BEBPA’s 2nd Annual

Neutralizing Antibody Assay Conference

February 11-12, 2016
Palm Springs, CA

Finally a conference about Neutralizing Antibody (Nab) assay development designed to help you accelerate the development of these critical assays. We have pulled together an experienced group of your colleagues and tasked them with clarifying best approaches, delineating unsuspected development problems and discussing the newest approaches. Last year Come learn how to develop, validate, establish cut points, and run you assay to support clinical trials. This conference is designed to help you.

Abstracts (Alphabetical by Speaker)

Developing a Cell-based Neutralizing Antibody Assay. How Hard Could it Be?

  • Speaker: Dr. Paul Caldwell, Covance
  • Abstract: (Pending)
  • (Day 2: Emerging Practices for NAb Assays Session)

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Achieving High Quality Cell-Based Bioassays Through Controlled Preparation of Bioassay-Ready Cells.

  • Speaker: Dr. Mei Cong, Promega
  • Abstract: Cells are critical reagents in cell-based bioassays. Many factors during cell culture and cell banking can cause batch-to-batch/run-to-run variations, which in turn make assay transfer very challenging. In this talk, I will describe the procedures we have established to successfully achieve cell line transfer and manufacturing. This includes thorough cell line characterization with functional stability tests, controlled cell seeding and harvesting densities for cell passaging and cell bank preparation, process development for cell line scaling up, DMSO tolerance test for cell bank size determination, and dry ice tests for shipping tolerance. The same practice is also extended into preparation of single-use vials of ‘thaw-and-use’ cells. The benefits of such ‘thaw-and-use cell reagents’ in bioassay development will be discussed.
  • (Day 1: Assay Component Development Session)

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Statistical Approaches Is it a Classical Calibration Line? Or is it a Four-parameter Logistic Model? Or is it Both?

  • Speaker: Dr. Stan Deming, Statistical Designs
  • Abstract: It’s a curious mathematical fact that a straight-line calibration line looks a lot like the sigmoidal four-parameter logistic relationship when plotted against log(x) instead of x. This short talk will suggest that for many Nab applications, an ordinary straight-line calibration line might be more appropriate (and accurate) than trying to use the “straight-line portion” of a four-parameter logistic sigmoid.
  • (Day 2: Statistical Approaches Session)

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Managing Critical Reagents in Cell-based Neutralization Assays: From Preparation to Bridging New Batches

  • Speaker: Dr. Bassam Hallis, Public Health England
  • Abstract: Reagents can play key role in the performance of assays and especially cell-based format. It is essential to have well defined and controlled procedures to manage critical reagents in such assays. This presentation will cover examples of how to manage and reduce impacts of reagents on assay endpoints.
  • (Day 1: Assay Component Development Session)

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Vaccines & Neutralizing Antibodies Vaccine Characterization and Evaluation: Assays for Real Correlates

  • Speaker: Dr. Bassam Hallis, PHE
  • Abstract: Regulators expect the use of appropriate assays in support of vaccine development, characterization and evaluation of efficacy and potency throughout the product cycle.
    The assays should accurately reflect the mechanism of action and be based on real correlates of protection. This presentation will describe actual examples used at different stages of product development cycle.
  • (Day 1: Vaccines & Neutralizing Antibodies Session)

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Recommendations for the Successful Transfer Between Laboratories of Cell-based Neutralizing Antibody Assays

  • Speaker: Dr. Richard Hughes, NovImmune
  • Abstract: (Pending)
  • (Day 1: Emerging Practices for NAb Assays Session)

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Development of a Functional Neutralizing Antibody Assay Using Engineered Immortal Reporter Cells and Sciences-Biologics Bristol-Comparison to a Primary Cell Antibody Dependent Cellular Cytotoxicity Assay

