited by logistical challenges and the time required for manufacturing, which has to be done for each patient. In addition, manufacturing may not be feasible in some patients. An allogeneic approach that utilizes engineered cells from a healthy donor could potentially expand patient access to these therapies by providing a readily available off-the-shelf product. We have previously described the screening of a library of single chain variable fragments (scFvs) with high affinity to human BCMA and the identification of candidate BCMA CARs with potent antitumor activity. Here we sought to further characterize ALLO-715, our lead allogeneic BCMA CAR T cell product, for its specificity to human BCMA, antitumor efficacy in vitro using a long-term killing assay and in xenograft mouse models with physiologic levels of human IL-7 and IL-15, and suitability for scale-up manufacturing.Allogeneic ALLO-715 CAR T cells were generated by lentiviral transduction with a second generation CAR construct incorporating a novel scFv derived from a fully-human antibody with high affinity to BCMA (KD value ~ 5 nM, determined at 37°C) and featuring a rituximab-driven off-switch. Transduced T cells were then transfected with mRNAs encoding Transcription Activator-Like Effector Nucleases (TALEN®) designed to specifically disrupt the T cell receptor alpha chain and CD52 loci. These modifications result in a cell product with a lower risk of TCR-mediated graft-versus-host disease and resistance to the CD52 antibody alemtuzumab, a lymphodepleting agent. BCMA CAR T cells exhibited robust cell expansion, with low levels of tonic signaling that resulted in minimal differentiation (> 50% Tscm/Tcm phenotype). In in vitro assays, ALLO-715 CAR T cells displayed potent cytotoxic activity when co-cultured with the target cell lines MM.1S, Molp-8, and BCMA-REH but negligible cytotoxicity against BCMA-negative REH cells.The high proliferative potential indicated by the high frequency of memory T cells was validated in long-term killing assays, where ALLO-715 CAR T cells showed substantial expansion in the presence of MM.1S cells with no evidence of exhaustion or diminished cytolytic activity after seven days of continuous exposure to target. The potency of ALLO-715 CAR T cells was unaffected by high concentrations of soluble BCMA (>10 ug/mL), which has been shown previously to interfere with the activity of some BCMA-specific CARs.In MM xenograft mouse models, ALLO-715 CAR T cells were highly efficacious at single dose. High serum IL-15 levels have been associated with CAR T cell expansion in clinical trials. To evaluate the impact of homeostatic cytokines on CAR T cell survival and antitumor activity in our xenograft models, mice were administered adeno-associated viruses (AAV) for the expression of human IL-7 and IL-15. In the presence of physiological concentrations of these cytokines, enhanced BCMA CAR T cell expansion and anti-tumor activity were observed.To assess potential off-target interactions of ALLO-715 CAR, tissue cross-reactivity studies were carried out on standard human tissue panels using a scFv-human IgG fusion protein. Consistent with the limited expression pattern of BCMA, reactivity was seen on scattered cells in lymphoid tissues such as tonsil and abundantly on BCMA-expressing cell lines, but no appreciable staining was detected in other tissues.We examined BCMA CAR T cells manufactured following a proprietary GMP-like clinical scale process and found that cell expansion and viability, T cell phenotype and in vivo antitumor efficacy were preserved.These results demonstrate the potential of ALLO-715 as a novel allogeneic BCMA CAR T therapy for the treatment of relapsed/refractory MM and other BCMA-positive malignancies. DisclosuresSommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boldajipour:Pfizer Inc.: Employment, Patents & Royalties. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Galetto:Cellectis SA: Employment, Equity Ownership, Patents & Royalties. Bentley:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Ni:Allogene Therapeutics: Employment, Equity Ownership. Leonard:Allogene Therapeutics: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Late-Stage Preclinical Characterization of Switchable CD123-Specific CAR-T for Treatment of Acute Leukemia

▪Application of autologous T cells genetically engineered to express CD19-specific chimeric antigen receptors (CAR-T) is highly effective in the treatment of B cell malignancies. To this date, application of CAR-T therapy beyond CD19 remains challenging due to the inability to control CAR-T reactivity in patients and the lack of tumor-associated antigens exclusively expressed by malignant cells. The interleukin-3 receptor alpha chain (CD123) is a promising immunotherapeutic target and associated with leukemia-initiating compartments in myeloid- or lymphoid derived diseases. However, in contrast to CD19, CD123 is a precarious target due to its prevalent expression on healthy hematopoietic stem and progenitor cells (HSPC) as well as endothelial cells. Thus, CAR-T lacking any fine-tuned control mechanisms are at risk to cause life threatening toxicities or can only act as bridging therapy to an allogeneic stem cell transplantation. To extend application of CAR-T therapy and safely redirect CAR-engineered T cells to challenging targets such as CD123, a switch-controllable universal CAR-T platform (UniCAR) was recently introduced. The UniCAR system consists of two components: (1) a non-reactive inducible second generation CAR with CD28/CD3ζ stimulation for an inert manipulation of T cells (UniCAR-T) and (2) soluble targeting modules (TM) enabling UniCAR-T reactivity in an antigen-specific manner.Here we provide late stage pre-clinical data for UniCAR-T in combination with a CD123-specific TM (TM123) for treatment of acute leukemia. Primary patient-derived CD123-positive leukemic blasts were efficiently eradicated by TM123-redirected clinical-grade manufactured UniCAR-T in vitro and in vivo. Activation, cytolytic responses and cytokine release were proven to be strictly switch-controlled. Moreover, anti-leukemic responses of UniCAR-T were demonstrated to be comparable to conventional CD123-specific CAR-T in vitro. In contrast to conventional CD123 CAR-T, TM123-redirected UniCAR-T discriminate between CD123high malignant cells and CD123low healthy cells with negligible toxicity towards HSPC in vivo. As 4-1BB mediated co-stimulation is known to enhance CAR-T activity in vivo, a novel CD123-specific targeting module bearing a covalently bound trimeric 4-1BB ligand (4-1BBL) was developed and characterized for co-stimulation at the leukemic site in trans. Specific binding of TM123-4-1BBL was demonstrated against native 4-1BB as well as CD123-positive leukemic blasts. In long-term tumor eradication models, TM123-4-1BBL ameliorated the killing capability of UniCAR-T in vitro. Additionally, the increased hydrodynamic radius of trimeric 4-1BBL-coupled TM123 prolonged plasma half-life and enhanced bioavailability in vivo. In conclusion, UniCAR-T maintain high anti-leukemic efficacy, while adding a sophisticated mechanism for immediate control to improve safety and versatility of CD123-directed CAR-T therapy. Moreover, switching between several TMs from short to moderate plasma half-life allows for an individualized treatment of various leukemic settings while minimizing potential adverse effects. DisclosuresLoff:GEMoaB Monoclonals GmbH: Employment. Meyer:Cellex Patient Treatment GmbH: Employment. Dietrich:Cellex Patient Treatment GmbH: Employment. Spehr:Cellex Patient Treatment GmbH: Employment. Julia:Cellex Patient Treatment GmbH: Employment. Gründer:GEMoaB Monoclonals GmbH: Employment. Franke:GEMoaB Monoclonals GmbH: Employment. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership. Ehninger:Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership. Ehninger:GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Equity Ownership. Cartellieri:Cellex Patient Treatment GmbH: Employment.

