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May 18th, 201611 am to 12 pm ET
Measuring the binding kinetics and affinity of small molecule drugs and biologics to their target receptors is very important in the drug development process in order to model pharmacodynamics, pharmacokinetics, dosing regimens, and efficacy. The BiOpitx 404pi is an enhanced surface plasmon resonance (eSPR) instrument that allows for low resolution kinetic screening of both small molecule drugs, and biologics from crude mixtures, to high resolution kinetic determinations on purified molecules. The 404pi also finds uses in epitope binning and active concentration analysis.
This webinar will highlight multiple biophysical applications of the use of SPR biosensors in drug discovery research. In the first application, the 404pi is used to compare the quality of a series of the same antigen purchased from various vendors by measuring binding kinetic parameters. Once the optimal antigen was identified, it was then used in the screening of several lead probodies, proteolytically-activatable antibody prodrugs.
In the second application, the BiOptix 404pi biosensor played an important role in the optimization and characterization of aptamer-based therapeutic and affinity reagents called SOMAmers, or “Slow Off Rate Modified Aptamers”. In this example two lead SOMAmers to IL-6 are optimized for higher affinity. SPR shows different off rates even though they both have similar affinities providing new granularity over KD measurements alone, allowing more informed selection of lead compounds and better understanding of the impact of modifications during affinity optimization.
Learning Objective Bullet Points:
Segment Title: “Comparison of Protein Variants to Obtain Ligand Binding Kinetics for Antibody Drug Discovery”
Antibodies have emerged as versatile, potent therapeutics with targets spanning cancer to infectious disease. CytomX has developed a new class of antibody therapeutics called Probody™ therapeutics that are recombinant, proteolytically-activatable antibody prodrugs. Probody therapeutics are designed to widen the therapeutic window by minimizing interaction with healthy tissue and maximizing interaction with diseased tissue. Probody therapeutics are “masked” to reduce binding to antigen in healthy tissue, but can become “unmasked” in the tumor microenvironment by tumor-specific protease activity.
In early pre-clinical development, we seek to identify sets of antibodies with a range of affinities and binding kinetics. Since biological mechanisms of tumor target antigens are a function of both equilibrium binding affinity and differences in dwell-time, measuring binding kinetic parameters becomes a critical component in selection of antibodies for further development. A recurring challenge for characterizing these antibodies is development of sensitive assays to compare epitopes on target antigens. The functional integrity of recombinant, purified protein antigens used in antibody development are often verified by low-sensitivity and/or highly amplified signaling assays such as SDS-PAGE, ELISA or FACS-based cell binding assays, which can be inadequate for optimal antibody selection. These protein antigens are often significantly misfolded and aggregated, limiting their use for analyzing antibody binding kinetics. Here, we compare the quality of a series of the same antigen purchased from various vendors utilizing eSPR and binding kinetic parameters. The antigens were also evaluated for percent monomer in solution. Once the optimal antigen was identified, it was then used in the screening of several lead antibodies. The ease of use and throughput of the BiOptix 404pi to measure these binding kinetic parameters accelerated the Probody therapeutic optimization process.
Sherry L. LaPorte, R.Ph., Ph.D.
CytomX Therapeutics, Inc.
Sherry LaPorte, Ph.D., is currently a Senior Scientist in the Department of Cancer Immunology and has been working in biotech industry for 10 years. She leads the T cell engaging Bispecific Probody™ team and is responsible for optimizing the platform and selection of lead candidate Probody™ therapeutics for this highly potent modality for cancer treatment. She has ten years of experience developing and analyzing antibody leads for diseases including neurodegeneration, inflammation/autoimmunity and oncology. She did her post-doctoral training at Stanford in Structural Immunology as well as at UCSF-San Francisco General Hospital with Dr. James Marks performing antibody engineering. Her thesis work at UCSF was performed under Dr. Robert Stroud in x-ray crystallography performing both structure determination and de novo design protein engineering.
Segment Title: “Slow Off-Rate Modified DNA Aptamer Inhibitors of IL-6 Signaling”
IL-6 is a pleiotropic cytokine that is of pharmacologic interest due to its involvement in inflammatory pathways. We have recently identified a novel class of modified aptamers called SOMAmer reagents (Slow Off-rate Modified Aptamers) that inhibit the biological activity of IL-6 as well as many other biologically interesting targets. SOMAmer reagents exploit the chemical diversity of protein-like side chains, leading to novel intra- and intermolecular interactions not possible with conventional aptamers. The co-crystal structure of a high-affinity SOMAmer reagent (Kd =0.20 nM) with IL-6 is presented, revealing a G-quartet domain and a stem-loop domain. This structural motif interacts with IL-6 over an extended surface via substantial hydrophobic interactions, exhibiting close shape complementarity with the protein, and the interface is characterized by overlapping the binding surfaces of the IL-6Rα and gp130 receptors. After identifying a lead sequence, a single modified nucleotide substitution led to a 37-fold enhancement in binding affinity of the G-quartet fragment. The measurement of on- and off-rates via surface plasmon resonance (SPR) creates new granularity over Kd measurements alone in the discovery of lead SOMAmer reagents for therapeutic programs, allowing more informed selection of lead compounds and better understanding of the impact of modifications during affinity optimization. These measurements can also aid in the development of SOMAmer affinity reagents for purification or diagnostic applications.
Steve Wolk, Ph.D.
Director of Analytical Chemistry
Steven Wolk, Ph.D., is currently the Director of Analytical Chemistry at SomaLogic, Inc. His group is responsible for biophysical characterization of SOMAmer reagents, QC and stability testing, as well as various research and product development projects. He received his Bachelor’s degree in Chemistry from UC San Diego, and his Ph.D. in Biophysical Chemistry from UC Berkeley, under Dr. Ignacio Tinoco, using 2D-NMR and other spectroscopic techniques to characterize nonstandard DNA structures. He has also led analytical groups at a number of other biotechnology companies (NexStar, Invenux, Amgen). He has a broad background in analytical techniques used for the characterization of biomolecules, including HPLC/UPLC, NMR, LC/MS, CGE, PAGE, SPR, FTIR, CD, and UV/Vis spectroscopy.
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