9^th European Immunology Conference associated with Antibody Engineering Meeting June 14-16 2018 Rome, Italy

Erik Wambre received his PhD in Immunology from the University Paris-6 and his Master’s degree in Business Engineering (MBE) from the Ecole de Biologie Industrielle (Cergy). He joined the Benaroya Research Institute in 2009 and completed his Postdoctoral fellowship, subsequently became an Associate Member. The projects in his laboratory are focused on understanding cellular and molecular mechanisms contributing to pathogenesis, regulation and functions of the adaptive immune system in disease pathways. The overarching aim of his lab is to generate the knowledge necessary to inform better vaccine design and to develop clinically meaningful biomarkers.

Allergic diseases are complex immunological disorders with multiple cellular and molecular alterations in pathways involving both activation and effector function. To rationally evaluate the mechanistic impact of candidate therapies in these diseases, it is essential to illuminate stages of pathogenesis with the help of informative biomarkers. Specific opportunities to develop correlates of immune-mediated disease outcome include inappropriate expansion of proinflammatory cells and alterations in gene expression pathways reflecting defective homeostasis. There is now extensive support for the concept that allergen-specific TH2 lymphocytes initiate and drive allergic sensitization. However, a major impediment to the use of allergic disease-causing T cells as both therapeutic targets and clinically useful biomarkers is the lack of an accepted methodology to identify and differentiate these cells from overall nonpathogenic TH2 cell types. We have recently described a proinflammatory human TH2 cell subpopulation that includes all allergen-specific TH2 cells. These cells are terminally differentiated CD4+ T cells characterized by coexpression of CR TH2, CD49d, and CD161 and exhibit numerous functional attributes distinct from conventional TH2 cells. In addition, we demonstrated that these cells are confined to allergic individuals and their disappearance is indicative of clinical responses induced by allergen-specific immunotherapy. Hence, we have denoted these cells with this stable allergic disease-related phenotype as the TH2A cell subset. Further detailed studies focusing on the TH2A cell subset may prove useful in the diagnosis, molecular characterization, or the discovery of novel therapeutic targets to enhance the power of allergen vaccines.

Loredana Frasca obtained her Biological Science degree in 1992 and her PhD in Cellular Immunology in 1996. She has expertise in cellular immunology with the particular focus on T-cells, antigen presentation, and T-cell tolerance. She has also expertise in innate immunity, in particular in regulation of dendritic cells functions. In the last ten years, she worked on activation and homeostasis of T-cells and dendritic cells in normal responses and autoimmunity. Most recent research is focused towards the identification of pathogenic pathways that activate both innate and adaptive immunity in autoimmune diseases such as psoriasis, lupus, systemic sclerosis and arthritis.

Background: Infl amed lupus skin/kidney over-express LL37, an antimicrobial peptide (AMP) with immuno-modulatory functions. By binding to nucleic acids, LL37 triggers plasmacytoid dendritic cells (pDCs) activation and Type I interferon (IFN-I), a master factor in lupus, via TLR7/9. In silico/experimental data indicate that LL37 sequence harbors T-cell promiscuous epitopes behaving as T-cell autoantigens in psoriasis, an autoimmune disease characterized by high skin LL37 levels. In Systemic lupus erythematosus (SLE), LL37 is released during NETosis and becomes target of anti-LL37 antibodies that correlate with serum IFN-I and lupus disease activity (SLEDAI).

 

Objectives: We addressed LL37 capacity to act as T-cells autoantigen in SLE patients.

 

Methods: We assessed circulating SLE T-cell proliferation and cytokine production to LL37 and control AMPs by BrdUincorporation- assay and ELISA/intracellular staining, respectively. We characterized SLE LL37-specifi c T-cell lines/clones and detected LL37-specifi c T-cells in blood by peptide-HLA-ClassII-tetramers; we addressed antibody reactivity to LL37 and DNA

in SLE patients’ sera and in in vitro-PBMC-cultures stimulated with LL37 or control AMPs by ELISA. We addressed presence of LL37 in lupus tissues by confocal microscopy.

