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

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.