Extracellular Epitope Research Articles

Islet autoantibodies in disease prediction and pathogenesis.

Type 1 diabetes (T1D) is now predictable by measuring specific islet autoantibodies (IAbs). Almost all children who developed multiple IAbs will progress to T1D with time, while individuals with single IAb have a very low risk although it is an important earlier biomarker. The poor prediction of single IAb has been found to be associated with IAb affinity. Majority of single IAb generated in current standard IAb radio-binding assay (RBA) are of low affinity, which have been demonstrated low risk in T1D development. New generation of nonradioactive IAb assay with electrochemiluminescence (ECL) technology has been shown to discriminate high-affinity from low-affinity IAbs and greatly improve sensitivity and disease specificity. With a high-affinity IAb assay, like ECL assay, single IAb will be expected to be a reliable biomarker for T1D early prediction. Although appearance of IAbs is most reliable biomarkers for T1D, there are no direct evidences that IAbs contribute to β-cell damage. With recent studies on ZnT8, a merging protein on β-cell surface membrane associated with insulin secretion, a subclass of ZnT8 autoantibodies directed to extra-cellular epitopes of ZnT8 on β-cell surface has recently been identified in T1D patients and these cell surface autoantibodies have been found to appear very early, before other IAbs. These findings lead us to a hypothesis that the immunogenic epitopes on β-cell surface might be early targets for autoimmune disease and IAbs to cell surface epitopes might be involved in β-cell destruction, which will change the paradigm of IAbs in T1D pathogenesis.

CS1 Targeted Chimeric Antigen Receptors (CAR) for treatment of multiple myeloma (MM)

BCMA-CAR-Ts induce complete responses in MM but not cure, indicating a need for other CAR targets. CS1 (SLAMF7) is uniformly expressed on MM with limited expression on normal cell types, so is a good CAR target. CS1-CAR-Ts expressing the scFv derived from Elotuzumab antibody (clone HuLuc63) show efficacy in preclinical MM models. Here, we report efficacy of CS1-CAR-Ts with an scFv derived from an antibody that binds a separate, more distal extracellular epitope on CS1 (clone Luc90; CS1-90). The MM.1S human myeloma cell line (CS1+; expressing luciferase and GFP; MM.1S-CG) were used as target cells in 4hr Chr51 release killing assays.

PB2299 AN INVESTIGATION INTO THE PRESENCE OF COMPLEMENT PROTEINS ON ERYTHROCYTE VESICLES IN SICKLE CELL DISEASE

Background:A single amino acid substitution in the globin gene segment is the underlying genetic defect in sickle cell disease (SCD) creating a haemoglobin molecule which polymerizes on deoxygenation. Recent insights into the pathophysiology of the disease have pointed to the roles inflammation and hypercoagulability play in disease manifestation and severity caused, in part, by increased phosphatidylserine exposure and extracellular vesiculation by sickle red cells.Current works have identified some of these vesicles as those released from reticulocytes in the process of maturation to the red blood cell. These vesicles are termed ‘inside‐out’ autophagic vesicles as they are the result of the fusion of endocytosed parts of the cell membrane with autophagic endosomes and are positive for intracellular glycophorin A (GPA) epitope using BRIC163 antibody. Further analysis has shown that some of these vesicles are also positive for extracellular GPA epitope using BRIC256 antibody raising the possibility that the vesicle membrane may have become permeabilized. One possible permeabilizing mechanism is the membrane attack complex of the complement system.Aims:The aim of this project was to investigate the presence of complement proteins on vesicles isolated from plasma or attached to erythrocytes using flow cytometry and confocal microscopyMethods:Vesicles were isolated from SCD plasma (n = 2) and healthy control (n = 1) using 2 different techniques: membrane‐based isolation technique and fluorescent‐activated cell sorter (FACS) and studied with flow cytometry using BRIC163, BRIC256, C3, C4, C3c and C3d antibodies. Cultured reticulocyte (n = 2), sickle red cells (n = 3) and healthy control red cells (n = 2) were analyzed with confocal microscopy using Annexin‐V, BRIC163, C3, C4, C3c and C3d antibodies.Results:Results show the presence of complement proteins on vesicles in both SCD plasma and healthy control in levels as high as 100% for BRIC163 vesicles in healthy control. Confocal analyses reveal this deposition is also present on vesicles prior to release and is associated with PS exposure.Summary/Conclusion:Complement proteins are present on vesicles in plasma and erythrocytes and may suggest activation and formation of the MAC complex. Further work is required to determine the involvement of these proteins in vesicle formation and release.

