Specialized Workshop F2Friday October 1, 2021: 7am - 5:30pm - Lecture List

"Gene Therapy, Cell Therapy and Vaccine Bioanalysis: Recent Issues in Scientific Advancements and Regulations"

Part 1: Cell Therapy

Part 2: Vaccines

Part 3: Gene Therapy

Part 4: Evolving Science & Regulations

Finale: ASK THE REGULATORS!

  • Panel Discussion with Regulators:

    Regulatory Feedbacks on Submitted Studies and Regulations on Gene & Cell Therapy and Vaccine

    • Dr. Nirjal Bhattarai (US FDA CBER)
    • Dr. Heba Degheidy (US FDA CBER)
    • Dr. Vijaya Simhadri (US FDA CBER)
    • Dr. Leslie Wagner (US FDA CBER)
    • Dr. Marco Cavaleri (EU EMA - invited)
    • Dr. Therese Solstad Saunders (Norway NoMA/EU EMA)
    • Dr. Elana Cherry (Health Canada)
    • Dr. Akiko Ishii (Japan MHLW)

 

Lecture DETAILS of F2 – Gene Therapy, Cell Therapy and Vaccine Bioanalysis

Part 1: Cell Therapy
  • Lecture 1
    Overcoming Matrix Interference for a Triplex CAR-T NAb assay: Advanced strategies in the development of Immunogenicity assays for Cell Therapy
    - Dr. Bonnie Wu, Assoc. Scientific Director Biologics Development Sciences, Janssen
    • Building on recent Industry/Regulators' recommendationson ADA/NAb Assays and Immunogenicity Strategy for CAR-T
    • Focus on challenges and strategy for the assessment of NAb Response to a bi-paratopic CAR-T cell therapy
      • NAb bioanalytical strategy for CAR-T therapy
    • Unique feature of the bi-paratopic CAR molecule
      • Recognizing two different epitopes on the same target molecule
        • Increase the binding affinity
        • Try to maintain drug efficacy when cancer cells escape therapy through mutations
    • Rationale for designing a Triplex Assay based on MSD U-Plex platform
      • Assessment ofNAb to the full length CAR and two binding domains respectively
      • Strategy to address severe matrix interference for the assay
    • Chimeric antigen receptor T-Cell immunotherapy (CAR-T) development challenges due to potentialhost immune responses to
      • Non-human sequences in the CAR construct
      • Suicide domainof the CAR-T
      • Residual viral proteins
      • Non-human proteins as part of the gene editing step of CAR-T production
    • New Insights onanti-CAR-T neutralizing antibody assay (NAb Assay)
      • Immunogenicity induction risk factors and neutralization effect
      • Advancement in NAb Assay development for Cell Therapy
      • CAR-T expansion and persistence reduced byHumoral Immunogenicity and Cellular Immunogenicity
      • Detection of the potential development of antibodies to an entire cell
    • Immunogenicity assessments and current regulatory expectations for Cell therapy NAb Assays
    • Case Studies: Overcoming challenges in development of Triplex NAb assays to measure neutralizing responses specific to CAR-T therapy
  •  

  • Lecture 2
    Advanced Immunogenicity characterization by Flow Cytometry & ELISpot for CAR-T programs
    - Dr. Bernd Potthoff, Senior Scientist II, Novartis
    • Building on recent Industry/Regulators' recommendations on applications of Flow Cytometry & ELISpot for CAR-T Immunogenicity assessment
    • Recent development and new data in evaluation of CAR-T Humoral immune response by Flow Cytometry
      • Strategies for direct measurement of real-time immune cell monitoring in vivo and Measurement of ADA to the CAR in their natural cell environment
      • Flow cytometry approaches to measures CAR expression on T cells using anti-CAR antibody staining and gating strategy
      • Advantages of using Flow Cytometry to detect antibodies directed against thesingle-chain variable fragment (scFv) of the CAR in human serum
      • Application of transduced cells with a vector containing an anti-target CAR
      • Approaches to detect CAR transduced cells by the bound anti-CAR surrogate Positive Control (PC)
      • Consideration to define Pre-existing ADA and Cut Points in disease specific human samples. Evaluation of the impact of Pre-existing antibodies against the assay cell line and CAR
    • CAR-T Cellular Immunogenicity response by ELISpot
      • Advantages of using ELISpot as very sensitive technique for detection of IFN-gamma-secreting CAR-specific CD4+ & CD8+ T cells
      • Strategies for Isolation and stimulation of PBMC using peptides from CAR and measurement of cytokine production
      • Quantitation of CAR-T cells Activation and Expansion using accurate and sensitive ELISpot assay to identify and measure the frequency of IFN-gamma-secreting cells
      • ELISpot Data Analysis and Gating Strategy
    • Limitation of Bridging LBA with labeled CAR fragments
      • Insoluble extracellular receptor andAggregation & Precipitation
      • Soluble CAR differences from extracellular domain
      • Potential No detection of an immunogenic response to missing epitopes and/or masking epitopes by labeling
      • Labeled recombinant CAR fragment potential differences from the membrane-bound CAR expressed on CAR-T cells
      • Interferences due to immunogenic interactions with other membrane proteins
    • Case Studies: Flow Cytometry & ELISpot assays development for CAR-T humoral and cellular immunogenicity assessment
  •  