  • Speaker: Dr. Marina Juhel, Bristol-Myers Squibb
  • Abstract: In vitro antibody dependent cellular cytotoxicity (ADCC) assays using primary peripheral mononuclear blood cells (PBMCs) as effector cells are regularly performed to support the development of therapeutic antibodies. However, ADCC assays with primary effector cells present many drawbacks (e.g., donor availability and day-to-day variability) in their implementation for routine testing. To resolve these problems, we evaluated a recombinant Jurkat T cell line stably expressing the FcγRIIIa receptor and the luciferase reporter gene as an alternate effector cell. Previous reports have shown the utility and comparability of using reporters cells in assessing drug potency. In the work presented here, we assessed whether the ADCC reporter assay is comparable to the PBMC-based ADCC assay with regards to characterizing the neutralizing potential of anti-drug antibodies. First, we evaluated the activity of the drug in the ADCC reporter assay and in the PBMC-based ADCC assay. We confirmed that in presence of the drug, the reporter cells responded by inducing luciferase activity in a dose-dependent fashion. These results show that the ADCC reporter assay is a suitable alternative for measuring ADCC activity for therapeutic antibodies. To demonstrate that the ADCC reporter assay can be used for evaluating the neutralizing potential of anti-drug antibodies, the inhibitory effect on the drug activity of four antibody controls was evaluated. The two anti-idiotypic control antibodies, a monkey polyclonal antibody (pAb) and a murine monoclonal antibody (mAb), neutralized the drug in both assays. To further establish the correlation of the antibody biological activity in both assays, the impact of two mouse monoclonal anti-human antibodies, one specific to the CH2 domain and one specific to the CH3 domain was evaluated. In both assays, only the mouse monoclonal anti-human antibody specific to the CH2 domain neutralized the ADCC activity. These data confirm the biological equivalence of the two assays and demonstrate the utility of using engineered immortal reporter cells for assessing the neutralizing potential of anti-drug antibodies in human samples.
  • ( Day 1: Case Studies Session)

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Interactive Survey & Mini-Course: Use of Design of Experiments (DOE) to Jump Start Method Development–New

  • Speaker: Dr. Laureen Little, Principal Consultant, Quality Services
  • Abstract: (Pending)
  • (Day 2: Surveys Session)

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Biochemical Characterization of Neutralizing Anti-idiotype Antibodies Directed Against Mirvetuximab Soravtansine (IMGN853)

  • Speaker: Dr. Sven Loebrich, ImmunoGen Inc
  • Abstract: IMGN853 is an antibody-drug conjugate (ADC) that represents a potential new treatment for patients with cancer highly expressing folate receptor alpha (FRα), including many ovarian cancers. The US Food and Drug Administration (FDA) granted orphan drug designation to IMGN853 for the treatment of ovarian cancer. IMGN853 comprises a highly specific monoclonal antibody (M9346A) directed against FRα, to which the cytotoxic small molecule maytansinoid, DM4, is attached via the sulfo-SPDB linker. As with the antibodies in a number of other ADCs, the M9346A antibody has been humanized through ImmunoGen’s proprietary resurfacing technology, developed to minimize anti-antibody immune responses in patients. Nevertheless, among the safety evaluations performed, patients receiving IMGN853 are rigorously monitored for potential immune responses against this protein moiety. Here we describe a method to assess patient immune responses in phase I clinical trials in an anti-drug-assay (ADA) using a highly specific neutralizing anti-idiotype antibody (anti-Id M) directed against M9346A. Using a direct ELISA format we show that M9346A and anti-Id M bind to each other with double-digit picomolar affinity. The association of M9346A with FRα –expressing H2110 cells in flow cytometry assays can be suppressed by >99% through preincubation of M9346A with increasing amounts of anti-Id M. Consistent with our cell-based flow cytometry data, anti-Id M is also capable of blocking association of M9346A with a soluble, purified form of FRα antigen in non-cell based ELISA assays. Moreover, pre-incubation of M9346A with increasing concentrations of soluble FRα antigen suppresses binding to immobilized anti-Id M in ELISA, suggesting that the anti-idiotype antibody either targets or sterically hinders the paratope on M9346A and therefore acts as a neutralizing antibody.
  • (Day 1: Case Studies Session)

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Strategies for Development of a Suitable Platform for Detection of Anty-Therapeutic Neutralizing Antibody

  • Speaker: Dr. Mark O’Dell, Covance
  • Abstract: Two types of assay platforms have been used to measure neutralizing antibody (NAb) activity: cell-based biologic assays and non-cell-based competitive ligand-binding assays (CLB). Cell-based assays employ a cellular model that reflects the mode of action (MOA) of the drug and represents a more physiological system for NAb detection. Generally FDA considers that bioassays are more reflective of the in vivo situation and are recommended. The perceived preference for bioassay made it a logical first approach in the development of a NAb assay. However, bioassays have significant variability and a limited dynamic range for their activity curves. Such problems make development and validation of neutralization bioassays difficult. When neutralizing bioassays are not feasible to be developed, a non-cell based CBL assay format becomes an alternative for cell-based assay for NAb evaluation. Non-cell-based CLB assays utilize direct or competitive binding events between drug and target (and ligand if the drug is an antagonist), which tend to be simpler and faster to develop and validate and can overcome some technical limitations of cell-based assays. This presentation will describe steps taken to develop a suitable assay format for the detection of neutralizing antibodies against a monoclonal antibody therapeutics.
  • (Day 1: Case Studies Session)