162-OR: Identification of Autoantibodies to ZnT8 Extracellular Epitope(s) in Patients with T1D

Previous studies demonstrate ZnT8 is displayed on the β-cell surface upon insulin secretion and IgG from ZnT8A-positive T1D patient sera stains the surface of live pancreatic β-cells. To further identify autoantibodies (AAb) to the ZnT8 extracellular epitope(s) (ZnT8ec) and to develop a ZnT8ec AAb assay, we first stabilized its native structure without the N-terminal domain (a.a 66-369; R325 or W325) by forming ZnT8-intracellular domain-Ab (ZnT8ic) complexes prior to release from liposome membranes. Sulfo-tagged ZnT8-Ab complexes were used as antigen in our electrochemiluminescence (ECL) AAb assay to analyze 130 sera (96 T1D patients, 22 T2D patients and 12 normal controls). Autoantibodies to ZnT8ec were identified in T1D patient sera by cross-reactivity of sera previously identified as exclusively ZnT8R or W positive, and ZnT8A negative sera. The prevalence of ZnT8ec AAbs in T1D patients was 21% (22/96: 5/37 of R+ sera, 7/24 of W+ sera and 8/35 of ZnT8A- sera) compared to 1/22 T2D patients and 0/12 normal controls. These are the first biochemically defined AAbs from T1D sera reported to target the β-cell surface. Further analysis of the role of these autoantibodies in β-cell autoimmunity and T1D etiopathology will be interesting and important for future studies. Disclosure Y. Gu: None. C. Merriman: None. J.M. Wenzlau: None. L. Yu: None. D. Fu: None.

Epitope Selection for HLA-DQ2 Presentation: Implications for Celiac Disease and Viral Defense.

We have reported that the major histocompatibility molecule HLA-DQ2 (DQA1*05:01/DQB1*02:01) (DQ2) is relatively resistant to HLA-DM (DM), a peptide exchange catalyst for MHC class II. In this study, we analyzed the role of DQ2/DM interaction in the generation of DQ2-restricted gliadin epitopes, relevant to celiac disease, or DQ2-restricted viral epitopes, relevant to host defense. We used paired human APC, differing in DM expression (DMnull versus DMhigh) or differing by expression of wild-type DQ2, versus a DM-susceptible, DQ2 point mutant DQ2α+53G. The APC pairs were compared for their ability to stimulate human CD4+ T cell clones. Despite higher DQ2 levels, DMhigh APC attenuated T cell responses compared with DMnull APC after intracellular generation of four tested gliadin epitopes. DMhigh APC expressing the DQ2α+53G mutant further suppressed these gliadin-mediated responses. The gliadin epitopes were found to have moderate affinity for DQ2, and even lower affinity for the DQ2 mutant, consistent with DM suppression of their presentation. In contrast, DMhigh APC significantly promoted the presentation of DQ2-restricted epitopes derived intracellularly from inactivated HSV type 2, influenza hemagglutinin, and human papillomavirus E7 protein. When extracellular peptide epitopes were used as Ag, the DQ2 surface levels and peptide affinity were the major regulators of T cell responses. The differential effect of DM on stimulation of the two groups of T cell clones implies differences in DQ2 presentation pathways associated with nonpathogen- and pathogen-derived Ags in vivo.

Encefalitis autoinmune: experiencia de una serie de casos

La encefalitis límbica autoinmune se define como un desorden inflamatorio severo en el cerebro que genera una encefalopatía rápidamente progresiva por lo regular en menos de 6 semanas. Se han descrito anticuerpos contra epítopes de proteínas sinápticas de superficie o contra proteínas intracelulares. Se reportan seis casos de encefalitis autoinmune con la descripción de cada uno incluyendo las manifestaciones típicas, alteraciones imagenológicas, curso clínico, complicaciones durante la hospitalización y respuesta al tratamiento. En dos de los casos se logró confirmar mediante el aislamiento de los anticuerpos. Uno falleció durante su hospitalización. Nuestra experiencia muestra un alto riesgo de secuelas y refractariedad al tratamiento que dependen del tiempo de inicio de la terapia.