ALLO-819, an Allogeneic Flt3 CAR T Therapy Possessing an Off-Switch for the Treatment of Acute Myeloid Leukemia

▪Patients with relapsed acute myeloid leukemia (AML) have poor prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented clinical efficacy in hematological malignancies, leading to durable responses in heavily pretreated patients. Adoptive immunotherapies using T cells redirected against AML cells are being pursued as one option with potential curative intent. However, the development of autologous CAR T therapies presents a significant logistical and clinical challenge in a rapidly progressing disease setting such as AML due to the lag time of cell manufacturing. Additionally, harvesting sufficient numbers of healthy T cells from patients with AML may not always be possible. For these reasons the development of an off-the-shelf CAR T cell product may be of benefit. This work details the preclinical evaluation of ALLO-819, an allogeneic CAR T therapy targeting the receptor tyrosine kinase Flt3 (CD135), an AML target with high prevalence in all AML subtypes and limited expression outside of the hematopoietic tissue.To construct a Flt3 CAR, a panel of high affinity (KD values of 0.19 to 233 nM, determined at 37°C) fully-human antibodies was generated using phage display technology. Single-chain variable fragments (scFvs) recognizing different immunoglobulin domains of the extracellular region of Flt3 were inserted into second-generation CAR constructs and tested for their ability to redirect T cell specificity and effector function towards AML cells. A lead CAR exhibiting minimal tonic signaling and potent antitumor activity in orthotopic mouse models of AML (2.5x106 and 1x107 CAR T cells for Eol-1 and Molm-13, respectively) was selected for further engineering to incorporate a safety off-switch in cis. To accomplish this, short amino acid stretches mimicking epitopes for the FDA-approved antibody rituximab were inserted between the hinge and target-binding regions of the CAR. The CAR T cell phenotype and antitumor efficacy were not affected by the presence of the off-switch. In the presence of rituximab, Flt3 CAR T cells were efficiently lysed via complement-dependent cytotoxicity (~ 80 % CAR T cell depletion in 3 hours) in vitro and eliminated in peripheral blood and bone marrow of NSG mice (>100-fold and >300-fold, respectively).Allogeneic ALLO-819 Flt3 CAR T cells with a lower risk of TCR-mediated graft-versus-host disease and resistant to anti-CD52 antibody (alemtuzumab)-mediated lysis were generated by disruption of the T-cell receptor alpha chain (TRAC) and the CD52 loci using TALEN® gene-editing technology. Transient expression of TALEN® in Flt3 CAR T cells resulted in high-efficiency inactivation of both loci and had no impact on T cell phenotype or antitumor efficacy. ALLO-819 Flt3 CAR T cells co-cultured with primary AML blasts ex vivo displayed target-dependent activation, cytokine secretion and cytotoxic activity.Consistent with previous reports, we detected Flt3 expression on a subset of normal hematopoietic stem and progenitor cells (HSPCs) which also showed susceptibility to CAR T cell cytotoxicity. To evaluate off-tumor effects of Flt3 CAR T cells in vivo, NSG mice were administered T cells expressing a CAR with similar affinity to both mouse and human Flt3. Mouse-cross-reactive Flt3 CAR T cells exhibited off-tumor activity that was limited to a subset of bone marrow multipotent progenitors and correlated with antitumor efficacy. Administration of rituximab led to effective depletion of CAR T cells in peripheral blood that was followed by a rapid repopulation of HSPCs to levels observed in naïve mice.In summary, these results support the development of ALLO-819 Flt3 CAR T as a novel immunotherapy for the treatment of AML. DisclosuresSommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Nguyen:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

A Next-Generation Fc-Bearing CD3-Engaging Bispecific DART® Platform with Extended Pharmacokinetic and Expanded Pharmacologic Window: Characterization As CD123 x CD3 and CD19 x CD3 DART Molecules