 

Results: T-helper-17(Th 17)/T-follicular-helper(Tfh )-like LL37-specifi c T-cells were present in 45% of SLE patients, correlated with SLEDAI and declined during stable remissions. LL37-specifi c T-cells correlated with (and help production of) NETinducing anti-LL37 and anti-DNA antibodies.

 

Conclusions: LL37 is target of T-cells and autoantibodies with pathogenic functions in SLE. Th e data suggest that T-cell and anti-LL37-antibody reactivity can represent novel SLE disease markers and highlights the role of NETosis in inducing autoreactive T-cells in SLE and, possibly, in other autoimmune diseases.

Ena Ray Banerjee has completed her PhD from IICB under Jadavpur University with a CSIR fellowship. She is the first lady DSc in a 100 years in Department of Zoology, University of Calcutta where her lab works on Immunobiology and Regenerative Medicine under Translational Outcomes Research. She has published more than 50 papers in reputed journals and serving as an Editorial Board Member of repute. She is a Founder-Director of consort Consulting and Research LLP, and founder member of Consortium for Life working on poverty alleviation through conservation and sustainable bioprospecting.

Background: Idiopathic pulmonary fibrosis is characterized by excessive matrix deposition that disrupts the normal architecture of the lung parenchyma and causes airway remodeling. It is a progressive and fatal lung disorder with high mortality rate. The current treatments provide only minimal benefits and have significant side effects, highlighting the need for novel treatment approaches to pulmonary fibrosis.

 

Objective: The aim of this study was to investigate the therapeutic potential of umbilical cord-derived MSC (uMSC) bleomycin induced fibrosis.

 

Methods: A mouse model of bleomycin induced pulmonary fi brosis was used in the study. Wharton jelly derived mesenchymal stem cells were injected intravenously and inflammation, fibrosis and regeneration was examined using a series of assays such as an assessment of total cell count, inflammation, hydroxyproline, cell proliferation and clonogenic potential and histology.

 

Results: We found the increase in the total cell count (p<0.001) and collagen content and a decrease in clonogenic potential (p<0.01) in the lung after bleomycin treatment as compared to the control group. Interestingly, intravenous administration of umbilical cord derived MSC showed reversal of these eff ects by decrease in collagen content, a decrease in total inflammatory cell count (p<0.01) and increase in clonogenic potential (p<0.05) in the lung. Upon umbilical cord derived MSC administration, reactive oxygen species and reactive nitrogen species generation in the lung decreased as compared to only bleomycin treated group. Histological study of bleomycin treated lung revealed extracellular matrix decomposition, abnormal collagen degradation and

distorted lung morphology compared to control groups; stem cell treatment assisted in restoration of lung morphology.

 

Conclusion: The present research suggests that administration of umbilical cord derived mesenchymal stem cells led to reduction in inflammation and collagen content, increased proliferative ability of the cells and restored lung morphology. Thus these cells may be used for future reference to formulate effective therapeutic protocols in managing bleomycin induced IPF.

Dupilumab, a monoclonal antibody against the IL-4-receptor α subunit has been developed and used in clinical trials to treat atopic dermatitis (AD). We performed a meta-analysis to assess the overall effi cacy and safety of dupilumab treatment in moderate to severe AD. PubMed, Embase, Cochrane library databases and the Chinese biological medicine published up to September 2017 were searched. All randomized controlled trials of dupilumab treatment on adult patients with AD were included. Fixed- or random-effects models were used to calculate pooled standard mean differences or relative risks (SMD or RR, respectively). Six trials involving 2447 patients were identified. Pooled analysis revealed significant improvements in eczema area and severity index (EASI) scores (SMD= -0.89, 95% CI: -1.0 to -0.78), percentage of body surface area (BSA) (SMD= -0.83, 95% CI: -0.90 to -0.75), pruritus numeric rating scale (NRS) scores (SMD= -0.81, 95% CI: -0.96 to -0.66), and dermatology life quality index (DLQI) scores (SMD= -0.78, 95% CI: -0.89 to -0.66). Dupilumab treatment was also associated with significantly increased the proportion of patients achieving investigator’s global assessment (IGA) response (RR=3.82; 95 % CI: 3.23 to 4.51) and a similar incidence of adverse events (RR=1.0; 95 % CI: 0.96 to 1.04). Our analysis provided evidence that dupilumab had an acceptable safety profile and resulted in clinically relevant improvements in signs and symptoms of AD. 300 mg weekly (qw) and every 2 weeks (q2w) of dupilumab seemed to have the similar benefi t for patients.