  • Lecture 3
    Biodistribution studies for CAR-T Cell Therapy by qPCR and Flow Cytometry: Are Biodistribution studies necessary for CAR-T Therapy? What do they tell you? What Bioanalytical Techniques to use for biodistribution characterization?
    -Dr. Timothy Mack, Scientific Director, Bristol-Myers Squibb
    • Building on recent Industry/Regulators' recommendations on CAR-T Biodistribution by qPCR and Flow Cytometry
    • Importance of performing Biodistribution in tracking CAR-T Cell due to potential cytokine storms, B cell hypoplasia and encephalopathy as side effects and antigen-independent proliferation (on/off-target toxicity)
      • Tracking CAR-T cells animal and human
      • Understanding CAR-T biodistribution to define delivery route, dosing regimenand durability of effects
      • Approaches to study T cell migration and distribution to tumor tissues
      • Bioanalysis of T cell phenotype and response to tumor microenvironment
      • Quantitative evaluation of CAR-T cell response inhibitory factors
      • Determination of replicative lifespan of CAR-T cells in vivo expansion,proliferation, survival and long-term persistence
    • Bioanalytical techniques for Biodistribution
      • Flow cytometry
      • qPCR
      • Combining bioanalytical results (Flow Cytometry & qPCR) with other analysis Bioluminescence imaging (BLI) Positron Emission Tomography (PET) and Histologic
    • CAR-T biodistribution bioanalysis of
      • T cell suppressive cytokine (TGF-beta and IL-10)
      • Myeliod-derived suppressor cell (MDSCs)
      • Immune checkpoint (PD-L1)
      • Regulatory T cells (Tregs)
    • Case Studies: Advanced techniques and strategies for Biodistribution studies using multiple assays to define the characteristics of CAR-T constructs
  •  

  • Lecture 4
    ddPCR Assay Development, Validation and Standardization for Cell Therapy
    -Dr. Xiaodong Fang, Associate Director / Group leader LBA, Immunogenicity, qPCR, and Flow Cytometry, Takeda
    • Building on recent Industry/Regulators' recommendations on applications ddPCR Assay Development, Validation and Standardization in bioanalysis
    • US FDA Guidance recommendations on conventional qPCR bioanalytical methods for Transgene
      • qPCR as current gold standard for quantification of transgene
      • Absolute quantification of targeted transgene
      • Use of Calibration with synthetic external transgene calibrator ina sample matrix
      • LLOQ of ≤50copies of product per 1 μg genomic DNA
    • ddPCREmerging Technology vs. conventional qPCR
      • Advantages of ddPCR over qPCR due to unique characteristic
      • Overcoming qPCR calibration and normalization issues
      • Partitioning the target gene analyte into nano droplets
      • Distribution of transgene in the partitioned sample is calculated by Poisson distribution
      • Sensitive, selective, and reproducible Absolute Quantification without an external calibrator, by end-point PCR, limiting dilutions and Poisson statistics
    • Overcoming ddPCR limitations
      • Risk of underestimation the actual T cell expansion
      • Strategies for normalization of transgene copy number
      • Use of copy/μL blood unit and comparison Flow Cytometry, cell surface expression data blood
    • Fit-for-purpose (FFP) Validation of ddPCR assay for cellular kinetics and biodistribution
      • Compliance with current regulatory requirements
      • Fit-for-purpose approach based on 2018 FDA BMV Guidance
      • Quantification of Transgene Copy Number
      • Assay Performance for Cellular Kinetics
      • Evaluation of selectivity, linearity, intra- andinter-assay precision, accuracy, and robustness
    • Updated recommendations on current best practice to implement ddPCR in bioanalysis
      • Plan to consolidate ddPCR emerging technology both in Industry and with Regulatory Agencies
      • No regulatory guidance available for ddPCR validation
    • Case Studies: ddPCR Assay Development,Validation and Standardization for AbsoluteQuantification of CAR-T and Reference Gene