Background: CD3-engaging DART molecules direct T cells to tumor-expressed antigens. Flotetuzumab, a CD123 x CD3 bispecific DART molecule that targets the differential expression of the IL-3 receptor alpha chain (CD123) on acute myeloid leukemia (AML) blasts and leukemic stem cells, is currently being investigated in a phase 1 study in relapsed/refractory AML and myelodysplastic syndrome (MDS), with initial evidence of clinical activity1. A common adverse event in redirected T-cell killing interventions is cytokine release syndrome (CRS), a Cmax-associated event that may limit their therapeutic window. Owing to its short circulating half-life, flotetuzumab is administered as continuous infusion, which affords prolonged exposure with limited Cmax excursions. In contrast, long-acting Fc-bearing, CD3-engaging molecules would eliminate the need for continuous dosing, but would be especially impacted by CRS, given the high Cmax levels required to maintain adequate trough concentrations over several days or weeks. We previously reported the development of a CD3-engaging DART molecule with reduced affinity for CD3 that maintained maximal target cell killing and T-cell proliferation, albeit at higher concentrations than its wild-type (WT) counterpart, but with reduced cytokine release2. We report here further optimization and validation of the approach.Methods: Anti-CD3 variants spanning a range of affinities were formatted as Fc-bearing DART molecules with CD123- or CD19-targeting arms and evaluated for cell binding as well as for antigen-dependent induction of T-cell proliferation, cytolytic activity and cytokine release. A therapeutic index (TI) was determined from the ratio of in vitro cell killing activity to that of target cell-induced cytokine release. In vivo anti-tumor activity was assessed in human immune cell-reconstituted mouse tumor models or in mice expressing the epitope recognized by the CD3 arm of the DART proteins (human CD3 k/i mice). Safety, pharmacokinetic (PK) and biological activity of selected molecules was evaluated in cynomolgus monkeys.Results: Six Fc-bearing DART molecules (from a panel of 23 CD123 x CD3 DART variants) were selected for characterization. These molecules spanned a relatively small interval of low CD3 affinities, but varied greatly in activity, from inactive at all concentrations tested to being capable of lysing tumor cells, including primary leukemic and MDS blasts, to the same maximal level of killing observed with the parental WT DART molecule. Higher concentrations of the active variants were needed, owing to a deliberate design incorporating decreased CD3 binding. At levels capable of complete tumor cell lysis, active DART variants showed greatly reduced pro-inflammatory cytokine release (e.g., IL-2, IFN-γ and TNF-α) in vitro and in human PBMC-reconstituted mice compared to the WT version. Molecules were ranked based on their TI and a version with a highly favorable index was engineered as a B-cell targeting CD19 x CD3 DART molecule to conveniently ascertain its biological activity in cynomolgus monkeys. The CD19 x CD3 DART variant demonstrated good tolerability at single doses up to 30 mg/kg. B-cell depletion in the circulation and lymphoid tissues occurred at 1 mg/kg and achieved maximal depletion at ≥10 mg/kg, equivalent to the degree of reduction observed with 0.1 mg/kg of the WT molecule. However, only minimal circulating pro-inflammatory cytokines (including IL-6) were observed at all doses of the DART variant, while a substantial increase in IL-2, IL-6 and IFN-γ (among others) was observed with 0.1 mg/kg of the WT counterpart. The PK profile of the DART variant in cynomolgus monkeys appeared linear in the 1-30 mg/kg interval, with a half-life of 5 to 10 days, consistent with possible clinical dosing at weekly or longer intervals.Conclusions: A next-generation CD3-engaging Fc-bearing DART platform was generated through CD3 arm affinity modulation. These DART variants feature extended PK and an expanded TI suitable for potential clinical development in hematological malignancies.1. Uy et al, Blood 2017, vol. 130, Suppl 1 6372. Huang et al, Keystone Symposia: Antibodies as Drugs, 2/25-3/1/2018, Whistler, BC, Canada DisclosuresBonvini:MacroGenics: Employment, Equity Ownership. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Huang:MacroGenics, Inc.: Employment, Equity Ownership. Lam:MacroGenics, Inc.: Employment, Equity Ownership. Kaufman:MacroGenics, Inc.: Employment, Equity Ownership. Liu:MacroGenics, Inc.: Employment, Equity Ownership. Alderson:MacroGenics, Inc.: Employment, Equity Ownership. Stahl:MacroGenics, Inc.: Employment, Equity Ownership. Brown:MacroGenics, Inc.: Employment, Equity Ownership. Li:MacroGenics, Inc.: Employment, Equity Ownership. Sharma:MacroGenics, Inc.: Employment, Equity Ownership. Tamura:MacroGenics, Inc.: Employment, Equity Ownership. Johnson:MacroGenics, Inc.: Consultancy, Equity Ownership. Moore:MacroGenics, Inc.: Employment, Equity Ownership.

Synergistic Anti-Leukemic Activity of PARP Inhibition Combined with IMGN632, an Anti-CD123 Antibody-Drug Conjugate in Acute Myeloid Leukemia Models