Tjie Kok is doing his PhD at University of Groningen, The Netherlands (2014-2018). He is previously the vice dean of Faculty of Biotechnology, University of Surabaya, Indonesia (2011-2014). In 2005-2010, he was a member of American Chemical Society (ACS). He has published more than 15 papers in national journals in Indonesia and 2 papers in reputed journals (Protein Expression and Purification journal, and Bioorganic and Medicinal Chemistry journal); in addition, one paper is now under review in Drug Discovery Today journal. At present, he is serving as a reviewer of African Journal of Pharmacy and Pharmacology.

The IF receptor, CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from endoplasmic reticulum to Golgi complex. It is also part of a receptor complex binding to macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. Inhibition of MIFCD74 complex formation is regarded as a potentially attractive therapeutic target in inflammation and cancer. In order to be able to produce large quantities of the extracellular moiety of human CD74, which has been reported to be unstable and protease-sensitive, different constructs were made as fusions with two solubility enhancers: the well-known maltose-binding domain and Fh8, a small protein secreted by parasite Fasciola hepatica. The fusion proteins could be purified with high yields from Escherichia coli and were demonstrated to be active in binding to MIF. In order to identify MIF binding compound that potentially interferes with MIF-CD74 interaction, we performed a substitution-oriented screening on a library of chromene compounds (inspired by Orita-13). MIF tautomerase inhibitors with IC50’s in low micromolar range were identifi ed. These novel inhibitors of the MIF tautomerase activity may ultimately support the development of novel therapeutic agents against diseases in which MIF is involved.

Wen Qiu is currently an Associate Professor at Department of Immunology of Nanjing Medical University. He is exploring the roles and mechanisms of complement especially C5b-9 in the induction of glomerular mesangial cell (GMC) apoptosis, inflammation and proliferation in rat Thy-1 nephritis as a widely used model of human mesangial proliferative glomerulonephritis (MsPGN) as well as the roles and mechanisms of IL-17 in astrocyte pathological changes in EAE mice. These include signal transduction, microRNA regulation, and transcriptional factor regulation. He is also exploring the effects of post-transcriptional regulation such as ubiquitination and acetylation on the activation of signaling molecules, transcription factors and histones.

The apoptosis of glomerular mesangial cells (GMCs) in the early phase of rat Th y-1 nephritis (Th y-1N), a model of human mesangio proliferative glomerulonephritis (MsPGN), is primarily triggered by sublytic C5b-9. However, the mechanism of GMC apoptosis induced by sublytic C5b-9 remains unclear. In this study, we demonstrate that expressions of TNFR1-associated death domain-containing protein (TRADD) and IFN regulatory factor-1 (IRF-1) were simultaneously upregulated in the renal tissue of Th y-1N rats (in vivo) and in GMCs under sublytic C5b-9 stimulation (in vitro). In vitro, TRADD was confirmed to be a downstream gene of IRF-1, because IRF-1 could bind to TRADD gene promoter to promote its transcription, leading to caspase 8 activation and GMC apoptosis. Increased phosphorylation of p38 MAPK was verified to contribute to IRF-1 and TRADD production and caspase 8 activation, as well as to GMC apoptosis induced by sublytic C5b-9. Furthermore, phosphorylation of MEK kinase 2 (MEKK2) mediated p38 MAPK activation. More importantly, three sites (Ser153/164/239) of MEKK2 phosphorylation were identified and demonstrated to be necessary for p38 MAPK activation. In addition, silencing of renal MEKK2, IRF-1, and TRADD genes or inhibition of p38 MAPK activation in vivo had obvious inhibitory effects on GMC apoptosis, secondary proliferation, and urinary protein secretion in rats with Th y-1N. Collectively, these findings indicate that the cascade axis of MEKK2-p38 MAPK-IRF-1-TRADD-caspase 8 may play an important role in GMC apoptosis following exposure to sublytic C5b-9 in rat Th y-1N.

