2015 Archived Content
As the pharmaceutical and biotech industries increasingly shift attention to biologics, much more attention is being paid to the prospect of membrane-bound proteins as drug targets for antibodies and other protein scaffolds. For the large GPCR and ion channel target classes, biologics offer improved selectivity, an alternative for targets with known function that have not been amenable to small molecule drugs and the potential for using antibodies for the targeted delivery of therapeutics.
The first meeting in the set, Antigen Optimization; Generation and Selection of Antibodies and Protein Scaffolds, offers a comprehensive examination of the range of methods available for producing appropriate membrane protein antigens, selecting the best screening methods for a specific target or target classes and how to develop and optimize the discovery and screening campaigns to identify functional antibodies. Part One also reviews other scaffold options, including single domain antibodies, as alternative therapeutic modalities for these targets.
Final Agenda
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Tuesday, September 22
7:00 am Registration and Morning Coffee
8:00 Chairperson’s Opening Remarks
Matt Holsti, Ph.D., Principal Scientist, Global BioTX Technologies, Pfizer
8:10 Keynote Presentation: Selection of Membrane Protein Targets: GPCRs as a Paradigm
Paul Insel, M.D., Ph.D., Vice-Chair and Distinguished Professor, Pharmacology; University of California, San Diego
GPCRs are the largest superfamily of membrane signaling receptors and the largest group of targets of approved drugs, but are the optimal GPCRs being targeted? Using a GPCRomic approach to define the GPCRs expressed by individual cell types from healthy and diseased subjects, we have discovered “novel” GPCRs, including potential therapeutic targets for diseases that are unmet medical needs.
8:40 Computational Design of Epitope Scaffolds to Induce Antibodies against Membrane Proximal Epitopes
William R. Schief, Ph.D., Professor, Immunology & Microbial Science, Scripps Research Institute; Director, Vaccine Design, International AIDS Vaccine Initiative
Our HIV vaccine design work involves immunogen design projects aiming to induce antibodies against membrane proximal structural epitopes – with challenges including engineering membrane proteins, optimizing epitope conformation and designing antigens to select for specific H-CDR3 loops. This talk presents lessons that may assist induction of antibodies against other membrane targets.
9:10 Cell-Free Expression of G-Protein Coupled Receptors: New Pipelines for Challenging Targets
Frank Bernhard, Ph.D., Group Leader, Institute of Biophysical Chemistry, Goethe University, Germany
The unique variability of cell-free expression reactions allows systematic optimization screens to improve the quality of synthesized GPCRs. Preparative scale amounts of ligand binding active receptor can thus be obtained and structural approaches come into focus. Strategies for GPCR quality optimization will be exemplified and characteristic biochemical properties of the samples are presented.
9:40 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing
10:25 Immunization Strategies for Antibody Generation against Multi-Spanning Proteins
Jane Seagal, Ph.D., Senior Scientist, AbbVie Bioresearch Center
Hybridoma generation against multi-spanning proteins is challenging. Expression levels of proteins on the cell surface may not be sufficiently high, and peptides corresponding to extracellular loops do not have natural conformation. We utilized cDNA immunizations to elicit immune response against multi-spanning proteins, leading to successful generation of hybridomas producing functional mAbs.
10:55 Identification of Biased Antagonists by Screening Single Domain Antibodies against GPCRs in Lipoparticles
Mick Foley, Ph.D., CSO, AdAlta, Australia
i-bodies are human single domains with a long CDR3 that enables access to proteins such as GPCRs. High affinity i-bodies specific for CXCR4 were obtained by screening on lipoparticles and each of the i-bodies have different functional profiles. These blocked SDF-1-induced leukocyte recruitment in a mouse model of inflammation and did not mobilize stem cells from bone marrow.
11:25 VelocImmune Mice for Generation of Antibodies against Complex Membrane Protein Targets
Nicole Alessandri-Haber, Ph.D., Senior Staff Scientist, Pain & Neurology, Regeneron Pharmaceuticals
The performance of the VelocImmune® platform is reflected in the pipeline of 14 fully-human antibodies (Abs) we have in clinical development and the 50 other Abs we have in preclinical development. VelocImmune® mice can generate antibodies with different binding characteristics and superior specificity against highly conserved and challenging targets. One example is the generation of specific functional mAbs against human ACCN2, an H+ gated ion channel implicated in pain transduction.
11:55 Discovery of MAbs against Difficult GPCRs, Ion Channels, and Transporters
Joseph Rucker, Ph.D., Vice President, Research and Development, Integral Molecular, Inc.