BackgroundCD123 (IL-3 receptor alpha-chain) is a therapeutic target for hematological malignancies based on high expression levels in acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), and other cancers. The anti-CD123 antibody-drug conjugate (ADC), IMGN632, comprises a humanized monoclonal antibody covalently linked to a DNA - alkylating cytotoxic payload which is currently in phase 1 evaluation for relapsed/refractory CD123-positive hematological malignancies (NCT03386513). Novel approaches to enhance the efficacy of ADCs are of significant therapeutic interest. Our laboratory has previously demonstrated that the Poly ADP Ribose (PARP) inhibitor, olaparib, synergistically enhances the activity of the CD33-targeted ADC, IMGN779, in preclinical AML models (Portwood S et al, ASH 2016). Based on the hypothesis that PARP inhibition will synergize with DNA damaging mechanism of IMGN632, we investigated the ability of olaparib and other PARP inhibitors (PARPi) in clinical development (talazoparib, niraparib, rucaparib, and veliparib) to enhance the therapeutic efficacy of IMGN632 across diverse human AML cell lines and primary relapsed/refractory AML samples.Materials and MethodsCD123 expression on human AML cell lines (HEL, HL60, MV411, Molm13, EOL-1, THP-1, and Kasumi-1) was quantified by flow cytometry using QuantriBrite beads. AML cells were continuously cultured for 72-96 hours with varying doses of IMGN632 (range 100pM - 100nM) and specific PARP inhibitors (range 100pM -15μM) alone and in combination. Cell viability was measured using a WST-8 colorimetric assay. Primary clinically annotated CD123+ AML cells from patients with relapsed/refractory disease were obtained under IRB-approved protocols from the Roswell Park Hematologic Procurement Shared Resource and cultured short-term in the presence of multiple cytokines plus IMGN632 +/- PARP inhibitors. Apoptosis (Annexin V/PI), cell cycle, and DNA damage (H2AX) were evaluated by flow cytometry. Additive vs. synergistic effects were determined by combination indices using Compusyn software. PARP trapping was evaluated by Western blot analysis in nuclear lysates obtained from IMGN632 +/- PARP inhibitors treated AML cells.ResultsHigh expression levels of CD123 (range 937 - 2231 CD123 molecules/cell) were detected on multiple human AML cell lines (HEL-luc, MV411, Molm13, EOL-1, and THP-1) relative to unstained negative controls. Western blot analysis of nuclear lysates from AML cells demonstrated that all PARP inhibitors had varying degrees of PARP trapping on DNA. Continuous single agent 5-day treatment with all tested PARP inhibitors resulted in dose dependent in vitro inhibition of AML cell line growth with IC50 values ranging from 360 nM (talazoparib, most potent) to 78uM (veliparib, least potent). Combination therapy using PARP inhibitors (doses ranging from 300nM - 15uM) and IMGN632 (10nM) consistently resulted in enhanced anti-leukemic effects over monotherapy (Figure 1 for example). Synergistic anti-proliferative effects were obtained across all tested AML cell lines (n=5) with combination indexes ranging from 0.3-0.7 by Compusyn analysis. Combination therapy correlated with enhanced DNA damage, tumor cell apoptosis, and cell cycle arrest of AML cells. Moreover, IMGN632 and PARPi (olaparib or talazoparib) resulted in single agent activity against clinically annotated primary relapsed/refractory AML patient samples with evidence of synergistic effects when combined in vitro.ConclusionsAddition of PARP inhibitors to IMGN632, a novel anti-CD123 antibody-drug conjugate, further enhances DNA damage effects and consistently results in synergistic in vitro anti-leukemic effects across multiple CD123+ AML cell lines and primary AML patient samples. Further studies investigating this novel combinatorial approach in specific molecular subtypes of AML with variable baseline sensitivities to PARPi are currently ongoing. Our results strongly support future investigation of PARPi as a novel class of agents with the potential to significantly enhance the efficacy of DNA-alkylating ADCs and/or cytotoxic chemotherapy for hematological malignancies. [Display omitted] DisclosuresSloss:ImmunoGen: Employment. Watkins:ImmunoGen Inc.: Employment. Kovtun:ImmunoGen Inc.: Employment. Adams:ImmunoGen Inc.: Employment. Wang:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Jazz: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Pharmacologic IL-6Rα inhibition in cholangiocarcinoma promotes cancer cell growth and survival

Biliary tract cancer (BTC) represents a malignant tumor of the biliary tract including cholangiocarcinoma (CCA) and the carcinoma of the gallbladder (GBC) with a 5-year survival rate between 5 and 18% due to late diagnosis and rapid disease progression. Chronic inflammation is one of the main risk factors for CCA and GBC in particular. IL-6, as a mediator of inflammation, can act through a membrane-bound receptor alpha-chain (mIL-6R, “IL-6 classic signaling”) or via soluble forms (sIL-6R, “IL-6 trans-signaling”). However, little is known about the impact on cellular responses of IL-6 trans-signaling on BTC. We analyzed primary tumors as whole sections and as tissue microarrays, and also searched The Cancer Genome Atlas database. Compared to non-neoplastic, non-inflamed gallbladder tissue, IL-6Rα was downregulated in GBC, and this correlated with the patients' overall survival. Furthermore, different CCA cell lines and compounds for activation (IL-6 and Hyper-IL-6) or inhibition (Tocilizumab and sgp130Fc) of IL-6 classic signaling and trans-signaling were used to determine their effects on cellular processes between the two modes of IL-6 signaling. Inhibition of IL-6 trans-signaling by sgp130Fc reduced CCA cell line viability and apoptosis, whereas migration and proliferation were increased. We conclude that IL-6Rα expression is a good prognostic marker for GBC, and that the blocking of IL-6 trans-signaling and activation of IL-6 classic signaling have tumor promoting activity. These findings warrant the exclusion of patients with GBC or other malignancies associated with bile metabolism from IL-6R inhibitor therapy.

IMMU-20. SINGLE CELL CYTOMICS OF PERIPHERAL BLOOD MONONUCLEAR CELLS REVEALS NEW AVENUES FOR GLIOMA IMMUNOTHERAPY

Glioblastoma is the most common primary brain tumor with a dismal prognosis and a lack of effective treatment options after relapse. Despite striking successes that were achieved in the last decade in other types of cancer, evidence of sustainable responses to T cell directed immunotherapy in glioblastoma remains limited. This might be explained by an emerging hypothesis that glioblastomas are cold tumours with poor lymphocyte infiltration. Therefore, some recent efforts have been focused on increasing cytotoxic lymphocyte infiltration. Here we present our workflow for subpopulation frequency, marker expression, and correlation network (INFERENCE) analysis, in this study applied to screen for possible new targets to enhance anti-glioblastoma immune responses. Using high-dimensional, single-cell mass cytometry (CyTOF) we profiled peripheral blood mononuclear cells derived from 160 patients with glioblastoma, lower grade glioma, or metastasis, meningioma and epilepsy as controls. Based on a CyTOF panel composed mainly of lymphocyte markers and check-point inhibition markers we could effectively dissect blood leukocytes into 100 phenotypically separate subsets of CD4 T cells (35 subsets), CD8 T cells (17 subsets), monocytes (12 subsets), NK cells (14 subsets), and B cells (15 subsets), and map their frequencies, phenotypes and correlation statistics. We thereby created an in depth human atlas of peripheral blood mononuclear cells in patients with different types of intracranial tumors. Interestingly, a subset of B cells characterized by high expression of the Interleukin-2 receptor alpha chain (CD25, a typical marker of regulatory T cells) was significantly increased in glioblastoma patients. This immune suppressive B cell subset has previously been associated with enhancing the suppressive effect of regulatory T cells. Further research is warranted to investigate the role of this aberrant B cell subset in the context of glioblastoma and its potential as target for immunotherapy.