 

Part 2: Vaccines
  • Lecture 5
    Bioanalytical Paradigm for Selecting COVID-19 Vaccine candidates: Protein & Cell-based Bioanalysis and Genomics opportunities & challenges
    -Dr. Shabnam Tangri, Vice President / Head of BioPharma Division, Navigate/Novartis
    • Complexity of Vaccine Development and Candidates Selection
      • Bioanalytical impact on crucial stages of vaccine development and testing
      • Exploratory stage to identify antigens
      • Determination of COVID-19 Vaccine Candidates immunogenicity & safety in cell culture and animal models
    • Bioanalytical challenges & solution in vaccine immunogenicity, efficacy, exposure, and safety
      • Importance of understanding the vaccine candidate mechanism of action (MoA)
      • Modulating or priming the immune response to a pathogen
    • How Bioanalytical methods and Genomics can provide insight into Vaccine Clinical Development
      • Strategies for selecting the appropriate assay COVID-19 Vaccine candidates development
      • How to choose the most relevant bioanalytical assay that correlates with protection against infection or disease
      • Challenges and considerations due to polyclonal and multidimensional immune responses
    • Development and Validation of a specific set of specialized bioanalytical assays to demonstrate the immunogenicity, efficacy, exposure, and safety
      • Integration of therapeutic insights
      • Application of state-of-the-art technologies
      • Development of custom reagents
    • Case Studies: Advanced application of Ligand-binding, Cell-Based and Genomic Assays to help the selection of COVID-19 Vaccine Candidate
  •  

  • Lecture 6
    Modernizing Vaccine Bioanalytical Assays: Taking Vaccine Bioanalytical Assays into a "New Frontier"
    - Dr. Rocio Murphy, Senior Principal Scientist Regulated Global PPDM, Merck
    • Building on recent Industry/Regulators' recommendations on Innovation in Vaccine Bioanalytical Assays and Multiplex Testing
    • Use of Emerging Technologies to replace the traditional gold standard assays
      • New technologies and solutions
      • Advanced bioanalytical Technologies as novel essential tools for optimal assays for vaccine development
      • Multiplexing assays implementation and choosing the best platform for a multivalent vaccine
      • Innovation by simplification with multi-parameter assays rather than multiple assays for the same analyte
      • Adaptable Vaccine LIMS to support globally
      • Importance of automation-friendly technologies due to bioanalysis of a large number of samples
      • Novel LBA to replace obsolete antigen-specific ELISA and inhibition ELISA
      • Different technologies comparison for Vaccine Assays
    • Development of High Throughput Neutralization assays (NAb Assays)
      • Plaque Reduction Neutralization Test(PRNT)
      • Application of Plaque-forming assay as infectivity assay based on cell monolayers overlaid with agar to prevent the virus from spreading in the medium
      • Virus Reduction Neutralization Test (VRNT)
    • Case Studies: Development of Specific bioanalytical assays for Vaccine using Emerging Technologies to replace the traditional gold standard assays
  •  

