For the 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 targeted delivery of therapeutics. But for the field to advance, challenges in optimizing antigen quality and presentation, discovery methodologies, protein engineering and target identification must be resolved. This two-part forum brings discovery biologists and protein engineers together to discuss next generation strategies and technologies that will allow protein therapeutics directed against membrane protein targets to advance into the clinic and beyond. Part Two, Structural Analysis, Characterization and Development, explores new developments in structural biology, screening campaigns and characterization assays used to support research against these challenging targets.

Final Agenda

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Wednesday, September 21

11:20 am Conference Registration Open

11:25 Enjoy Lunch on Your Own

2:40 Refreshment Break in the Exhibit Hall with Poster Viewing

STRUCTURAL BIOLOGY STUDIES FOR DEVELOPMENT OF ANTIBODIES AGAINST MEMBRANE PROTEIN TARGETS

3:20 Chairperson’s Opening Remarks

Debra T. Hansen, Ph.D., Associate Research Professor, Center for Applied Structural Discovery, Arizona State University

3:25 KEYNOTE PRESENTATION: PRODUCTION OF HUMAN MEMBRANE PROTEINS FOR ANTIBODY GENERATION, X-RAY AND SERIAL FEMTOSECOND CRYSTALLOGRAPHY, AND ELECTRON MICROSCOPY

Liz Carpenter, Ph.D., Professor, Structural Genomics Consortium, Oxford University, United Kingdom

For human integral membrane proteins (IMPs), producing the protein samples is often the limiting factor for structural and functional studies, and for antibody generation. The SGC has created a pipeline for human IMPs production that has allowed us to solve structures of seven human IMPs, using a range of structure determination techniques. We use these methods to produce and study proteins that are identified as genetic hits for human diseases.

4:05 Conformational Dynamics of a Neurotransmitter Sodium Symporter (NSS) in a Membrane Bilayer

Satinder Kaur Singh, Ph.D., Assistant Professor, Cellular & Molecular Physiology, Yale University School of Medicine

Neurotransmitter sodium symporters are dynamic proteins that exploit pre-existing ion gradients to transport a diverse array of substrates across the lipid bilayer. Eukaryotic members are established targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric diseases. We applied a novel biophysical approach to dissect the conformational behavior of NSS members in customizable lipid microdomains (nanodiscs), allowing us to probe global conformational changes induced by mutations and/or ligand binding.

4:35 Computational Advances in Antibody Design: Toward Improved Optimization, Selection and Formulation

David Pearlman, Ph.D., Senior Principal Scientist, Schrödinger

Recent computational advances hold significant promise both for improved prediction of antibody structure from sequence, and for the ability to precisely calculate physically relevant properties such as affinity and stability. When combined with additional theoretical approaches to identify liabilities such as aggregation propensity and immunogenicity, we can use these tools to variously optimize a lead antibody candidate, triage among multiple potential leads, and improve the formulation process.

5:05 Refreshment Break in the Exhibit Hall with Poster Viewing

5:40 Speaker has cancelled. Delegates may attend sessions of concurrent meetings. Visualization Functional Motions of Membrane Transporters Using Advanced Simulation Technologies

Emad Tajkhorshid, Ph.D., Professor, Biophysics, Biochemistry, and Pharmacology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign

Describing structural changes of membrane transporters continues to pose a major challenge to both experimental and computational approaches. I will describe recently developed methodologies in our lab, and their application to structurally and mechanistically diverse membrane transporters. We can not only capture inter-conversion of major functional states, but also successfully characterize how chemical details such as ion/substrate binding modulate the associated free energy landscapes.

6:10 Speaker has cancelled. Delegates may attend sessions of concurrent meetings. Engineering Membrane Proteins and Their Ligands for Structural and Functional Studies

Christopher Koth, Ph.D., Senior Scientist, Structural Biology, Genentech

6:40 End of Day

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Thursday, September 22

7:30 am Registration Open and Morning Coffee

SCREENING AND CHARACTERIZATION

8:30 Chairperson’s Remarks

Joseph Rucker, Ph.D., Vice President, Research and Development, Integral Molecular, Inc.

8:45 Assay Development for High Throughput and Biased Agonist Screening of GPCR Targets

Patricia McDonald, Ph.D., Associate Scientific Director, Translational Research Institute, The Scripps Research Institute

The development of an autocrine-based assay system for the selection of GPCR agonists from large intracellular combinatorial peptide libraries is described. One out of ~108 different peptides and a GPCR are co-localized in the plasma membrane. When the co-localized peptide activates the neighboring receptor a fluorescent signal is generated and each cell becomes a reporter unto itself. The system was validated by selection of highly potent agonists for the GLP-1R.

9:15 Cell-Based Characterization of a Conformationally and Topologically Sensitive Epitope of Human Gpr34: Implications for GPCR Immunogen Design

Haruki Hasegawa, Ph.D., Principal Scientist, Therapeutic Discovery - Biologics, Amgen, Inc.

We identified and characterized an epitope that reports the correct conformation, membrane topology, and cell surface trafficking competency of GPR34. The epitope formation required the oxidation of four cysteine residues located individually in the four separate extracellular regions of GPR34. The underlying biochemical properties of the conformational epitope not only illustrated the intrinsic challenges of raising mAbs against GPCRs, but also suggested preferred strategies for GPCR antigen design.

