Epibase™ In Vitro Screening for Biotherapeutics

T cells, B cells and cytokines in the blood stream
  • Epibase™ In Vitro Platform

    Lonza’s Epibase® In Vitro Platform evaluates the immunogenicity risk of biotherapeutic proteins by directly measuring the immune response in the human population using peripheral blood mononuclear cells (PBMCs).  By using PBMC in vitro, the Epibase® In Vitro platform allows a direct comparison of immunogenicity risk for multiple biotherapeutic leads in a safe and cost effective manner. When combined with our Epibase® In Silico tool, these cellular assays allow for a complete immunogenicity risk assessment of your lead candidates. Understanding the immune response against your leads in a fully human system in early development will allow you to manage any potential safety and immunogenicity risk prior to human clinical trials.

      

    Epibase® In Vitro Assays:

    • Dendritic cell activation 
    • Cytokine release
    • MHC-associated peptide proteomics (MAPPs) 
    • T cell responses
    • B cell responses
    • Customized potency assays (e.g. cell binding and cell killing assays)
    • Clinical immunoprofiling 

     

    Designed for a Wide Range of Targets:

    • Antibodies and antibody fragments
    • Enzyme replacement therapies
    • Plant and bacterial proteins 
    • Viral vectors
    • Synthetic peptides

     

    Advantages of Epibase® In Vitro Assays:

    • Multi-parameter measurement of human primary immune cell activation by flow cytometry, FluroSpot and Luminex® allows for a detailed characterization of the immune response
    • High throughput processes capable of running large panels of samples
    • Stringent quality control to guarantee assay consistency and traceability of each sample
    • Consistent supply of highly diverse, high quality PBMC samples from healthy donors
    • PBMC can also be isolated from specific patents to characterize the immune response in the disease background (taking into account patient genetics, immune status and concomitant medication)  

     

  • Epibase Cell Banking for Clinical Monitoring

    The isolation and cryopreservation of high quality cells during clinical trials is crucial for the subsequent characterization of the immune response. PBMC and subsets of immune cells need to be isolated from whole blood and frozen as quickly and as consistently as possible after withdrawal from the patient. Lonza has experience with acting as a central lab for multi-center clinical trials to ensure the rapid and consistent banking of patient cells for subsequent ex vivo assessment.

    PBMC Banking  

    • All necessary ethical documents and HTA (Human Tissue Authority) license in place for the storage of human tissue for research use
    • Dedicated laboratory for the processing and cryopreservation of patient samples
    • Controlled rate freezing of cells to ensure high viability and functionality post-thaw
    • Full traceability and electronic tracking of all samples
    • Opportunity to optimize the process depending on the intended use of the samples
    • Both standard and customized assay design for samples analysis

     

     

  • Epibase® T Cell Assays

    T cell response induced by a biotherapeutic protein is now commonly used as a surrogate marker for the risk of developing anti-drug antibodies (ADA) in the clinic. The Epibase™ In Vitro T cell assay platform takes into account not just the amino acid sequence of the protein, but also a host of other factors that can contribute to immunogenicity risk, such as the 3D structure of the protein, PTMs, aggregates, production contaminants and formulation components. T cell responses against whole proteins, protein fragments, short synthetic peptides or peptide pools can be assessed in healthy donor PBMCs. Additionally, these assays can be used to monitor the strength and quality of the immune response during clinical trials.

     

    T cell Assay Formats

    • PBMC-based assays using flow cytometry to assess CD4+ and/or CD8+ T cell proliferation
    • Highly sensitive DC:CD4 and DC:CD8 re-stimulation assays using a dual-color FluroSpot readout of T cell activation (IFNγ/IL5 and IFNγ/Granzyme B readouts respectively)
    • Multiplex cytokine analysis using Luminex® to characterize the T cell response
    • Memory assays for the assessment of T cell responses during clinical trials

     

     Flow cytometry for monitoring T cell response

    Immune cells pass through the light source for detection and identification in a flow cytometry instrument.