  • Lecture 7
    GSK experience in Immune Monitoring of Vaccine Responses - Using multiple assay/technologies to understand the highly complex immune system responses: Building on WRIB Recommendations, New Case Studies and Evolving Trends
    -Dr. Sylvie Bertholet, Scientific Advisor & Director Preclinical Immunology, GlaxoSmithKline
    • Building on recent Industry/Regulators' recommendations on Immune Monitoring of Vaccine Responses
    • Vaccine Assay Development process for Immune Monitoring
      • Pre-clinical/early clinical specializedassays development/qualification
      • Late phases clinical trials Validation and parameters and criteria characterization
      • Monitoring the highly complex immune system responses by measuringGeometric mean antibody titers (GMTs), Seroconversion rates (SCRs), Seroprotection rates (SPRs), functional antibodies, antibody avidity, B and T cell activation, lymphoproliferation, and cytokine responses
    • Immunogenicity assays for Vaccines
      • Understanding the multiple assays required to measure immune responses
      • Strategies to develop bioanalytical assay to provide confidence that the candidate vaccine is capable of eliciting a robust immune response
      • Assays to determine immune stimulation
      • Development of Inhibition assays to show the immune response can reduce the growth of the pathogen
      • Considerations on Antigen-specific, inhibition and neutralization assays
    • Humoral Immune response
      • LBA development and validation for antibody-mediated response on B-cell recognition of antigens and subsequent antibody release
      • Measurement of total antibody
    • Cellular Immune response
      • Flow Cytometry and ELISpot development and validation for on T-cells
      • Measurement of immune cells response upon exposure to specific antigens
      • Approaches for Cell-based neutralization assays for functional antibody measurement for detection of antibodies capable of inhibiting viral replication
      • Current experience in T-cell ELISpot assay in clinical settings to monitor the number of reactive antigen-specific T cells indicative of a patient’s immune response
    • Case Studies: Vaccine Immune Monitoring by collection and evaluation of multiple parameters using multiple assays/technologies
  •  

  • Lecture 8
    RT-PCR and Sequencing Assays for Vaccines - Scientific and Regulatory Perspective
    - Dr. Frank Taddeo, Director Clinical Assay Development, Takeda
    • Building on recent Industry/Regulators' recommendations on development and validation of Molecular Assays (RT-PCR) for Vaccine
    • Validation of qPCR Assays to support Vaccine Clinical Trials
      • Parameters assessed and acceptance criteria
      • Limit of Detection vs Limit of Quantification
      • Relevance for clinical testing
      • Determination of False Positive rate and relevance
    • Application of highly sensitive real-time reverse transcription polymerase chain reaction (RT-PCR) for Vaccines
      • Quantification of amount of virus shedding
      • Differentiation of viral strains
      • Tools for increasing primer sensitivity & specificity (S&S)
      • gel-based RT-PCR assays
      • Determination for assay Sensitivity, specificity and reproducibility
    • Evaluation of the variables that affect Ct values (Cycle threshold)
      • Optimizing the number of cycles required for the fluorescent signal to cross the threshold
      • Concentration of the target
      • Artifacts from the reaction mix
      • Changes in the fluorescence measurements associatedwith Ct calculation
    • Development & Validation of qPCR assays for Vaccine
      • Assay sensitivity & specificity for specific vaccine efficacy on clinical settings
      • Evaluation of Vaccine Efficacy,Exposure and Safety
      • Measurement of Infectivity and Biodistribution
    • Case Studies: Application of highly sensitivereal-time RT-PCR assay inVaccine development, Vaccine Efficacy, Exposure and Safety

 

Part 3: Gene Therapy
  • Lecture 9
    UCB Experience in qPCR Assay Validation for Gene Therapy
    - Dr. Wibke Lembke, Associate Director Translational Biomarker & Bioanalysis, UCB
    • Building on recent Industry/Regulators' recommendations on qPCR Assay Validation for Gene Therapy Viral Shedding and Biodistribution
    • Current challenges with the bioanalytical support for AAV GT using quantitative qPCR
      • Lesson learned from 2018-2020 White Paper in Bioanalysis recommendations
      • Consideration on the challenges in Biodistribution
    • Assay Development: beyond analyte evaluation via quantitative qPCR
      • Primer and Probe Design
      • Sample collection and Handling
      • DNA Extraction and unique challenges with each matrix (target / non-target tissue, secretory / excretory matrices)
      • Data Reporting
      • Assay Life Cycle
    • Evolution of qPCR-based assay
      • Best practice in level of assays validation considering development stage
      • Acceptance Criteria
      • Assay performance controls and QCs to monitor accuracy, precision, range of quantification, analytical sensitivity and specificity
      • Current status of Regulatory Requirements for qPCR validation
    • Case Studies: Applying recent Industry/Regulators' recommendations to qPCR unique challenge in assay Development and Validation
  •  