Development of Cirmtuzumab Antibody-Drug Conjugates (ADCs) Targeting Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1)

ROR1 is an onco-embryonic surface antigen expressed on chronic lymphocytic leukemia (CLL) and a variety of other cancers, but not on most normal adult tissues. We generated a humanized IgG1 monoclonal antibody (mAb) cirmtuzumab (formerly UC-961) that binds with high affinity to a specific extracellular epitope of human ROR1 and that can block Wnt5a-induced ROR1 signaling (Yu, J et al, J Clin Invest126:585, 2016; Yu, J et al, Leukemia31:1333, 2017). Preclinical studies found that cirmtuzumab did not react with normal post-partem cells and had a pharmacokinetic (PK) volume distribution in primates consistent with a lack of off-target binding to normal tissues. We evaluated cirmtuzumab in a phase I clinical trial involving patients with relapsed-refractory CLL (Choi MY, et al, Cell Stem Cell22:951, 2018); the drug was well-tolerated at doses ≤20 mg/kg (highest dose tested) without dose-limiting toxicity. PK studies showed cirmtuzumab had a half-life of 32.4 days with no evidence for development of neutralizing antibodies or off-target sequestration of infused antibody. Furthermore, cirmtuzumab effected partial down-modulation of leukemia-cell ROR1 in patients treated with doses ≥2 mg/kg. In vitro confocal microscopy studies showed that this down-modulation was caused by internalization of cirmtuzumab-ROR1 complexes into lysosomal compartments and concomitant steady-state re-expression of nascent surface ROR1. Because of its high specificity, in vivo stability, long serum half-life, and potential capacity to concentrate conjugated drugs into lysosomal compartments, cirmtuzumab appeared ideally suited to serve as the targeting moiety in anti-ROR1 ADCs. We therefore examined cirmtuzumab-based ADCs in collaboration with VelosBio Inc., evaluating multiple linker/payload chemistries, both as single agents and in combinations. We selected for further testing cirmtuzumab-ADC-7, a cirmtuzumab-linker-monomethyl auristatin E (MMAE) ADC that preserves the high-affinity binding specificity of cirmtuzumab and allows for ROR1-targeted intracellular release of MMAE. We found cirmtuzumab-ADC-7 was selectively cytotoxic for ROR1+ CLL and mantle-cell lymphoma (MCL) cell lines at nM concentrations in vitro. Moreover, cirmtuzumab-ADC-7 caused dramatic and sustained in vivo clearance of adoptively-transferred ROR1+ leukemia cells generated from ROR1xTCL1 transgenic mice (Widhopf G, et al, PNAS111:793, 2014), ROR1+ MCL-xenografts, or ROR1+ cancer patient-derived xenografts (PDX). Further, treatment caused dose-dependent and statistically significant decreases in total cancer burden with complete regressions of tumor in multiple animals; no effect on tumor-clearance was observed in mice treated with a control MMAE-ADC of irrelevant specificity. Recently we identified that miR-15a/16-1, which commonly are deleted/downregulated in CLL, target both BCL2 and ROR1, thereby accounting in part for the direct relationship we observed between the levels of BCL2 and levels of surface ROR1 expressed by CLL of different patients (Rassenti, LZ, et al,PNAS114:10731, 2017). Because high level expression of BCL2/ROR1 may mitigate the cytotoxic activity of the BCL2-antagonist venetoclax, but potentially enhance the cytotoxicity of cirmtuzumab-ADC-7, we treated ROR1+ leukemia/lymphoma cell lines with venetoclax and/or cirmtuzumab-ADC-7. Chou-Talalay combination indices were <0.5 in all ROR1+ cell lines tested, indicating strong antitumor synergy with these two agents. Collectively these data support the rationale for clinical development of a cirmtuzumab-based ADC for treatment of patients with ROR1+ malignancies. DisclosuresVo:VelosBio: Employment. Jessen:VelosBio: Employment. Kipps:Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Verastem: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 5759: Discovery of a novel claudin 6 (CLDN6) specific monoclonal antibody

