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Pre-Conference Training Course
Introduction to Antibody Engineering
TRAINING COURSE OVERVIEW
Today’s wealth of knowledge of protein structures will be reviewed along with the genetics of diversity generation of antibodies, to give insights into the best strategies for improving protein function.
There is particular emphasis on the choice of a functional assay to monitor effectively the changes in a desired property, and the use off unctional enrichment steps where a library approach is employed.Not only is amino acid sequence amenable to engineering, but glycan structures and other modifications may also be engineered.
The course will focus on the engineering and enhancement of antibodies and antibody-like scaffolds. Examples will include work on antibody fragment affinity improvement by 100-fold to low pM affinity. Also the engineering of bispecific antibodies by diverse approaches and the adaptation to generate Chimeric Antibody Receptor (CAR) constructs will be discussed.
Expression platforms for producing antibodies for testing and for manufacture will also be covered. A background in biochemistry and molecular biology is useful, as the course is designed to progress rapidly from simple to advanced concepts.
• Functions amenable to engineering: affinity, specificity, stability,solubility, immunogenicity
• The measure of success: functional assays• Engineering by design
• Engineering by random mutation
• Designed libraries
• Display technologies
• Improving manufacturing by protein engineering methods
• Glycosylation engineering – function and homogeneity
• Other protein modifications
• Immunogenicity engineering
• Bispecific antibodies
• Antibody-drug conjugates (ADCs)
• CAR-T strategies
• Expression of antibodies and fragments for discoveryand testing
• Manufacturing platforms for antibodies and fragments
Workshop A: The Nuts and Bolts of Antibody Development: Accelerating Antibody Drugs to the Clinic
1:00 Workshop Moderators’ Remarks
James Larrick, M.D., Ph.D., Managing Director and Chief Medical Officer,Panorama Research Institute and Velocity Pharmaceutical Development, Mark Alfenito, Ph.D., President and CEO, EnGen Bio, LLC
1:15 Looking at the Historical Antibody Landscape to Understand and Improve Developability
Close examination of the sequences and physical properties of antibodies that have reached advanced stages of development will likely provide useful guidelines for novel antibody developability. Antibodies with assigned non-proprietary names have the most reliable sequence information, and a significant subset of over 140 of them were expressed with the resulting samples analyzed in a variety of high-throughput assays. Results and trends of this analysis will be discussed.
Max Vasquez, Ph.D., Vice President, Computational Biology, Adimab LLC
1:45 Engineering Antibody Fabs for Long Acting Delivery to the Eye
Maximal clinical benefit for treatment of back of the eye disorders such as age-related macular degeneration (AMD) with protein therapeutics is currently obtained with intravitreal injections at 4-8 week intervals. Use of long acting delivery (LAD) technologies or molecules with increased vitreal half-life would benefit patients through reduced dosing frequency, less incidence of injection-related complications, and perhaps improved durability of treatment outcome. Challenges for LAD to the eye or clearance modification include restrictions on volume (≤100 μL) of injection necessitating high concentration formulations, a short list of formulation excipients approved for ocular use, stability of proteins to vitreous humor conditions, and a limited understanding of the mechanism of protein clearance from the eye. We have been using pharmacokinetic (PK) studies in a rabbit model to explore molecular attributes of antibodies and antibody Fab fragments affecting vitreal half-life. In contrast to systemic clearance, FcRn-dependent recycling does not contribute to the vitreal half-life of IgG and molecular charge variation does not influence the vitreal clearance of antibody Fab. We find that hydrodynamic size is a key contributor to the rate of vitreal clearance, suggesting that diffusion out of the vitreous cavity is a rate-determining step, with vitreal half-life showing a linear dependence on hydrodynamic radius calculated from light scattering measurements. Increasing the hydrodynamic size of a protein therapeutic slows clearance and is an effective means to achieve less frequent dosing. In addition, we present a case study involving the re-engineering of a therapeutic Fab to increase the stability to vitreous humor conditions yielding variants that are more suitable for LAD.