  • Lecture 10
    Mitigation Strategies for Overcoming Pre-existing Immunity to AAV Vectors: Current approaches, potential gaps and outstanding questions
    -Dr. Soumi Gupta, Senior Director / Head of Clinical Immunology, BioMarin
    • Building on recent Industry/Regulators' recommendation on Pre-existing Immunity to AAV Vectors
    • Updated data on High prevalence of pre-existing immunity to AAV vectors in humans exposed to natural Adeno-Associated Virus (AAV) infections resulting in an immune response against the virus prior to treatment
      • Current data reported and evaluations prior to treatment
      • Characterization of seroprevalence and titer of pre-existing antibodies by age and geographical location
      • Impact of pre-existing antibodies on current gene therapy efficacy and safety
      • Immunogenicity Risk Assessment for AAV Gene Therapies
    • Approaches to Remove Pre-existing Antibodies
      • Immunoadsorption plasmapheresis
      • IdeS treatment
    • Preventing the Development of treatment-induced antibodies
      • Prophylactic immunosuppression strategies
    • Next Generation Capsid, Mitigation strategies and impact on Pre-existing Immunity
      • How to enable immunogenicity evasion
      • Minimizing NAbformation to the viral vector
      • Reduce vector clearanceand transduction inhibition
      • Other solutions currently available to reduce the titers of pre-existing antibodies
    • Case Studies: Novel data on pre-clinical studies performed for the mitigation of Pre-existing Immunity to AAV Vectors
  •  

  • Lecture 11
    Advancements in Immunogenicity Monitoring related to AAV Gene Therapy - Correlation of Gene Mutation, Immunogenicity Response & Efficacy for Genetic Disorders
    -Dr. Luying Pan, Senior Director Clinical Biomarker Innovation and Development, Takeda
    • Building on recent Industry/Regulators' recommendations on Immunogenicity Response to AAV Gene Therapy
    • Addressing the unique and complex challengesassociated with Immunogenicity Monitoring related to AAV Gene Therapy
      • Understanding the mechanisms behind immune response to design an efficient immunogenicity mitigation strategy and monitoring for AAV Gene Therapy
      • Impact of immunogenicity on re-dosing and importance of correct monitoring of the immune response
      • Immune monitoring assays as critical component for understanding the correlation of Gene Mutation, Immunogenicity Response & Efficacy
    • Current strategies to more effectively mitigate the impact of immune response on the GT efficacy and safety
      • Consideration on Gene Mutation and the formation of an anti-AAV and anti-transgene immune response
      • Strategies for immunogenicity risk assessments to better evaluate the efficacy and safety of AAV Gene Therapyfor Genetic Disorders
      • How to measure, modulate and predict immuneresponse to gene therapy
    • Case Studies: Recent data on Immunogenicity monitoringfor AAV gene therapy strategy for Genetic Disorders

 

Part 4: Evolving Science & Regulations
  • Lecture 12
    Development & Validation of Companion Diagnostics (CDx) for AAV Gene Therapies and Point of Care (PoC) Immunoassays
    - Dr. Mark Milton, Global Head Gene Therapies PK Sciences, Novartis
    • Building on recent Industry/Regulators' recommendation on Companion Diagnostics (CDx) for AAV Gene Therapies
    • Uncommon use of Companion Diagnostics (CDx) for patient selection based on pre-existing
      • Guidelines for CDx assays differ between different Regulatory Agencies.
      • What would it take for a CDx to be accepted globally?
    • Important considerations on the Selection of the assay to be used in clinical trials for patient selection
      • Can the assay become a successful CDx?
      • Can Point of Care Assays be regarded as a CDx and not be performed in a CLIA approved laboratory?
    • Recent discussion on CDx for decision-making to qualify or exclude patientsfrom clinical trials
      • Using companion diagnostic for taking decision regarding immunogenicity
    • Updated recommendations and strategies for the development of Cell-Based Anti-Viral Vector NAb/TI Assays to support clinical trials
      • When to use / not to use CDx for treatment decisions in Gene Therapy clinical trials
      • How to develop CDx to help identify patients who may benefit from gene therapy
      • CDx and PoC to monitor of treatment responseto adjust therapy and achieve improved safety or efficacy
    • Current Regulatory Guidance for development & validation ofCDx assays for Gene Therapy
      • NAb/TI Assays as CDx for safety and efficacy
      • When Regulators require CDx
      • When to start turning a NAb/TI Assays into the development/submission of a CDx to evaluate pre-existing antibodies
    • Case Studies: Importance of developing and validating reliable NAb/TI Assays as CDx and Point of Care Immunoassays to identify patients who may experience treatment-related adverse events
  •  

