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

Dr. Jinhong Liu has completed his MD and PhD at the age of 30 years from University of South Florida and postdoctoral studies from H. Lee Moffitt Cancer Center. She is very active physician scientist in the clinical and translational research in the precision medicine.

We recently reported that, the accumulation of myeloid-derived suppressor cells (MDSCs), defi ned as CD33+HLA-DR− Lin−, play a direct role in the pathogenesis of myelodysplastic syndrome (MDS). In particular, CD33 is greatly expressed in MDSC isolated from patients with MDS and plays an important role in MDSC-mediated hematopoietic suppressive function through its activation by its ligand S100A9. Th erefore, we tested the hypothesis that blocking this interaction with a fully human, Fc-engineered monoclonal antibody against CD33 (BI 836858) suppresses CD33-mediated signal transduction and improves the bone marrow microenvironment in MDS. We found that BI 836858 can reduce MDSC by antibody-dependent cytotoxicity (ADCC), which correlated with an increase in granule mobilization and increased cytotoxicity. BI 836858 can also block CD33’s downstream signaling and prevent immune-suppressive cytokine secretion, which correlates with a signifi cant increase in the formation of CFU-GM and BFU-E colonies. Activation of the CD33 pathway can cause reactive oxygen species (ROS)-induced genomic instability but BI 836858 reduced both ROS and the levels of double strand breaks and adducts (measured by comet assay, γH2AX and 8-oxoguanidine). This work provides the ground for the development of a novel group of therapies aimed at MDSC and their signaling with the long term goal of improving hematopoiesis

Jung-Eun Kim has expertise in cancer immunotherapies. Aims to elucidate how antibodies and cytokines modulate immune cells that infi ltrate tumor through quantitative and qualitative assessment of immune cells in syngeneic mouse tumor models. Research is focused on cellular immune mechanisms of T lymphocytes activation and regulatory T cells suppression. Team also investigates rationales for combination of therapeutic antibodies targeting tumor cells and/or immunecheckpoint receptors. Their research will lead to the next generation of immune-based therapies in human cancer.

Statement of the Problem: Interleukin-12 (IL12) (p35/p40 complex) is a heterodimeric cytokine with potent anti-tumor activity. However, its short serum half-life and high dose-related toxicities limit its clinical efficacy. Methodology & Theoretical Orientation: In this study, we exploited heterodimeric Fc technology to develop mono-mIL12- Fc, which presents mIL12 in the naturally occurring heterodimeric form with an Fc-mediated extended serum half-life. We constructed heterodimeric Ig Fc-fused IL12 in the naturally occurring heterodimeric form of IL12, termed mono-mIL12-Fc, in which the p35 and p40 subunits were fused to the N-terminus of two diff erent Fc variants, respectively. We also generated Fc-fused bivalent IL12 with two IL12 units (bi-mIL12-Fc) by fusion of scIL12 (p40-linker-p35) to the N-terminus of wild-type Fc as a control. Findings: Mono-mIL12-Fc exhibited a much longer plasma half-life than recombinant mIL12, enabling twice-weekly systemic injections to remove established tumors in syngeneic mouse models. Mono-mIL12-Fc was more potent than wild-type Fc-based bivalent-binding IL12-Fc (bi-mIL12-Fc) for eradicating large established immunogenic tumors without noticeable toxicities by enhancing interferon-gamma production and the proliferation of immune eff ector cells in tumors. More importantly, monomIL12- Fc triggered weaker IL12 signaling than bi-mIL12-Fc, favoring the generation of functional and protective memory CD8+ T cells. Notably, our study illustrates that mono-mIL12-Fc triggers modest pSTAT4 activation and T-bet expression in eff ector CD8+ T cells, resulting in a switch from T-bet to Eomes activation for the diff erentiation into MPECs and eventually long-lived memory CD8+ T cells. However, the strong IL12 signaling mediated by bi-mIL12-Fc drives eff ector CD8+ T cells into terminally diff erentiated SLECs, thereby excluding the development of MPECs. Conclusion & Signifi cance: Our results demonstrate that heterodimeric-Fc-fused IL12 is a suitable format for augmenting adaptive CD8+ T cell immune responses, providing a practical alternative to the systemic administration of IL12 for anti-tumor therapy.

Ji-Sun Kim has developed a passion for antibody engineering to generate the extremely effective cancer drug. She is a second year PhD student in Antibody Engineering lab at the Ajou University in South Korea. She focuses on research for generating cytosol penetrating antibody targeting intracellular antigen and evaluating the developability of the cytosol penetrating antibody therapeutics. Also, she evaluated in vivo effi cacy of antibody to overcome the limitation of current therapeutics and therapy approach. She is also interested in the development of new-generation cytotoxic fusion protein using cytosol penetrating antibody platform technology. Her research achieves results including papers and intelligence properties in Korea and abroad.