Abstract Claudin 6 (CLDN6) is a tight junction molecule that is involved in cell to cell adhesion of epithelial and endothelial cell sheets. CLDN6 is considered an oncofetal protein which is not expressed in normal human tissue but is expressed in some cancers such as endometrial, ovarian and testis cancer. Expression of CLDN6 in endometrial and urothelial cancer leads to a poor prognosis. The problem of developing antibodies against CLDN6 is that the family member claudin 9 (CLDN9) is highly homologous, only varying by 2 amino acids in the extracellular domain. To address this need, Integral Molecular has developed the MPS Discovery Engine® to enable the isolation, characterization, and engineering of monoclonal antibodies for tight junction proteins, GPCRs, ion channels, and transporters. MPS utilizes a collection of technologies to address each of the barriers to monoclonal antibody development against the native extracellular epitopes of multispan membrane proteins. These include, antigen engineering to attain high levels of surface expression, DNA and Lipoparticle immunization to present native epitopes to the immune system, diverse immunization host species to deal with highly conserved proteins, Lipoparticles (high concentration native membrane proteins) to enable phage display, microfludic B-cell isolation to isolate rare MAbs, and shotgun mutagenesis (comprehensive alanine scanning) for epitope mapping. Using the MPS Discovery Engine® we were able to successfully screen a large panel of clones for claudin 6 specificity. From these clones there were 72 potential antibodies that reacted with either claudin 6 or 6/9. A subset of these antibodies reacted only to claudin 6 and not to claudin 9 which has led to our lead drug candidate. With our MPS Discovery Engine® platform, we have the ability to target intact, conformationally specific, and functional antibodies to multipass membrane proteins. Citation Format: Lewis J. Stafford, Brad Screnci, Chidananda Sulli, Erin Rosenberg, Nicholas Molino, David Tucker, Jonathan Sullivan, Trevor Barnes, Jennifer Pfaff, Tanmayee Hazarika, Thomas Charpentier, Samantha Gilman, Rebecca Rimkunas, Rona Wilf, Sharon Willis, Benjamin Doranz, Joseph Rucker, Ross Chambers. Discovery of a novel claudin 6 (CLDN6) specific monoclonal antibody [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 5759.

Human Stem Cell–Derived Models: Lessons for Autoimmune Diseases of the Nervous System

Autoimmunity of the peripheral and central nervous system is an important cause of disease and long-term neurological disability. Autoantibodies can target both intracellular and extracellular neuronal epitopes. Autoantibodies that target cell-surface epitopes infer pathogenicity through several distinct mechanisms, while patients often respond to immunotherapy. However, the underlying pathogenesis of these autoantibodies is yet to be fully understood. Human stem cell–based disease modeling, and the rise of induced pluripotent stem cell technology in particular, has revolutionized the fields of disease modeling and therapeutic screening for neurological disorders. These human disease models offer a unique platform in which to study autoimmunity of the nervous system. Here, we take an in-depth look at the possibilities that these models provide to study neuronal autoantibodies and their underlying pathogenesis.

S60. Case series of anti-GABAb receptor antibody autoimmune limbic encephalitis - clinical data, EEG, brain imaging and treatment outcome

Introduction Autoantibodies to extracellular epitopes of neurons and associated neurological disorders, especially autoimmune limbic encephalitis (LE), are increasingly recognized. GABAb receptor antibody associated autoimmune LE often presents with refractory seizures. Timely recognition and diagnosis are critical, as prompt immunotherapy achieves more favorable outcomes. Methods We identified 3 autoimmune LE cases associated with anti-GABAb receptor antibody (Ab). Clinical data was retrospectively obtained and evaluated. Broad neuronal autoantibody panels were also examined. Results The initial clinical presentation of LE was new onset seizures with altered mental status, two patients had prodromal infectious symptoms prior to the onset of seizures. When symptoms evolved, seizures became pharmacologically refractory to anti-epileptics. One patient had convulsive status, and two others had non-convulsive status confirmed by either repetitive EEG tests or continuous EEG monitoring. The patients also presented with behavioral symptoms including agitation, behavioral outbursts, and one patient had hypersomnia. Two patients were with diagnosed with small cell lung cancer prior to the seizure onset, one of whom also had recurrent refractory optic neuritis. The other patient had a negative malignancy workup. CSF studies showed mild pleocytosis with lymphocyte predominance, and two patients were found to have positive oligoclonal bands in CSF. GABAb receptor Abs were found in CSF from all three patients, two patients were also found to have positive GABAb receptor Abs in serum. Other neuronal autoantibodies were also detected, CRMP5 (in serum/CSF from patient with optic neuritis), P/Q, or N-type calcium channel antibodies were found in serum from all three patients. Brain T2/FLAIR signal changes were seen in the temporal lobe and hippocampus in two. The other one patient’s brain MRI was unrevealing. All patients underwent IV Steroid and IVIG or plasma exchange treatment, which helped seizure control and briefly improved mental status. Two patients received further treatment with Rituximab due to persistent cognitive deficits (short term memory loss, poor attention/concentration) or persistent hypersomnia. Rituximab treatment resulted in partial clinical improvement. Though there was no seizure recurrence in these patients followed for 3–9 months, both patients continued to have cognitive deficits and memory loss. Follow-up CSF study showed resolved CSF pleocytosis, however, serum and CSF serology showed persistent positivity of GABAb receptor Abs in these patients. Thus, the patients continued with long term immuno-therapies. Conclusion We report 3 cases of GABAb receptor antibody associated autoimmune LE including one unique case with concurrent CRMP5 associated paraneoplastic optic neuritis. Though coexistence of other neuronal autoantibodies were noted, the neurological presentations in our cohort were likely directly due to the pathogenic effects medicated by GABAb receptor antibodies.