Yong-Sung Kim has been a Professor in Department of Molecular Science and Technology at Ajou University, Korea since 2004. He received his BSc in Food Science and Technology from Seoul National University in 1996. He obtained his MSc in Biotechnology in 1998 from KAIST and PhD in Pharmaceutical Sciences in 2002 from the University of Colorado, Denver. After obtaining the PhD degree, he joined the lab of Prof. K. Dane Wittrup at MIT as a Post-doc for protein and antibody engineering using yeast surface display. He spent one year (2010-2011) during his Sabbatical at Genentech Inc. (SF, USA), where he worked with scientists at Department of Molecular Oncology and Antibody Enginnering. His research focuses on development of next-generation antibody platform technology for potent anti-cancer therapeutics, including heterodimeric Fc-based bispecific antibody, solid tumor-penetrating antibody, and cytosol-penetrating antibody to address cytosolic proteins. In August of 2016, he co-founded ORUM Therapeutics Company with Dr. Sung-Joo Lee for the commercialization of cytosol-penetrating antibody technology.

Ras proteins (KRas, HRas, and NRas) are small GTPases that function as molecular switches at the inner plasma membrane by alternating between GTP-bound active forms (Ras-GTP) and GDP-bound (Ras-GDP) inactive forms. Oncogenic mutations in Ras proteins, predominantly found at G12, G13, and Q61 residues, impair the GTPase activity rendering the mutants persistently GTP-bound active form, thereby promoting tumorigenesis and tumor malignancy. Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. In this talk, I will present the development of a human IgG1 format antibody, named iMab (internalizing & protein-protein interaction (PPI) interfering monoclonal antibody), which directly targets the intracellularly activated GTP-bound form of various oncogenic Ras mutants aft er internalization into the cytosol of living cells. iMab specifi cally binds to the PPI interfaces between activated Ras and eff ector proteins such as Raf and PI3K to block the associations, thereby suppressing downstream signaling and exerting anti-proliferative eff ects in a variety of tumor cells harboring oncogenic Ras mutants. When systemically administered, an iMab variant with an additional tumor-associated integrin binding moiety for tumor tissue targeting signifi cantly inhibited the in vivo growth of oncogenic Ras-mutated tumor xenograft s in mice, but not wild-type Ras-harboring tumors. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology.

Hongtao Zhang is a Research Associate Professor in the Department of Pathology and Laboratory Medicine at the University of Pennsylvania, Perelman School of Medicine. Currently his research focus is in the receptor- targeted therapies using antibodies, antibody-like proteins, and small molecules. The research projects in his lab are relevant to understand the functions of cytokines in the regulation of immune cells in tumor microenvironments, including the polarization of tumor associated  acrophagesm. He is also devoted to the identification of serum biomarkers that can help the diagnosis and therapeutic guidance for breast cancer and melanoma. He has published more than 70 articles in journals including Nature Medicine and Nature Biotechnology. He is listed as inventors in 17 approved patents and some other pending patents. He serves as an Editorial Board Member for several journals.