Parvovirus-Induced Adult Onset Stills Disease Presenting With Elevated Liver Transaminases

Adult-onset Still's disease (AOSD) is an inflammatory disorder affecting multiple systems characterized by polyarthritis, rash, fever, and an elevated erythrocyte sedimentation rate. Macrophage activating syndrome is a severe complication of AOSD leading to pancytopenia, hypofibrinogenemia, elevated triglycerides, lactate dehydrogenase, soluble IL-2 receptor alpha chain (CD25), and ferritin. AOSD is a very rare cause of markedly elevated liver chemistries. A 50-year-old female with a past medical history of asthma developed sore throat, diffuse joint aches, muscle weakness, and a fever of 104° F. She was evaluated at an outside facility and noted to have positive Parvovirus IgM/IgG. She was started on Prednisone 30 mg and with a taper, she experienced a reticular erythematous rash, arthralgias involving the wrist and ankle, myalgias, and cyclic fevers with a temperature max of 100.8° F. She had an aspartate aminotransferase (AST) of 853, alanine transaminase (ALT) of 866, and total bilirubin of 1.5 from prior normal baseline. Complete evaluation for viral hepatitis and hereditary liver disease was negative. Ultrasound with doppler showed parenchymal liver disease with patent vascular flow. Within 48 hours, her AST increased to 2,452, ALT 2,213, and total bilirubin of 3.3. Ferritin was elevated >30,000 ng/mL. Transjugular liver biopsy showed hepatic parenchyma with acidophil bodies, Kupffer aggregates, and preserved architecture with patchy collections of acute and chronic inflammatory cells. No bridging necrosis or iron deposition was appreciated. She was treated with high dose Methylprednisolone 250 mg IV daily with downtrend in transaminases. As steroids were tapered, AST rose from 71 to 198 and ALT from 390 to 900. Percutaneous liver biopsy showed mildly increased inflammation and confluent necrosis. She was discharged on anakinra 100 mg, a recombinant interleukin 1 receptor antagonist, and prednisolone 60 mg twice daily with continued improvement in transaminases to AST of 59 and ALT of 380. The patient developed AOSD likely induced by parvovirus which was complicated by MAS and transaminase elevation >5 upper limit of normal. The pathogenesis of liver involvement of AOSD is unknown but it is presumed that sustained macrophage activation with cytokine production play a role. This is evidenced in this case as transaminases decreased with steroid dosing and increased with the initial taper.

EFFECTS OF BENRALIZUMAB ON THE MANAGEMENT OF SEVERE ASTHMA

SESSION TITLE: Allergy/Airway Abstracts SESSION TYPE: Original Investigations PRESENTED ON: 10/09/2018 02:30 PM - 03:30 PM PURPOSE: Many patients with eosinophilic severe asthma despite taking medium to high dose inhaled corticosteroids and a second controller medication still have symptoms and exacerbation, and their asthma is not well controlled. Benralizumab is a humanized monoclonal antibody against the interleukin-5 receptor alpha-chain causing eosinophilic depletion. The aim of this systematic review and meta-analysis was to determine the impacts of Benralizumab on the management of eosinophilic severe asthma. METHODS: A comprehensive and systematic review of electronic data bases including PubMed, CENTRAL, CINAHL Plus, EMBASE, IPA, and ClinicalTrials.gov. were performed from the inception of Databases till June 16, 2017. Inclusion criteria were double-blinded, randomized, placebo-controlled, phase 3 clinical trials that measure the impacts of Benralizumab as add-on treatment on eosinophilic severe asthma patients. RESULTS: From1211 reviews articles, finally three articles with a combined 2730 patients were assessed the effects of Benralizumab on annual asthma exacerbation rate and four studies were determined it impacts on Asthma Control Questionnaire (ACQ) scores, Asthma Quality of Life Questionnaire (AQLQ) scores, and prebronchodilator FEV1% values in asthmatic patients with eosinophilia. There was a significant reduction in annual exacerbation rates (rate ratio vs. placebo = 0·62, 95% CI 0·49 to 0·78, p<0·0001) which is more pronounced in patients with elevated blood Eosinophil (rate ratio vs. placebo = 0·54, 95% CI 0·45 to 0·66, p<0·00001). ACQ scores, AQLQ scores, and FEV1% values were significantly improved in patients received Benralizumab in comparison to placebo (Mean Difference: -0.24, 95%CI: -0.32-0.16, p-value: <0.00001); (Mean Difference: 0.23, 95%CI: 0.14,0.32, p-value: <0.00001); (Mean Difference: 0.11, 95% CI 0.08,0.15, p-value: <0.00004) respectively. The adverse event rates were similar in the Benralizumab and placebo groups for many of the adverse events including any adverse events (rate ratio vs. placebo = 0·94, 95% CI 0·88 to 1·00), upper respiratory tract infection (rate ratio vs. placebo = 0·93, 95% CI 0·72 to 1·21), and nasopharyngitis (rate ratio vs. placebo = 0·94, 95% CI 0·78 to 1·13). In addition, only 38 out of 1808 (2·1%) patients discontinued treatment due to an adverse event in the Benralizumab group and 9 out of 922 (1%) in the placebo group. CONCLUSIONS: This systematic review and meta-analysis demonstrate that management of patients with eosinophilic severe asthma with Benralizumab significantly improves in asthma exacerbation rate, asthma control, asthma related quality of life and pre-bronchodilator FEV1 with minimal and acceptable adverse effects. CLINICAL IMPLICATIONS: This study provides evidenced-based recommendations for use of Benralizumab in asthmatic patients with eosinophilia and help direct current treatment guidelines. DISCLOSURES: no disclosure on file for Shabnam Asghari; No relevant relationships by Masoud Mahdavian, source=Web Response

CD16+NK-92 and anti-CD123 monoclonal antibody prolongs survival in primary human acute myeloid leukemia xenografted mice.