  • Lecture 13
    Recent Developments in the Safety/Efficacy Assessmentfor AAV5-encoded Factor VIII for treatment of Hemophilia: Thorough assessment of immune responses using multiple technologies/approaches/strategies, Cell-based, Ligan-Binding and ELISpot/ FluoroSpot Assays
    - Dr. Brian Long, Assoc. Director Translational Sciences, Immunogenicity Assessment, Biomarin
    • Immune responses to AAV5-encoded Factor VIII and impact on safety & efficacy prior to and following dose administration
    • Understanding the Clinical Immunogenicity of Valoctocogene Roxaparvovec & AAV5-Mediated Gene Therapy for Hemophilia A
      • Importance of Translational Science to bridge of non-clinical immunogenicity to clinical studies
      • Considerations on Humoral and Cellular immune responses to AAV5 vector and transgene-expressed hFVIII-SQ
    • Immunogenicity response to AAV5-encoded Factor VIIIand impact on Safety/Efficacy
      • Development of Assay to detect Pre-existing antibodies to AAV5 to excluded patients from clinical trial
      • Pros/Cons of using an orthogonal TAb & NAb/TI Assays approach to screen negative for pre-existing immunity to AAV5
      • Cell-based AAV5 transduction inhibition (TI) assay to measure the neutralizing capacity of patient plasma toward AAV5
      • Considerations on clinical relevance & high sensitive Cell-based NAb assays and risk of detecting of physiologically irrelevant ultra-low levels of antibodies
      • LBA - Bridging electrochemiluminescence assay measuring AAV5 total binding antibody (TAb)
      • Post-dosing humoral immune response, consideration on the strategies for the Assessment of Humoral Immune Response to FVIII and AAV5
      • Post-dosing cellular immune response
      • Assays for the determination of a neutralizing antibody response to the FVIII (Inhibitor)
      • ELISpot (FluoroSpot) measurement of Cellular immune responses specific for AAV5 capsid protein and FVIII protein
      • Method development of specific bioanalytical assays to detect cross-reactive antibodies against capsidsof diverse AAV serotypes
    • Case Studies: Challenges in the developmentof immunogenicity assays to detect pre-existing / post-dosing AAV5-encoded Factor VIIIImmunogenicity
  •  

  • Lecture 14
    Bioanalytical Challenges for CRISPR/Cas9 Genome Editing
    - Dr. Yuanxin Xu, Vice President Early Development and Translational Medicine, Intellia Therapeutics
    • Building on recent Industry/Regulators' recommendations on CRISPR/Cas9 Genome Editing Bioanalysis
    • CRISPR/Cas9 RNP complex is composedof a sgRNA and the Cas9 endonuclease
      • Potential for undesired editing in-vivo and/or immunesystem initiation, in the form of anti-cas9 antibodies or T-cell activation
    • Recent developments in the immune responses to CRISPR-associated nuclease Cas9
      • Unique challenges while assessing the immunogenicity to Cas9
      • Need for characterized reagents for evaluating the immunogenicity of Cas9
    • Advancements in CRISPR-Cas9 technology characterization & bioanalytical assays needed for
      • Efficacy assessment
      • Safety considerations
      • Specificity evaluation
      • Delivery to the target cells
    • Current industry standards in Bioanalytical evaluation of endogenous vs newly expressed or reduced expression of proteins
      • Detection & quantification of response differentiation
      • Immunogenicity assessment
        • Systemic delivery of Viral vector-CRISPR
        • Newly expressed proteins
    • Regulators current expectations for gene editing therapeutic
      • Exposure/biodistribution data
      • Measurement of both total and active ribonucleoprotein complex
      • Testing of tissue biopsies in clinical studies
    • Case Studies: Development and fit-for-purpose (FFP) Validation of bioanalytical assays in support of CRISPR/Cas9 Genome Editing




Agenda at a Glance Agenda at a Glance