Despite substantial clinical progress with targeted therapies, current antibody-based approaches have limited effi cacy at controlling HER2/neu-positive breast cancers, especially in the absence of chemotherapies. Previously we showed that the combination of IFN-γ and anti-HER2/neu antibody synergistically reduces tumor growth in an in vivo implanted mammary tumor model. Here, we report a recombinant approach to produce an HER2 AbZED-IFN-γ fusion protein, which contains anti-HER2 scFv, an engineered eff ector domain (EED) scaff old, and IFN-γ, as a novel way to treat HER2+ tumors. HER2 AbZED-IFN-γ induces in vitro apoptosis in an IFN-γ receptor dependent manner. In the in vivo xenograft ed tumor model, HER2 AbZED-IFN-γ at a very low dose demonstrates superior activity over the anti-HER2/neu antibody on the growth of HER2+ tumors. In the CT26-HER2 tumor model, which is resistant to anti-HER2 antibody trastuzumab, HER2 AbZEDIFN- γ remains to show activity to inhibit tumor growth. Examination of tumor infi ltrated macrophages and lymphocytes reveals that the fusion protein can induce changes in tumor microenvironment to support immune reactivity against tumors. Furthermore, we have humanized the EED domain to minimize immunogenicity of the therapeutic protein. Our studies have defined a targeted immunotherapy approach for the treatment of cancers.

 

Isaac Melamed has focused on clinical practice and research for over 25 years. He has published more than 100 papers and presented and lectured in national and

international settings. His major concentration is in primary and secondary immune deficiency, immune-related disease, crosstalk between the immune system and the central nervous system. His mission is early intervention to ensure his patients' optimal health.

Statement of the Problem: Pediatric acute-onset neuropsychiatric syndrome (PANS) is a broad diagnostic criterion characterized by a severe, sudden onset of neuropsychiatric changes. Recently, we acquired evidence that other infectious pathogens (eg, Mycoplasma pneumoniae, Epstein-Barr virus; and Borrelia burgdorferi) may play a role in a similar neuroinflammatory syndrome. Innate immunity is the first line of defense against pathogens and is comprised of effectors that provide rapid, robust, non-specifi c responses. Two aspects of innate immunity are toll-like receptors (TLRs) and the complement

system. C1 esterase inhibitor (C1-INH) is the major inhibitor of the classical pathway. Recently, we explored the role of C1INH in a subset of patients with common variable immunodeficiencies (CVID) as well as the role of mast cell (MC) activation, which may lead to microglial activation and may function as a partner to TLR and C1INH signaling in developing neuroinflammation. In this study, we explored whether TLR-3 signaling, C1INH function, and MC activation play a role in the pathogenesis of post-infectious neurological diseases.

 

Methodology & Theoretical Orientation: We reviewed clinical cases of patients who presented with neurocognitive changes and had evidence of neuro-inflammation based on autoimmune, neurological biomarkers such as anti-68kDa, anti-GAD antibody autoimmune panels. Patients with neurocognitive clinical presentations and positive biomarkers had an immune

workup that included TLR signaling, C1INH levels, and atopic markers including MC signals.

 

Findings: Th e response to poly (I:C), the synthetic analogue dsRNA (TLR 3), was decreased by 86±8% compared to normal control. Average levels of C1INH were 16+2 mg/dL, while control levels were 22+5. All patients had evidence of atopy based on IgE, RAST tests, and MC activation. 

 

Conclusion & Significance: We suggest that a complex of immune dysfunction, including TLR-3 signaling, C1INH levels, and atopic partners, specifically mast cell activation, are playing a crucial role in a neuro-inflammatory clinical presentation similar to PANS syndrome.

Ulrich Rothbauer is working on new technologies to study proteins and protein interactions in living cells. His main focus lies on the development of disease relevant assays and target specifi c nanoprobes for high content analysis based on the chromobody-technology. He has studied Biology at the Ludwig-Maximilian University (LMU). He received his PhD in Biochemistry in the group of Prof. Walter Neupert revealing the pathomechanism of a mitochondrial disease in 2003. After his Postdoctoral work on the regulation of the epigenetic key factors he became an independent group leader at the LMU-Biocenter in 2006. As a Junior Group Leader he directed R&D projects in a range of cellular diagnostic areas including cell cycle studies, apoptosis studies, proteomics and protein-protein interactions. In 2008, he founded the biotech spinoff company ChromoTek, which develops and commercializes new technologies to perform cellular diagnostics and proteomics. In 2011, he became Full Professor for Pharmaceutical Biotechnology at the University Tübingen, Germany.

