The process of monoclonal antibody discovery starts with identifying an antigen, usually a protein, that is involved in the disease process. Scientists search for antigens that are unique to the disease state versus healthy individuals. Once an antigen is discovered, antibodies can be generated against it for further study. Initially, polyclonal antibodies were produced by injecting antigens into animals like mice or rabbits. This generated a heterogeneous mixture of antibodies that targeted different regions of the antigen. However, these polyclonal antibodies suffered from batch-to-batch variability and purity issues which limited their use as treatments.
The Breakthrough Of Hybridoma Technology
In 1975, Georges Köhler and César Milstein revolutionized antibody research by developing the hybridoma technology that enabled production of monoclonal antibodies. Hybridoma cells are generated by fusing B cells that produce a single specific antibody with cancer cells known as myeloma cells. This fusion results in a hybrid cell called a hybridoma that is immortal and retains the antibody-producing ability of the B cell. These monoclonal hybridomas can be mass cultured indefinitely to produce large quantities of identical monoclonal antibodies targeting a specific region of the antigen. Antibody Discovery breakthrough technology enabled scalable production of pure antibodies with consistent specificities, paving the way for development of monoclonal antibodies as therapeutics.
Screening And Isolation Of Hybridomas
After generating hybridomas through cell fusion, screening steps are undertaken to isolate cell lines producing the desired monoclonal antibodies. Supernatants from hybridoma cultures are evaluated for binding to the target antigen using enzyme-linked immunosorbent assay (ELISA). Positive clones are subcloned to ensure monoclonality and pure colonies are expanded for further analysis. Secondary screening includes isotyping, determining antibody class and subclass, and characterization studies such as epitope mapping. Hybridomas with high antibody production and specificity for functional or disease-related epitopes are prioritized for further use in developing antibody therapies.
Phage Display: A New Discovery Method
While hybridoma technology remains a mainstay, alternative antibody discovery methods have emerged. One such method is phage display, developed in the 1980s, that utilizes bacteriophages to link antibodies with their encoding DNA sequences. In this technique, antibody repertoires from immunized mice or naive human donors are cloned into the genome of filamentous phage. The phage express the antibody fragment as a fusion to one of its coat proteins. Interacting phage are enriched through multiple rounds of panning on immobilized antigens. Individual phage clones are isolated and sequenced to determine the antibody sequences they display. This powerful in vitro technique allows rapid selection and high-throughput screening of antibody libraries without the need for animal immunization or hybridoma development.
Generation Of Full-Length Antibodies
Many therapeutic antibodies require full-length format containing both heavy and light chains paired together for effector functions. Following identification of antibody fragments using phage display or hybridoma technology, their sequences are reformatted into expression vectors. These vectors are transfected into mammalian cells lines like Chinese hamster ovary cells that have the ability to properly fold and assemble full-length antibodies. Stable cell lines are selected and scaled-up in bioreactors for production of gram quantities of therapeutic monoclonal antibodies. Analytical techniques like mass spectrometry and gel electrophoresis are used to extensively characterize critical quality attributes like size, charge variants and post-translational modifications of the manufactured antibodies.
Preclinical Testing Of Antibody Candidates
Once promising therapeutic monoclonal antibodies are produced at scale, rigorous preclinical evaluation is undertaken to select lead candidates for clinical development. Antibody candidates are profiled in vitro in biochemical and cell-based assays to measure antigen binding, epitope specificity, effector functions and ability to block disease processes. Efficacy is assessed in relevant animal models to gauge ability to neutralize disease targets and improve outcomes.
Parallel toxicology studies in animals evaluate potential side effects at intended clinical doses. Pharmacokinetic profiling is performed to understand antibody clearance rates and tissue distribution in vivo. Antibodies that demonstrate desired mechanisms of action, efficacy and safety are advanced to Investigational New Drug enabling studies and early phase clinical trials in humans.
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1. Source: Coherent Market Insights, Public sources, Desk research
2. We have leveraged AI tools to mine information and compile it
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