  • Epibase™ Cytokine Release Assay

    The Epibase™ Cytokine Release Assay (CRA) assesses the risk of a biotherapeutic protein inducing a dangerous cytokine response when administered to patients. The assay assesses the immediate interaction of the biotherapeutic protein itself (or other product-related factors such as formulation components and production contaminants) with the immune system. Rapid cytokine release is not traditionally associated to an adaptive immune response driven by T or B cell epitopes but more likely to be driven by the mode of action of the biotherapeutic protein.

    Cytokine Release Assay

    • Healthy donor PBMC are incubated with the test biotherapeutic protein for 24-48 hours
    • Supernatant samples are harvested and cytokine/chemokine levels assessed by Luminex®
    • A standard cytokine panel includes: IFNγ, IL1β, IL2, IL4, IL6, IL8, IL10, IL12 and TNFα
    • Customized cytokine/chemokine panels can also be used on demand
    • Cytokine release profiles compared to low and high risk biotherapeutic proteins
    • Additional controls can also be included on request           

     

  • Epibase™ Dendritic Cell Activation Assay

    The Epibase™ Dendritic Cell Activation Assay uses human dendritic cells (DC) to assess the impact of your Biotherapeutic protein on the innate immune response. Similar to the Epibase™ Cytokine Release Assay, cytokine profiles are assessed after incubation of the DC with the test biotherapeutic protein.  

    Dendritic Cell Activation Assay

    • Dendritic cells (DC) are generated from healthy donors and loaded with test biotherapeutic protein
    • DC phenotype is assessed before and after treatment with the biotherapeutic protein by flow cytometry (DC viability and CD11c, CD14, CD80, CD83, CD86, CD209, HLA-DR expression)
    • Supernatant samples are analyzed for the expression of key DC cytokines (e.g., GM-CSF, IFNy, IL1ß, IL6, IL10, IL12, MIP1α, MIP1ß, TNFα) 
    • Customized cytokine/chemokine panels can also be used on demand
    • LPS (lipopolysaccharide) is included as a typical control and potent activator of DC cytokine expression

  • Epibase™ B Cell Assays

    The Epibase™ In Vitro B cell FluroSpot assay is designed to identify immunoglobulin (IgA, IgE, IgG and IgM) secreting B cells. The assay can be used to assess the frequency of pre-existing B cell responses against a biotherapeutic protein in healthy donors, as well as monitoring B cell responses to a biotherapeutic protein during clinical trials.

     

    Recall B Cell FluroSpot Assay

    • Detection of total and antigen-specific IgA, IgG, IgE, and IgM secreting B cells
    • Blood samples (PBMC) or suspensions of lymphoid tissues can be tested
    • Identification and enumeration of total and antigen-specific secreting cells
    • Highly sensitive, single cell analysis and quantitation of the number of circulating antigen-specific memory B cells

     

  • Epibase™ MAPPS Assay

    The Epibase™ MAPPs (MHC-associated peptide proteomics) assay identifies HLA-binding peptides processed and presented from a whole biotherapeutic protein. The regions identifies can be targeted for removal of potential T cell epitopes or ‘de-immunization’.  

    MAPPs Assay

    • Dendritic cells (DC) are generated from healthy donors and loaded with test biotherapeutic protein
    • DC takeup, process and present peptides from the protein via HLA Class II molecules
    • DC lysis and immunoprecipitation of HLA-DRB molecules
    • Elution of peptides from HLA-DRB molecules
    • Peptides identified by mass spectrometry and mapped back on to biotherapeutic protein sequence
    • Peptide binding can then compared to donor HLA-DRB alleles

     

  • Epibase™ Customized Potency Assays

    Target binding and cell killing are key attributes for many biotherapeutic proteins, particularly monoclonal antibodies. Binding to membrane-bound targets on tumor cells and subsequent killing of target cells via ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity) or toxic payloads on ADC (antibody-drug conjugates) needs to be assessed during development. Other novel cell therapy products can also be assessed using CTL (cytotoxic T lymphocyte) killing assays. 

    Binding and Potency Assays

    • Target cell banking
    • Design and optimization of binding assays
    • Development of antibody-based cell killing assays with fluorescently labeled target cells and primary effector cells (PBMC or isolated NK (natural killer cells)
    • Flow cytometry and/or Fluorospot readout platforms to directly monitor cell killing
    • DC:CD8 assay to generate activated CTL which can be used as effector cells and target cell killing directly monitored