9:45 Speaker has cancelled - delegates may attend sessions of concurrent meetings. Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of G-Protein-Coupled Receptor Activation

Dean P. Staus, Ph.D., Research Specialist, Robert Lefkowitz Lab, Duke University Medical Center

Through the use of two different single domain antibodies (nanobodies), which stabilize an active and an unappreciated inactive conformation of the β2-adrenergic receptor, we combine multiple biophysical and pharmacological approaches to show that ligands differentially stabilize these receptor states to achieve their efficacy. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

10:15 Coffee Break in the Exhibit Hall with Poster Viewing and Poster Competition Winner Announced

11:10 New Technologies to Determine Specificity of mAbs Targeting Complex Membrane Proteins

Joseph Rucker, Ph.D., Vice President, Research and Development, Integral Molecular, Inc.

Integral Molecular has developed a panel of complementary technologies for detailed specificity and epitope profiling of therapeutic antibodies targeting complex membrane proteins. We have developed the Membrane Proteome Array™ to profile antibody binding across 4,500 membrane proteins, providing identification of off-target interactions, target deconvolution and detailed comparison of biosimilars. In addition, we have used a high-throughput comprehensive mutagenesis platform for high-resolution mapping of over 500 antibody epitopes on complex targets.

11:40 Single Domain Antibodies Targeting Complex Membrane Proteins Identified by Phage Display Screening

Mick Foley, Ph.D., CSO, AdAlta, Australia

Identifying monoclonal antibodies to complex membrane proteins such as GPCRs and ion channels is a notoriously difficult undertaking. By screening a large phage library displaying single domain antibodies (i-bodies), high affinity i-bodies specific for CXCR4 and TRPV4 were obtained. These binders have been shown to have valuable biological functions in vitro and in vivo. Indeed we are progressing the CXCR4 single domains as a treatment for fibrosis.

12:10 pm High Throughput Single Cell Antibody Discovery from Natural Immune Repertoires Against Membrane Proteins

Veronique Lecault, Ph.D., Co-Founder, AbCellera

12:40 Session Break

12:50 Luncheon Presentation: Hu-mAb Chickens: A New Path for Therapeutic Antibody Discovery

William Harriman, Ph.D., MBA, CSO, Crystal Bioscience

Crystal Bioscience has developed a line of genetically engineered chickens containing human V genes that have been targeted into the chicken immunoglobulin loci. Upon immunization hu-mAb chickens generate affinity matured human sequence chimeric antibodies that can be recovered through the GEM single-B cell cloning technology. Fully human recombinant antibodies from hu-mAb chickens are shown to bind therapeutic targets with the high affinities and diverse epitope coverage that is characteristic of wild-type chicken mAbs.

1:30 Refreshment Break in the Exhibit Hall with Poster Viewing

PROBLEM SOLVING AND BIOTHERAPEUTIC DEVELOPMENT

2:15 Chairperson’s Remarks

Xin Huang, Ph.D., Principal Scientist and Group Leader, Molecular Engineering, Amgen

2:20 Efficiency of Genetic Immunization for the Generation of Antibodies Against Membrane Proteins

Debra T. Hansen, Ph.D., Associate Research Professor, Center for Applied Structural Discovery, Arizona State University

We describe the first report of the efficiency of a DNA-based immunization approach to generate antibodies against membrane proteins. Genetic immunization relies on the immunized host to express, fold, and modify the antigen. We used micronanoplex gene gun immunization of mice to generate antibodies against BSL3 pathogens and a novel in vitro expression method that purifies the antigen. We will discuss method development and applications.
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2:50 Structural Studies of Human Glycine Receptors

Xin Huang, Ph.D., Principal Scientist and Group Leader, Molecular Engineering, Amgen

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in the central nervous system. Selective activation of GlyRs has been hypothesized as an alternative approach to treat neuropathic pain. Here we present crystal structures of GlyRa3 with both positive and negative modulators. Our structures provide new insights into molecular recognition of these modulators and their modulation mechanisms. These results also offer promise of rational structure-based design of new classes of GlyR modulators.

3:20 Session Break

3:30 Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies

Anatoly Shcherbatko, Ph.D., Associate Research Fellow, Rinat Laboratories, Pfizer, Inc.

Monoclonal antibodies inhibited P2X3 after short-term exposure binding to the inactivated state of the channel and potentiated the heteromeric P2X2/3 channel. Extending the duration of exposure resulted in a profound inhibition of both homomeric P2X3 and heteromeric P2X2/3 receptors by efficient antibody-induced internalization of the channel from the plasma membrane. The efficacy in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

4:00 Harnessing Venomics for Ion Channel Drug Discovery

Hongkai Zhang, Ph.D., Senior Scientist, Lerner Laboratory, The Scripps Research Institute

Animal venoms represent a rich source of active peptides for ion channel and GPCR drug targets. However, a challenge remains with the slow pace at which venom peptides are discovered and refined. Combining autocrine-based selection with proximity-based assay provides a robust and user-friendly solution. The talk will include discovery of novel Kv1.3 blockers from natural venom peptide library and selection of refined venom peptides from combinatorial library.
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4:30 Discovery, Generation, and Development of Therapeutic mAb Candidates against GPCR Targets

Shuqian Jing, Ph.D., Founder and CEO, Gmax Biopharm, LLC, China

Gmax Biopharm focuses on discovery and development of novel antibody therapeutics targeting GPCRs. Using our proprietary technologies, we have successfully generated functional mAbs against a number of GPCR targets, including endothelin receptor A (ETa), GLP-1 receptor (GLP-1R), and glucagon receptor (GCGR), etc. The most advanced programs of our GPCR mAb therapeutic development have reached various clinical or preclinical stages.

5:00 Close of Conference

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