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NAb Me Well – FDA Regulatory Perspectives on Neutralizing Antibody Assays

  • Speaker: Dr. João Pedras-Vasconcelos , Ph.D., OBP/CDER/FDA
  • Abstract: Biologics are a class of drugs of growing importance in the treatment of human diseases. The cost to the biotechnology industry of developing a biologic from early stage all the way to licensure is very high, and many factors can impinge upon approval. One potential obstacle impacting product development is the occurrence of neutralizing antibodies (NAb) against the biologic in treated patients. NAbs are a subset of the total anti-drug antibodies induced by the biologic, and are functionally defined by the assay that detects them. NAbs can impact both efficacy and safety of the product depending on patient-related and product-related factors. This presentation will provide an overview of two FDA guidances pertaining to immunogenicity risk management for biologics including risk assessment and mitigation, and neutralizing antibody detection and assay selection. We will discuss the importance of NAb assays to a well thought out immunogenicity plan.
  • (Key Note Speaker, Day 1: Regulatory Update)

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Growing Cell Stocks for Cell-based Nab Assays

  • Speaker: Dr. C. Jane Robinson, Consultant
  • Abstract: Cell-based assays better reflect the in vivo action of neutralizing antibodies (Nab) than do ligand binding assays, but are generally less precise and robust. Variability in the cell-based assays can be reduced by ensuring the quality and uniformity of the cell stocks. This requires rigorous control and appropriate quality control tests for preparation of the cell bank aliquots, their thawing and preparation for use, and for those cells that require expansion and maintenance, rigorous control of the growth and passage conditions. Useful measures and common problems will be discussed.
  • (Day 1: Assay Component Development Session)

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Assessment of a Dynamic Cut Point in a 384-well Cell-based Neutralizing Antibody Assay

  • Speaker: Dr. David Rusnak, BioAgilytix
  • Abstract: Cell-based assays are the current preferred platform for assessing the neutralizing capacity of anti-drug antibodies generated by subjects in preclinical toxicity programs or clinical trials. Cell-based assays are often impacted significantly by plate to plate and day to day variability in response (signal), and typically necessitate a floating assay cut point to accommodate the signal variability. Reliably establishing this cut point during validation is challenging, to the point that often cut points require adjustment during sample analysis. In practical terms, generating statistical confidence of a population range during the sample analysis phase of a study is not possible. Thus, current guidelines for NAB assays recommend establishing a cut point during validation using a minimum of 30 individual samples, with 100 individual samples preferred over multiple runs. While 30 to 50 samples can be readily tested in current 96-well formats, the need for assay controls limits the ability to test more cut point samples in a single assay. Higher density assay formats, e.g. 384-well assays, have been common in high throughput screening programs and preclinical discovery efforts for some time. Laboratories can take advantage of these formats to include many more samples and controls in a single assay plate. We are evaluating rapid assay development, qualification and validation with the potential to generate a plate-specific (dynamic) cut point), from 70 or more individual cut point samples in each assay, including sample analysis.
  • (Day 2: Statistical Approaches Session)

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Emerging Practices for NAb Assays Immunogenicity Assessment of Next-generation Therapeutic Protiens

  • Speaker: Dr. Michael Tovey, Biomonitor
  • Abstract: Numerous protein therapeutics in development including bi-specific monoclonal antibodies, antibody-drug conjugates (ADCs), or pegylated fusion proteins have several functional epitopes and/or a multifunctional action that present significant challenges for the development of assays that can assess the immunogenicity of each component. A cell engineering approach has been used to resolve these challenges. Case studies will be presented for the development of unique multiple readout reporter gene assays for the quantification of the neutralizing antibody response to a PD-1/PD-L1 inhibitor that targets the interaction of the inhibitory receptor PD-1 on T cells with PD-L1 expressed on tumor target cells and a monoclonal antibody that targets VEGFR2 and that exhibits ADCC activity.
  • (Day 2: Emerging Practices for NAb Assays Session)

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Selection of Ligand Binding NAb Assay to Support Benralizumab Clinical Development: Comparison with an ADCC MoA Cellbased Assay

  • Speaker: Dr. Yuling Wu, Medimmune
  • Abstract: (Pending)
  • (Day 1: Case Studies Session)