Patients with acute myeloid leukemia (AML) often relapse after initial therapy because of persistence of leukemic stem cells that frequently express the IL-3 receptor alpha chain CD123. Natural killer (NK) cell-based therapeutic strategies for AML show promise and we explore the NK cell lines, NK-92 and CD16+ NK-92, as a treatment for AML. NK-92 has been tested in phase I clinical trials with minimal toxicity; irradiation prior to infusion prevents risk of engraftment. The CD16 negative NK-92 parental line was genetically modified to express the high affinity Fc gamma receptor, enabling antibody-dependent cell-mediated cytotoxicity, which we utilized in combination with an anti-CD123 antibody to target leukemic stem cells. NK-92 was preferentially cytotoxic against leukemic stem and progenitor cells compared with bulk leukemia in in vitro assays, while CD16+ NK-92 in combination with an anti-CD123 mAb mediated antibody-dependent cell-mediated cytotoxicity against CD123+ leukemic targets. Furthermore, NK-92 infusions (with or without prior irradiation) improved survival in a primary AML xenograft model. Mice xenografted with primary human AML cells had a superior survival when treated with irradiated CD16+NK-92 cells and an anti-CD123 monoclonal antibody (7G3) versus treatment with irradiated CD16+NK-92 cells combined with an isotype control antibody. In this proof-of-principle study, we show for the first time that a CD16+NK-92 cell line combined with an antibody that targets a leukemic stem cell antigen can lead to improved survival in a relevant pre-clinical model of AML.

Abstract 1538: Analysis of the paired TCRα- and β-V(D)J full-length chains of single-cell sequence from human naïve and antigen-experienced T cells

Abstract The unbiased and paired characterization of T-cell receptor (TCR) alpha and beta chains is critical to understand the TCR repertoire and adaptive immunity. However, the direct cloning of the single human full-length TCRα- and TCRβ-chains has very few reports. We present a new methodology enabling capture of natively paired, full-length rearranged T-cell receptor (TCR) sequences at single-cell level of human T cells. More than 50% paired TCRα- and β- chains were captured in 100 single naïve human CD8+ T cells using 5'SMART and 3' TCRαβ constant region nested gene-specific primers (GSP). Furthermore, we sequenced the TCR of the specific T cells that were stimulated by representative tumor-associated antigens (TAA), virus-related antigens (VRA) and neoantigens. The highest proportion of TCR subtypes were extracted in the TCR abundance ranking by IMGT blast. Finally, we obtained the paired, full-length V(D)J TCR from melanoma antigen Melan A epitope 27-35 that was TRAV(12-2)J(47), TRBV(7-6)J(2-1), and the TCR from antigen EBV LMP2-FLY that was TRAV(26-1)J(7), TRBV(27)J(2-1). These results suggest that the method provides accurate identification of the paired, V(D)J rearrangements for each individual human naïve and antigen-experienced T cells. The information could be used in the construction of an engineered T-cell receptor for cancer immunotherapy and infectious diseases. Citation Format: Fei Wang, Qing Zhou, Cheng-chi Chao. Analysis of the paired TCRα- and β-V(D)J full-length chains of single-cell sequence from human naïve and antigen-experienced T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1538.

Abstract 987: Organoid-based characterization of patient tumors and microenvironments at single cell resolution

Abstract The advent of microenvironment directed cancer treatments such as antiangiogenic agents and recent immunotherapies have produced a pressing need for robust and systematic characterization of patient tumors and corresponding stromal populations. However, in vitro cultures of tumor biopsies do not typically preserve both the tumor epithelium and tumor microenvironment as an intact syngeneic unit. To address these limitations, we developed organoid cultures of surgically resected patient cancer biopsies that intrinsically retain diverse tumor microenvironmental cellular components without requiring reconstitution. We compared transcriptome profiles of fresh tumor cell populations to organoid cultures by droplet based single cell 5' RNA sequencing (scRNA-seq). Analysis of &amp;gt;50k single cell transcriptome profiles revealed tumor and stromal cell populations including tumor epithelia, fibroblasts and immune cells in fresh tumors. scRNA-seq of organoid cultures revealed a similar composition of native cancer cells and stromal components in both immune and non immune populations. In particular, 5' scRNA-seq enabled simultaneous characterization of paired T cell receptor alpha and beta chains as well as generalized gene expression in individual cells. We observed clonotype expansion of cytotoxic T cells in both fresh tumors and organoid cultures. The faithful recapitulation of tumor microenvironmental diversity within human organoid cultures should facilitate the in vitro exploration of immunotherapeutic agents and modeling of associated patient-specific responses. Citation Format: Ameen A. Salahudeen, Junjie Zhu, Jihang Ju, Arpit Batish, Ken Sutha, James T. Neal, Valeria Giangarra, Luz Montesclaros, Jerald Sapida, Osman Sharifi, Josephine Lee, Grace X. Zheng, Dhananjay A. Wagh, John A. Coller, Joel W. Neal, Sukhmani K. Padda, Chiara Sabatti, Calvin J. Kuo. Organoid-based characterization of patient tumors and microenvironments at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 987.

Abstract SY42-01: Targeting T cell malignancies with gene edited CAR-T

Abstract T-cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multiagent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-30% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy. Targeted therapy against T-cell malignancies represents a significant unmet medical need. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B-cell leukemia and lymphoma. Engineered T cells that express a chimeric antigen receptor (CAR) directed against T-cell malignancies are limited by several significant obstacles, but are a promising cancer immunotherapy. First, the shared expression of target antigens between T effector cells and T-cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients. We hypothesized that deletion of CD7 and the T-cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T-cell fratricide or induction of GvHD. We chose to target CD7 on malignant T cells because it is overexpressed on the vast majority of T-cell and NK-cell malignancies. Second, germline biallelic deletion of CD7 resulted in mice with normal T-cell numbers, T-cell subsets, and T-cell function. We generated a CD7 CAR using a anti-CD7 single chain variable fragment (scFv) created using commercial gene synthesis and cloned into the backbone of a 3rd-generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC was confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by FACS analysis. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. UCART7 effectively killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7 had no GvHD and unlike UCART19 controls, effectively cleared T-ALL blasts. Fratricide-resistant and allo-tolerant "off-the-shelf" UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T-cell lymphoma. Citation Format: John F. Dipersio, Matthew Cooper. Targeting T cell malignancies with gene edited CAR-T [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY42-01.