To enable the isolation, characterization, and engineering of MAbs against challenging membrane protein targets, Integral Molecular has developed the MPS Discovery Engine™ platform, encompassing lipoparticles for concentrating native membrane proteins and shotgun mutagenesis for membrane protein engineering and epitope mapping. Using MPS, we have generated functional MAbs against the ion channel P2X3 (neuropathic pain), GLUT transporter (diabetes), and CXCR4 (cancer), and have ongoing discovery programs against additional GPCR, ion channel, and transporter targets.
12:25 pm Towards Native and Stable GPCRs for Conformational Antibody Development. Case Study with Native and Functional Adenosine Receptor A2A
Anass Jawhari, Ph.D., CSO, CALIXAR
Adenosine receptor A2A plays an essential role in the central nervous system. Thanks to CALIXAR patented technology based on new chemistry approach to safely isolate membrane proteins, functional A2A was solubilized and purified without any single mutation, truncation or fusion for antibody development purposes and crystallization.
12:40 Session Break
12:45 Luncheon Presentation (Sponsorship Opportunity Available)
1:25 Refreshment Break in the Exhibit Hall with Poster Viewing
1:50 Chairperson’s Remarks
Barbara Swanson, Ph.D., Director, Research, Sorrento Therapeutics, Inc.
2:00 Selecting Antibodies Using a Combined in vitro and in vivo Approach
Andrew M. Bradbury, Ph.D., MB, BS, Staff Scientist, Biosciences, Los Alamos National Laboratory
The identification of antibodies suitable for in vivo use can be challenging. In this talk, we will present new data on the selection of antibodies against previously identified membrane protein targets using a combined in vitro/in vivo approach.
2:30 Protein Knockout Mice: A Novel in vivo Validation Approach for Membrane Targets
Stefan Dübel, Ph.D., Director, Institute of Biochemistry, Biotechnology and Bioinformatics, Technical University of Braunschweig, Germany
We present a novel drug discovery method which is applicable to all membrane and secretory proteins. We demonstrate that endoplasmatic reticulum retained antibodies (“intrabodies”) can induce a protein knockdown phenotype in transgenic mice. A single cloning step is needed from phage display selection of human antibodies to a target to induction of the mouse knockdown phenotype for this target.
3:00 Identifying Antibodies against Cell-Surface Receptors Using Live Cells within an Emulsion
Michael Weiner, Ph.D., CSO, AxioMx, Inc.
Our goal is to produce a fully recombinant IgG antibody within 3-4 weeks of receiving a suitable antigen. We will demonstrate the use of emulsion screening to identify affinity binders from a >1010 sized library against protein receptors displayed in situ on a living cell surface. Emulsion panning generates a greater number of unique clones by effectively eliminating the clonal expansion seen with traditional panning methods that use multiple rounds of enrichment.
3:30 Refreshment Break in the Exhibit Hall with Poster Viewing and Poster Winner Announced
4:10 Engineering Conformation Selective Antibodies to Membrane Protein Complexes
Marcin Paduch, Ph.D., Technical Director, Synthetic Antibody & Crystallography Core Facility, Department of Biochemistry and Molecular Biology, Recombinant Antibody Network, The University of Chicago
To address the challenges of membrane protein complexes and their conformational states, we have developed a suite of high-throughput technologies exploiting multiple antigen presentation formats, novel phage display libraries and improved biopanning techniques networking them together on a modern automation system. This integrated pipeline generated antibodies to nuclear pore complex and several Get3-tail anchored protein complexes.
4:40 Going Native: Direct High-Throughput Screening of Soluble, Secreted mAbs against Intact Target Cells
Karl Griswold, Ph.D., Associate Professor, Thayer School of Engineering, Dartmouth University
We describe an ultra-high throughput screening platform that enables identification and isolation of soluble, secreted, monoclonal antibodies that bind membrane proteins in their native environment: on the cell surface. Using gel microdroplet encapsulation combined with high-speed flow cytometry, we demonstrate recovery of rare clones producing IgG binders to targets such as EGFR.
5:10 Interactive Breakout Discussion Groups
This interactive session provides conference delegates and speakers an opportunity to choose a specific roundtable discussion group to join. Each group has a moderator to ensure focused discussions around key issues within the topic. This format allows participants to meet potential collaborators, share examples from their work, vet ideas with peers, and be part of a group problem-solving endeavor. The discussions provide an informal exchange of ideas and are not meant to be a corporate or specific product discussion.