Devin Tesar, Ph.D., Scientist, Drug Delivery Department, Genentech, Inc.
2:15 Optimization of Pre-clinical Cell Line Development – Current Progress on Antibody Production Cell Line Development
Isolation of stable, highly productive cell line remains to be a critical step in development of a large scale manufacturing process. Most protein therapeutics are produced from CHO cells with a few exceptions. Production titers have generally reached multi-gram/L levels. Facile processes have shortened the screening time from 6-12 months to 4-6 months. Product quality assurance and monoclonality documentation have garnered increasing attention in cell line development process.
Bo Yu, Ph.D., Co-founder and Chief Scientific Officer, Larix Bioscience LLC
2:45 Networking Refreshment Break
3:15 The Process of CMO Selection for Antibody Development: Matching Capabilities to Need
Financing realities of biotech start-ups require that companies outsource the development, scale-up and manufacture of therapeutic antibodies that are to be tested clinically. This is a substantial undertaking, both in terms of time and investment. In working with a small-company client to evaluate, select and manage a CMO to produce a recombinant human mAb expressed in CHO cells, we navigated to a successful outcome for the clinical Phase 1 program by matching the CMO’s capabilities to the company’s needs. Challenges in subcontracting biologics production will be discussed.
Steven Chamow, Ph.D., Principal, Biopharmaceutical Product Development, Chamow & Associates, Inc.
3:45 "Human-like” Treg Epitope Sequences in Biologics: Regulating Immune Responses to Biologics
Modulation of T cell responses in the context of protein therapy and inflammation may contribute to the design of improved biologic therapeutics. Tregitopes (natural T regulatory epitopes derived from IgG) have now been demonstrated to reduce immune response to antigens in an antigen-specific manner. We report on recent unpublished studies on Human-Like Non-IgG Tregitopes, and describe plans for the application of Human-like Treg epitope sequences to reducing the immunogenicity of gene therapy vectors, transplantation, and to the treatment of autoimmune disease. Adaptation or incorporation of T reg epitopes may aid in the design of safer, more effective protein therapeutics.
Guilhem Richard, Ph.D., Computational Immunologist, Bioinformatics, Epivax, Inc.
4:15 Open–Access Integrated CMC Platforms for Accelerating Biologics Development to the Clinic
Open-access integrated CMC platforms have been established from cell line development to cGMP manufacturing of biologics. These platforms include state-of-art continuous cell culture processing technology and innovative manufacturing using single-use disposable bioreactors. Several case studies will showcase how their applications have greatly expedited development of diversified biologics modalities including monoclonal antibodies, antibody drug conjugates (ADCs) and recombinant proteins from DNA to IND and BLA filing.
Weichang Zhou, Ph.D., Senior Vice President, Biologics Development and Manufacturing, WuXi Biologics, China
4:45 Closing Remarks
5:00 Close of Workshop
Workshop B: Overview and Applications of Next-Generation Sequencing (NGS) in Antibody Discovery and Engineering
Next-generation sequencing (NGS) provides a quantitative approach to measuring the diversity and distribution of antibody libraries. This workshop will introduce and enable researchers on how to design, analyze, and perform antibody NGS studies and how these can be applied for discovery and engineering of monoclonal antibodies from both synthetic and immune libraries.
We will address how to practically construct libraries for NGS, with an emphasis on using proper standards for quality control, data processing, and bioinformatic analysis. We will also provide information on advanced NGS strategies, such as paired heavy-light chain sequencing, error correction, bias correction, machine learning and modeling for identifying antigen-specific clones.
• Overview and introduction to antibody NGS and applications in antibody engineering
• Design and construction of antibody library generation for NGS (including in vivo repertoires from mice and humans)
• Quantitative analytical tools for quality control of antibody sequencing libraries
• Preprocessing and quality assessment of antibody NGS data
• Annotation and bioinformatic analysis of antibody NGS data
• Paired VL-VH sequencing using single-cell approaches
• Error and bias correction with molecular tagging and clustering algorithms
• The use of machine learning and statistical modeling for identifying potential antigen-specific clones