Abstract 5565: Potency-reduced IL15/IL15Rα heterodimeric Fc-fusions display enhanced in vivo activity through increased exposure

Abstract IL15 and IL2 are two similar cytokines that stimulate the proliferation of lymphocytes, and their therapeutic potential has been well established in animal models and human trials. Both cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain (γc) and IL2Rβ, as well as an alpha chain receptor unique to each cytokine: IL2Rα or IL15Rα. Both cytokines share a similar biology, with the exception that IL2 has a greater preference for Tregs due to their high constitutive expression of IL2Rα. IL15 functions as a stabilized heterodimeric complex with membrane-bound IL15Rα on the surface of monocytes and DCs, and this IL15/IL15Rα complex is presented in trans to lymphocytes expressing IL2Rβ and γc. It has been shown that recombinant IL15/IL15Rα heterodimer is stable and highly active. As potential drugs, both IL2 and IL15 are extremely potent and suffer from low tolerability and very fast clearance that limits therapeutic window. Seeking to engineer a more druggable version of IL15, we created various IL15/IL15Rα heterodimeric Fc-fusions (IL15/IL15Rα-Het-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and prolong half-life. We engineered IL15/IL15Rα-Het-Fc by fusing IL15 to one side of a heterodimeric Fc-region, and the sushi domain of IL15Rα to the other side, or by creating a single-chain IL15/IL15Rα that was attached to one side of a heterodimeric Fc-region. These Fc-fusions were tuned for optimal activity by engineering amino acid substitutions in IL15 - at the IL2Rβ or γc interface - that reduced overall in vitro potency. In vitro proliferation of lymphocytes in normal human PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days and by measuring signaling in a STAT5 phosphorylation assay for 15 minutes. In vivo activity was evaluated using a huPBMC-NSG mouse model by measuring the extent of human leukocyte engraftment by flow cytometry and IFNγ. Tolerability, immune stimulation, and pharmacokinetics were evaluated in non-human primates (NHP). IL15/IL15Rα-Het-Fc were produced with good yield and purity. The Fc-fusions enhanced proliferation of CD8+ T and NK cells in vitro, and potency of variants with substitutions at the IL2Rβ and/or γc interface was reduced up to ~700-fold compared to wild-type IL15/IL15Rα-Het-Fc. Treatment of huPBMC-NSG mice with IL15/IL15Rα-Het-Fc promoted enhanced T cell engraftment and elevated IFNγ in a dose dependent manner. NHP studies indicated half-lives of several days for IL15/IL15Rα-Het-Fc, which are significantly longer than the &amp;lt;1 hr half-life of IL15. In both in vivo settings, a marked inverse correlation of pharmacodynamics and clearance was observed, with weaker potency variants allowing higher doses and enhanced in vivo lymphocyte proliferation as a result of more sustained exposure. Citation Format: Matthew J. Bernett, Rajat Varma, Christine Bonzon, Rumana Rashid, Liz Bogaert, Ke Liu, Suzanne Schubbert, Kendra N. Avery, Irene W. Leung, Nicole Rodriguez, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. Potency-reduced IL15/IL15Rα heterodimeric Fc-fusions display enhanced in vivo activity through increased exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5565.

Selectively Agonizing Treg in Type 1 Diabetes with IL-2 Muteins

Regulatory T cells (Treg) play a pivotal role in maintaining self-tolerance. For their development and function, Treg rely on interleukin-2 (IL-2) and efficiently compete with other immune cells for IL-2 by expressing the highest levels of the IL-2 receptor alpha-chain (IL-2Ra or CD25). Treg deficiencies and alterations of the IL-2 pathway have been reported in several autoimmune diseases including type 1 diabetes (T1D) where the loss of self-tolerance leads to the destruction of insulin-producing beta cells in the pancreas. Low-dose IL-2 therapy has been shown to be successful in several autoimmune and inflammatory conditions; however, the potential for IL-2 to activate pro-inflammatory immune cells raises concerns regarding its widespread utility, particularly in type 1 diabetes (T1D) where pathogenic autoreactive T cells are activated by IL-2. Hence, there is a need to develop novel IL-2 based therapeutics with enhanced Treg-specificity. We have generated a series of Fc-fused murine IL-2 muteins (Fc.IL-2 mutein) to optimize Treg-selectivity, tissue distribution and PK. Twenty-eight Fc.IL-2 muteins were screened for their signaling potency and surface retention on Treg. Eight of the mutein candidates, showing a reduced signaling in Treg but prolonged association with CD25, were further characterized in vitro. Two of the weakest muteins were tested in vivo and showed a greater enrichment and Ki-67 induction in Treg compared to wild type (WT) IL-2, and less effect on CD4+ T effector cells, CD8+ cells and NK cells. Finally, in the non-obese diabetic mouse model of T1D, we observed a significantly improved protection in mice treated infrequently with Fc.IL-2 mutein compared to WT IL-2, and, unexpectedly, differences in efficacy between WT and mutein were more pronounced at a very high dose level. Altogether, our data suggest that Fc.IL-2 mutein treatment represent a safer and more efficient alternative for the WT IL-2 therapy, allowing a selective Treg enrichment and a targeted control of the autoimmune response. Disclosure L. Khoryati: None. S. Kumari: None. T. Pham: None. M. Bogdani: None. M. Gavin: None.