Process Development for the Cell-Free Production of Membrane Proteins
Frank Bernhard, Ph.D., Group Leader, Institute of Biophysical Chemistry, Goethe University, Germany
- Troubleshooting, applications, general experience
- Targets and template design
- Variation of cell lysates and reaction conditions
- Improving yield
- Selection of suitable microenvironments
Is it Better to Immunize or Screen Libraries to get Good Antibodies to GPCRs and Ion Channels? Or Does it Really Matter?;
Mick Foley, Ph.D., CSO, AdAlta, Australia;
- What is the best format for the GPCR or ion channel to be in to elicit good antibodies?
- Immunization versus screening naive libraries does it really matter. Do these different approaches produce noticeably different antibodies?
- What is a good antibody? Is it some combination of affinity, agonist/antagonist activity, specificity etc.
- Once we have an antibody what is the best way to optimize it for desired activity against a GPCR or ion channel?
Characterization of Antibodies Against Membrane Proteins
Joseph Rucker, Ph.D., Vice President, Research and Development, Integral Molecular, Inc.
- Affinity and Kinetics: Useful approaches for characterizing antibody binding; interpreting complex binding
- Epitopes: Different techniques for epitope mapping; binning versus mapping; does epitope matter?
- Cell Function: Integrating functional assays into antibody discovery and development; ion channel assays beyond electrophysiology
- Endocytosis: What are the best approaches for measuring antibody endocytosis? Recycling versus degradation
6:10 Welcome Reception in the Exhibit Hall with Poster Viewing
7:15 Close of Day
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Wednesday, September 23
7:30 am Registration and Morning Coffee
8:00 Chairperson’s Remarks
Matthew Gardener, Ph.D., Senior Scientist, Research, MedImmune, United Kingdom
8:10 Affimers – Novel Scaffolds for Molecular Recognition of Membrane Targets
Darren Tomlinson, Ph.D., University Academic Fellow, University of Leeds, United Kingdom
Novel scaffold proteins represent a promising alternative to antibodies in applications ranging from research reagents to therapeutics. Affimers are highly stable scaffold proteins that show great versatility. We describe the development of these scaffolds and give examples of generating highly specific reagents against membrane proteins that can inhibit function directly or through allosteric mechanisms.
8:40 Rational Design of Humanized Agonist Antibodies
Yong (Tiger) Zhang, Ph.D., Assistant Professor, Pharmacology & Pharmaceutical Sciences, University of Southern California
Inspired by antibodies with an ultralong heavy chain CDR3H that exhibits a unique architecture, functional polypeptides were genetically fused into the CDR regions of a humanized antibody. The resulting chimeric antibodies show excellent physical, biological, and pharmacological properties, and support the use of this approach for the generation of innovative antibody therapeutics for the treatment of human diseases.
9:10 Novel Long VH CDR3 Antibodies Targeting the FPR Receptor
Matthew Gardener, Ph.D., Senior Scientist, Research, MedImmune, United Kingdom
Using an immunization approach and subsequent phage display-based engineering, Fpro0165 was generated which completely neutralizes the formyl peptide-mediated activation of human neutrophils. This antibody contains a long, protruding VH CDR3 of 24 amino acids and the critical requirement for this long VH CDR3 in determining the antibodies binding profile will be discussed.
9:40 Coffee Break in the Exhibit Hall with Poster Viewing
10:25 Identification of Potent Nanobodies® against Ion Channels and GPCRs
Marie-Ange Buyse, Ph.D., Director, Discovery, Ablynx, Belgium
Nanobodies® are therapeutic proteins based on single-domain antibody fragments derived from naturally occurring heavy-chain only antibodies. They can be easily engineered via genetic fusion into multivalent and multispecific molecules. Case studies on the identification of potent Nanobodies with superior target-selective ion channel binding as well as potent GPCR Nanobodies will be presented.
10:55 Antibody-Mediated Blockade of Human Orai1 Inhibits T Cell Activation In Vitro and In Vivo
Stefan Zahn, Ph.D., Principal Scientist, Antibody Technology, Novo Nordisk A/S, Denmark
Ion channels are widely expressed on cells and tightly regulate the flow of ions between the extracellular and the intracellular environment. Dysregulation has been linked pain, epilepsy and even autoimmune inflammatory diseases. We will present our recent work on targeting T-cell specific ion channels like CRAC by antibodies inhibiting T-cell activation in vivo and in vitro.
11:25 Enjoy Lunch on Your Own
12:55 pm Plenary Keynote Program
2:40 Refreshment Break in the Exhibit Hall with Poster Viewing
3:25 Close of Conference
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