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3.

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.

Monoclonal antibody targeting the β-barrel assembly machine of Escherichia coli is bactericidal

The folding and insertion of integral β-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect β-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli β-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its β-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying β-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

Selective Targeting of CD133-Expressing Glioblastoma Stem Cells Using Lentiviral Vectors.

Several lines of evidence suggest a cellular hierarchy in glioblastoma (GBM). In this hierarchy, GBM stem-like cells (GSCs) play critical roles in tumor progression and recurrence, by virtue of their robust tumor-propagating potential and resistance to conventional chemoradiotherapy. Therefore, targeting GSCs holds significant therapeutic promise. Expression of CD133 (PROM1), a cell surface glycoprotein, has been associated with the GSC phenotype and used as a GSC marker. Here, we describe a protocol that allows the selective lentiviral transduction of CD133-expressing GBM cells. This selectivity is conferred by pseudotyping the lentiviral envelope with a single-chain antibody against an extracellular epitope on CD133. We previously demonstrated the efficacy and specificity of this lentiviral vector using patient-derived GBM cultures. This chapter outlines the preparation of the vector and the transduction of human GBM cells.

Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells.

The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, e.g., MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.

Development of fluorescence imaging probes for nicotinic acetylcholine α4β2∗ receptors

Nicotinic acetylcholine α4β2∗ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4β2∗ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4β2∗ nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the α4β2∗ nAChRs in [3H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in α4β2∗ nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface α4β2∗ nAChRs, labeled with antibodies specific for a β2 subunit extracellular epitope indicating that nifrofam labels α4β2∗ nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of α4β2∗ nAChRs in brain regions.

Imaging PD-L1 Expression with ImmunoPET.

High sensitivity imaging tools could provide a more holistic view of target antigen expression to improve the identification of patients who might benefit from cancer immunotherapy. We developed for immunoPET a novel recombinant human IgG1 (termed C4) that potently binds an extracellular epitope on human and mouse PD-L1 and radiolabeled the antibody with zirconium-89. Small animal PET/CT studies showed that 89Zr-C4 detected antigen levels on a patient derived xenograft (PDX) established from a non-small-cell lung cancer (NSCLC) patient before an 8-month response to anti-PD-1 and anti-CTLA4 therapy. Importantly, the concentration of antigen is beneath the detection limit of previously developed anti-PD-L1 radiotracers, including radiolabeled atezolizumab. We also show that 89Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. 89Zr-C4 detects mouse PD-L1 expression changes in immunocompetent mice, suggesting that endogenous PD-1/2 will not confound human imaging. Lastly, we found that 89Zr-C4 could detect acute changes in tumor expression of PD-L1 due to standard of care chemotherapies. In summary, we present evidence that low levels of PD-L1 in clinically relevant cancer models can be imaged with immunoPET using a novel recombinant human antibody.

Autoimmune headache in anti-nmda receptor encephalitis

Background: Anti-NMDAreceptor (NMDAR) encephalitis is an autoimmune encephalitis caused by autoantibodies against the extracellular epitope on the NR1 subunit of the NMDAR. Headache is often initially reported by patients; however, a few studies have addressed possible immune-mediated headache in this disorder.

Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action.Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.