Sex-steroids and hypolipidemic chemicals impacts on brown trout lipid and peroxisome signaling — Molecular, biochemical and morphological insights

Lipid metabolism involves complex pathways, which are regulated in a similar way across vertebrates. Hormonal and hypolipidemic deregulations cause lipid imbalance from fish to humans, but the underlying mechanisms are far from understood. This study explores the potential of using juvenile brown trout to evaluate the in vivo interferences caused by estrogenic (17α-ethinylestradiol – EE2), androgenic (testosterone – T), and hypolipidemic (clofibrate – CLF) compounds in lipidic and/or peroxisomal pathways. Studied endpoints were from blood/plasma biochemistry, plasma fatty acid profile, ultrastructure of hepatocytes and abundance of their peroxisomes to mRNA expression in the liver. Both T and CLF caused minimal effects when compared to EE2. Estrogenized fish had significantly higher hepatosomatic indexes, increased triglycerides and very-low density lipoproteins (VLDL) in plasma, compared with solvent control. Morphologically, EE2 fish showed increased lipid droplets in hepatocytes, and EE2 and T reduced volume density of peroxisomes in relation to the hepatic parenchyma. Polyunsaturated fatty acids (PUFA) in plasma, namely n−3 PUFA, increased with EE2. EE2 animals had increased mRNA levels of vitellogenin A (VtgA), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor alpha (PPARα), PPARαBa and acyl-CoA long chain synthetase 1 (Acsl1), while ERβ-1, acyl-CoA oxidase 1–3I (Acox1–3I), Acox3, PPARγ, catalase (Cat), urate oxidase (Uox), fatty acid binding protein 1 (Fabp1) and apolipoprotein AI (ApoAI) were down-regulated. In summary, in vivo EE2 exposure altered lipid metabolism and peroxisome dynamics in brown trout, namely by changing the mRNA levels of several genes. Our model can be used to study possible organism-level impacts, viz. in gonadogenesis.

T Cell Receptor Alpha Chain Genes in the Teleost Ballan Wrasse (Labrus bergylta) Are Subjected to Somatic Hypermutation.

Previously, somatic hypermutation (SHM) was considered to be exclusively associated with affinity maturation of antibodies, although it also occurred in T cells under certain conditions. More recently, it has been shown that SHM generates diversity in the variable domain of T cell receptor (TCR) in camel and shark. Here, we report somatic mutations in TCR alpha chain genes of the teleost fish, Ballan wrasse (Labrus bergylta), and show that this mechanism adds extra diversity to the polymorphic constant (C) region as well. The organization of the TCR alpha/delta locus in Ballan wrasse was obtained from a scaffold covering a single copy C alpha gene, 65 putative J alpha segments, a single copy C delta gene, 1 J delta segment, and 2 D delta segments. Analysis of 37 fish revealed 6 allotypes of the C alpha gene, each with 1–3 replacement substitutions. Somatic mutations were analyzed by molecular cloning of TCR alpha chain cDNA. Initially, 79 unique clones comprising four families of variable (V) alpha genes were characterized. Subsequently, a more restricted PCR was performed to focus on a specific V gene. Comparison of 48 clones indicated that the frequency of somatic mutations in the VJ region was 4.5/1,000 base pairs (bps), and most prevalent in complementary determining region 2 (CDR2). In total, 45 different J segments were identified among the 127 cDNA clones, counting for most of the CDR3 diversity. The number of mutations in the C alpha chain gene was 1.76 mutations/1,000 bps and A nucleotides were most frequently targeted, in contrast to the VJ region, where G nucleotides appeared to be mutational hotspots. The replacement/synonymous ratios in the VJ and C regions were 2.5 and 1.85, respectively. Only 7% of the mutations were found to be linked to the activation-induced cytidine deaminase hotspot motif (RGYW/WRCY).

024 Exposure of murine Langerhans cells (LCs) to IL-6 biases antigen (Ag) presentation toward an IL-17A response

Exposure of endothelial cells (ECs) to the neuropeptide calcitonin gene-related peptide endows ECs with the ability, acting as bystanders, to bias the outcome of LC Ag presentation to T cells away from Th1 responses and toward Th17 responses, and IL-6 production by ECs appears to mediate most of this effect. To determine if IL-6 action on LCs alone, or T cells alone, is sufficient for this phenomenon, we exposed epidermal BALB/c LCs or, separately, T cells from DO11.10 mice (BALB/c background; DO11.10 mice have T cells that respond to a fragment of chicken ovalbumin, cOVA323339) to IL-6 or medium alone for 3 h. Then, all cells were washed x 4. IL-6-exposed or medium-exposed LC were cultured with DO11.10 T cells not exposed to IL-6 along with Ag. Supernatants were harvested 72 h later and analyzed by ELISA for cytokine content. Exposure of LCs to IL-6 led to significantly enhanced production of IL-6 and IL-17A with significantly reduced IFNγ production. When analogous experiments were set-up treating responding T cells with IL-6 instead of LCs, no effect was observed. Exposure of LCs to monoclonal anti-CD126 antibodies (Abs) before and during treatment with IL-6 significantly inhibited enhancement of IL-17A and IL-6 production, as well as decreased IFNγ production, by responding DO11.10 T cells, suggesting that presentation of IL-6 by the IL-6 receptor (IL-6R) alpha chain is important for this effect. Similar experiments exposing LCs to Abs against the type I cytokine receptor subunit CD130 instead of anti-CD126 Abs showed a much smaller, but still significant, effect on production of IL-6 and IFNγ and no significant effect on IL-17A. These experiments show that exposure of LCs to IL-6 polarizes the outcome of Ag presentation to T cells away from an IFNγ response and toward an IL-17A response and suggest that IL-6 must be presented by the IL-6R alpha chain for the effect observed. These findings may have implications for a greater understanding of the pathophysiology of inflammatory skin disorders.

Benralizumab: First Global Approval.

Kyowa Hakko Kirin, AstraZeneca and subsidiaries are developing benralizumab (Fasenra™)-a humanised anti-interleukin-5 receptor alpha chain (IL-5Rα) monoclonal antibody-as a treatment of severe eosinophilic asthma and chronic obstructive pulmonary disease (COPD). Eosinophilia is a characteristic of certain asthma and COPD phenotypes and depletion of eosinophils has demonstrated therapeutic benefit. Benralizumab was recently approved by the US FDA as add-on maintenance therapy for patients with severe asthma who have an eosinophilic phenotype. This article summarizes the milestones in the development of benralizumab leading to this first approval for the treatment of severe eosinophilic asthma.

An "off-the-shelf" fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies.

T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here we describe a fratricide resistant ‘off-the-shelf’ CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide resistant, allo-tolerant ‘off-the-shelf’ CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin’s T cell lymphoma without a requirement for autologous T cells.