Considering the number of cytosolic proteins associated with many diseases, development of cellular internalization molecules from outside of living cells is highly in demand. To reach the cytosol aft er cellular uptake, cell-penetrating molecules should be escaped from early endosomes prior to the lysosomal degradation. However, it is very challengeable to separate the pool of cytosolic localized molecules from those trapped in the endocytic vesicles. In this study, we described a method to directly demonstrate the cytosolic localization and quantifi cation the cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP11 fragment and separately established HeLa cells expressing the other GFP1-10 fragment in the cytosol such thatcomplemented GFP fl uorescence is observed only when the extracellularly-treated TMab4 reaches the cytosol aft er cellular internalization. Th e high affi nity interactions between streptavidin-binding peptide 2 (SBP2) and streptavidin (SA) was employed as respective fusion partner to the GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration and endosomal escape effi ciency of TMab4 was estimated to be about 170 nM and 1.3-4.3% aft er extracellular treatment of HeLa cells with 1μM of TMab4 for 6h. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amounts of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

Se-Yeon Park is studying for a Master’s course at Ajou University.

Necroptosis is a type of programmed cell death that usually occurs under apoptotic-defi cient conditions. Receptorinteracting protein kinase-3 (RIP3, or RIPK3) is a central player in necroptosis and its kinase activity is essential for downstream necroptotic signaling events. Since RIP3 kinase activity has been associated with various diseases, development of specifi c RIP3 inhibitors is an attractive strategy for clinical application. In this study, we identifi ed a potent RIP3 inhibitor, HS-1371 using an extensive kinase cross-screening of our chemical libraries. Th e mechanism of action of this compound is via direct binding to RIP3 in an ATP-competitive and time-independent manner. HS-1371 inhibited TNF-induced necroptosis but did not inhibit TNF-induced apoptosis, indicating that this novel inhibitor has a specifi c inhibitory eff ect on RIP3-mediated necroptosis via suppression of RIP3 kinase activity. Our results suggest that HS-1371 could serve as a potential preventive or therapeutic agent for RIP3 hyperactivation-mediated diseases.

Jee Min Chung has completed her B.S at the age of 22 years from Wonkwang University and a master's and doctorate integrated course studies from Ajou University School of Medicine.

Cetuximab, a chimeric monoclonal antibody developed for targeting the epidermal growth factor receptor (EGFR), has been intensively used to treat cancer patients with metastatic colorectal cancer and head and neck cancer. Intact immunoglobulin G (IgG) antibody like cetuximab, however, has some limitations such as high production cost and low penetration rate from vasculature into solid tumor mass due to its large size. In attempt to overcome these limitations, we engineered cetuximab to create single chain variable fragments (scFv-CH3; Minibody) that were expressed in bacterial system. Among three engineered minibodies, we found that MI061 minibody, which is composed of the variable heavy (VH) and light (VL) region joined by an 18-residue peptide linker, displays higher solubility and better extraction properties from bacterial lysate. In addition, we validated that purifi ed MI061 signifi cantly interferes ligand binding to EGFR and blocks EGFRs hosphorylation. By using a protein microarray composed of 16,368 unique human proteins covering around 2,400 plasma membrane associated proteins such as receptors and channels, we also demonstrated that MI061 only recognizes the EGFR but not other proteins as compared with cetuximab. Th ese results indicated that engineered MI061 retains both binding specifi city and affi nity of cetuximab for EGFR. Although it had relatively short half-life in serum, it was shown to be highly signifi cant anti-tumor eff ect by inhibiting ERK pathway in A431 xenograft model. Taken together, our present study provides compelling evidence that engineered minibody is more eff ective and promising agent for in vivo targeting of solid tumors.

Yingwei Wang is currently a Professor at Department of Immunology of Nanjing Medical University. She is conducting research in exploring the mechanisms of sublytic C5b-9-induced glomerular mesangial cell (GMC) apoptosis, infl ammation and proliferation in mesangial proliferative glomerulonephritis (MsPGN) by using rat Thy-1 nephritis model. These include signal transduction, microRNA regulation, and transcriptional factor regulation. She is also exploring the effects of ubiquitination, acetylation and phosphorylation modifi cation on the activation of signaling molecules, transcription factors and histones.