Abstract P320: Cooperative Signaling and Membrane Recruitment of the Dopamine-1 (D 1 R) and Angiotensin Type 2 (AT 2 R) Receptors in Human Renal Proximal Tubule Cells (RPTC)

The D 1 R and AT 2 R are interdependent natriuretic receptors that are critical for the regulation of renal sodium balance and are implicated in both hypertension and salt-sensitivity. D 1 R and AT 2 R stimulation with the D 1 R agonist fenoldopam (FEN) and Angiotensin III (AngIII), selectively increases cAMP and cGMP, respectively, leading to D 1 R and AT 2 R recruitment to the cell surface (measured by fluorescent extracellular receptor epitope specific antibodies). The interdependence of the D 1 R and AT 2 R recruitment was investigated following AT 2 R stimulation with AngIII (10 nmol/L 1 hour) which leads to D 1 R cell surface recruitment using a normally D 1 R/Gs coupled cell line (i22) (VEH 8209±863, AngIII 19485±3425 RFU, n=6, p&lt;0.05) but not in a D 1 R uncoupled from Gs cell line (i19). These data were verified by increasing intracellular sodium with monensin (100 μM, 1 hr) and measuring cell surface binding with a fluorescent labelled D 1 R agonist SKF83566 (10 nmol/L, 1 hr, i22 1.67±0.7 fold, n=5, p&lt;0.0001, i19, NS). The importance of dopamine in regulating these receptor actions was shown by stimulating the AT 2 R with AngIII and measuring D 1 R recruitment followed by blockade of amino acid decarboxylase using either carbidopa or benzeraside (100 μM each, 1 hour). Additionally, FEN stimulated D 1 R surface recruitment is blocked by the AT 2 R inhibitor PD123319 (1 μM, 1 hour) and the coupling defect is fully rescued by 8Br-cGMP (1 mmol/L, 1 hour), a cell permeable second messenger normally thought to signal specifically through the AT 2 R. We then show that FEN stimulation leads to increased production of AngIII only in the D 1 R/Gs coupled i22 cell line (5.82±0.43 fold, n=6, p&lt;0.05) but not in i19 the D 1 R/Gs uncoupled cell line and this production was blocked with an aminopeptidase A inhibitor, EC-33 (10 μmol/L, 1 hour). Addition of 8Br-cAMP (1mmol/L 1 hour) not only leads to an increase in AngIII production, but also cGMP production (4.33±0.36 fold, n=6, p&lt;0.05) that is blocked by PD123319 (potent, selective, non-peptide angiotensin AT 2 R antagonist, 1 μM, 1 hour). In summary we not only show that dopaminergic stimulation leads to increased AngIII production in a D 1 R coupled manner, but also that AngIII stimulation of AT 2 R leads to dopaminergic activation in a D 1 R coupled manner.

Psychosis: an autoimmune disease?

Psychotic disorders are common and disabling. Overlaps in clinical course in addition to epidemiological and genetic associations raise the possibility that autoimmune mechanisms may underlie some psychoses, potentially offering novel therapeutic approaches. Several immune loci including the major histocompatibility complex and B-cell markers CD19 and CD20 achieve genome-wide significance in schizophrenia. Emerging evidence suggests a potential role via neurodevelopment in addition to classical immune pathways. Additionally, lymphocyte biology is increasingly investigated. Some reports note raised peripheral CD19+ and reduced CD3+ lymphocyte counts, with altered CD4 : CD8 ratios in acute psychosis. Also, post-mortem studies have found CD3+ and CD20+ lymphocyte infiltration in brain regions that are of functional relevance to psychosis. More specifically, the recent paradigm of neuronal surface antibody-mediated (NSAb) central nervous system disease provides an antigen-specific model linking adaptive autoimmunity to psychopathology. NSAbs bind extracellular epitopes of signalling molecules that are classically implicated in psychosis such as NMDA and GABA receptors. This interaction may cause circuit dysfunction leading to psychosis among other neurological features in patients with autoimmune encephalitis. The detection of these cases is crucial as autoimmune encephalitis is ameliorated by commonly available immunotherapies. Meanwhile, the prevalence and relevance of these antibodies in people with isolated psychotic disorders is an area of emerging scientific and clinical interest. Collaborative efforts to achieve larger sample sizes, comparison of assay platforms, and placebo-controlled randomized clinical trials are now needed to establish an autoimmune contribution to psychosis.