There is a continual need in biomedical research for reliable binding molecules that recognize cellular targets with high affinity and specificity. Single-domain antibodies - referred to as nanobodies - have emerged as an attractive alternative to traditional antibodies and became highly valuable tools for numerous bio-analytical and biotechnical applications. Recently we have identified novel nanobodies for protein purification, protein-protein interaction analysis, crystallization studies and mass spectrometry approaches. For in cellulo studies we developed a novel format of intracellular functional nanobodies (chromobodies) to target and trace endogenous components in living cells. In combination with high-throughput microscopy and automated image analysis we applied chromobodies as intracellular biosensors for phenotypic screening and high content imaging (HCI) in real time. To date chromobody-based cellular models have been established to monitor cell cycle, signal transduction or to trace epithelial mesenchymal transition upon compound treatment within living cells. Due to their extraordinary properties nano- and chromobodies are versatile binding molecules offering a unique opportunity to combine biochemical, microscopic and functional analyses of cellular targets in flexible settings.

Loredana Frasca obtained her Biological Science degree in 1992 and her PhD in Cellular Immunology in 1996. She has expertise in cellular immunology with the particular focus on T-cells, antigen presentation and T-cell tolerance. She has also expertise in innate immunity, in particular in regulation of dendritic cells functions. In the last ten years she worked on activation and homeostasis of T-cells and dendritic cells in normal responses and autoimmunity. Most recent research is focused towards the identification of pathogenic pathways that activate both innate and adaptive immunity in autoimmune diseases such as psoriasis, lupus, systemic sclerosis and arthritis.

Psoriasis is a chronic infl ammatory and immune-mediated skin disease of unclear etiology, aff ecting 2-3% of individuals in Europe. A third of patients develop psoriatic arthritis (PsA), an infl ammatory arthritis associated with psoriasis. Th e exact mechanisms that lead to development of PsA in psoriasis patients are elusive. We previously uncovered that the antimicrobial peptide LL37, that is over-produced by keratinocytes in psoriasis skin, plays a crucial role in sustaining infl ammation in the skin by binding to nucleic acids and stimulating plasmacytoid and myeloid dendritic cells (pDC, mDCs) to release IFN-a and other pro-infl ammatory factors production. In psoriasis, LL37 also becomes the target of autoimmune T cells with a Th 1/ Th 17 phenotype. On the other hand, anti-LL37 antibodies are frequently detected in systemic lupus erythematosus, a systemic autoimmune disease characterized by increased neutrophil NETosis in target tissues. Furthermore, it is known that LL37 is the substrate for irreversible post-translational modifications (PTM), such as citrullination or carbamylation. In these work we showed the presence of native and modified LL37 and related anti-LL37 antibodies both in circulation and synovial fluids of psoriasis and PsA subjects as compared to healthy donors (HD) and osteoarthritis (OA) patients by ELISA. Moreover, we found the presence of inflammatory factors that can either recruit neutrophils and/or induce neutrophil activation/degranulation or NETosis, or citrullination of proteins (GM-CSF, C5a, IFN-a). In synovial biopsies from very early and untreated PsA patients, expression of the IFN-related gene MX1 is highly up-regulated in close proximity of myeloperoxidase and LL37 staining. Synovial anti-LL37 autoantibodies recognizing the citrullinated form of LL37 correlate with IFN-α expression, and both anticitrullinated and anti-carbamylated LL37 do correlate with GM-CSF. These results suggest that serum IFN-a, GM-CSF and anti-LL37 antibodies, especially those directed to carbamylated LL37 are potential disease markers in PsA.