The apoptosis of glomerular mesangial cells (GMCs) is considered to be an important contributor to the initiation and development of rat Th y-1 nephritis (Th y-1N) and is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 triggers GMC apoptosis has not been elucidated. In this study, functional and histological examinations were performed on GMCs treated with sublytic C5b-9 (in vitro) and renal tissues of Th y-1N rats (in vivo). Th e in vitro studies found that sublytic C5b-9 could trigger GMC apoptosis through upregulating Egr-1, ATF3, and Gadd45 expression. Egr-1-mediated post-transcriptional modulation of ATF3, Egr-1/ATF3-enhanced Gadd45 promoter activity, and p300-mediated ATF3 acetylation were all involved in GMC apoptosis. More importantly, the eff ective binding elements for Egr-1 and ATF3 to Gadd45β/γ promoters and the ATF3 acetylation site were identifi ed. In vivo, silencing renal p300, Egr-1, ATF3, and Gadd45β/γ signifi cantly decreased GMC apoptosis, secondary GMC proliferation, and urinary protein secretion in Th y-1N rats. Together, these fi ndings implicate that sublytic C5b-9-induced activation of Egr-1/p300-ATF3/Gadd45 axis plays a critical role in GMC apoptosis in Th y-1N rats.

Eun Hee Han has completed his PhD at the age of 29 years from Chosun University and postdoctoral studies from Hormel Institute of Minnesota University in USA. I am senior researcher of Korea Basic Science Institute in South Korea. I have published more than 60 papers in reputed journals. Based on confocal microscopy and flow cytometry/sorter equipment, I am focusing on cancer biology and immunotherapy through cell imaging studies.

Lung cancer is among the most deadly cancers for both men and women. Immunotherapy is a breakthrough treatment in oncology that uses the body’s own natural defence system to fi ght off cancer. Higher numbers of T cells, natural killer cells, and/or dendritic cells are associated with better patient survival. In particular, NK cell therapy has the advantages of safety, economy, and versatility. Several studies have been undertaken to increase the target and effi cacy of NK cell therapy, such as antibody or CAR-T therapy. We observed that when NK cells co-cultured with lung cancer cell line A549, NK cells entered A549 to form a cell-in-cell (CIC) structure and immune cells die. Previous reports have suggested that CIC structures may be formed depending on the characteristics of immune cells, but we assumed that CIC-associated cancer cells may have different characteristics from those of other cancer cells. In order to prove this, CIC-engraft ed cancer cells and non-CIC cancer cells were sorted at the same time and proliferation, NK killing eff ect, foci, soft agar, migration, and invasion assays were performed. As a result, a more malignant phenotype was observed in CIC-engraft ed cancer cells. To confirm the reason, proteomics and transcriptomics analysis showed that the epithelial mesenchymal transition (EMT) property in CIC-engraft ed cancer cells. EMT marker analysis and phalloidin staining validated these results. Th e population of CIC-engraft ed cancer cells aft er sorting CIC-engraft ed cancer cells repeatedly co-cultured with A549 at the 2nd, 3rd and 4th cycles showed no signifi cant change. Therefore, the EMT properties of CIC-engraft ed cancer cells seem to be a temporary phenomenon that occurs dynamically in the cancer environment, not permanently maintained. In order to increase the efficacy of NK cell therapy, it is necessary to consider the study of targeting CIC-engraft ed cancer cells.

Currently, Hyun Jeong Kwak is particularly interested in signaling pathways mediated by reactive oxygen species (ROS) on various inflammation models. We utilize a wide variety of approaches ranging from basic molecular and cell biology methods to high throughput chemical genetic screening and animal inflammation models to elucidate these pathways.

Hematopoietic stem cell transplantation (HSCT) is used to treat hematological and immunological diseases. The success of HSCT is dependent on their ability of homing and engraftment to the bone marrow (BM) of recipients. Reactive oxygen species (ROS) is traditionally play a central role as second messengers in the signal transduction of many cell type including hematopoietic stem cells (HSCs), however high levels of ROS exhausted hematopoietic stem cells (HSCs). Therefore, in this study we evaluated whether maintaining a low levels of ROS in HSCs may augment the engraftment potential of HSCs. Transplantation of donor BM cells (CD45.1+) into recipients mice (CD45.2+) with a GSH biosynthesis inhibitor, buthionine sulfoximine (BSO) displayed a significant increase in HSC engraftment, and this was perturbed in the presence of ROS scavenger, N-acetyl-L-cysteine (NAC). In comparison with control mice, BSO-treated recipients displayed a 9.5 fold increase in hematopoietic engraftment. Specifically, engraftment efficiency was lower in 25 mg/kg BSO-treated mice than in 5 mg/kg BSO. Moreover, granulocyte/macrophage progenitors (GMPs) populations was increased in recipient mice, suggesting that enhancement of cell differentiation and proliferation of myeloid progenitor cells. Taken together, our results suggest a distinct role of ROS that regulates HSC functionality. Therefore, low ROS has the potential as a therapeutic approach in hematopoietic transplantation.