The term “target indifferent approach” describes the Center for Therapeutic Antibody Development's novel protocol for the production of monoclonal antibodies (mAbs) in cases where the target protein is unknown.
Protocol Offers Advantages
This protocol is advantageous when a reagent is needed that:
- Differentiates functional phenotypes when novel surface proteins are unknown (i.e. differentiation of islet cells)
- Acts as a diagnostic marker/prognostic indicator (i.e. cancer cells)
- Performs a function, but the mechanism is unknown (i.e. blocking viral entry by binding to a host protein)
Our target indifferent approach relies on the observation that mice can be efficiently immunized to complex or multiple antigens of a cell simultaneously by the injection of cellular vesicles. These vesicles, comprised of transport vesicles as well as other small orgenelles with a lipid bilayer, display membrane proteins in their native tertiary structure. Our experience is that immunization in this manner results in monoclonal antibodies that have superior binding to the native proteins.
After the antibodies of interest are established, their targets can be identified by immunoprecipitation and mass spectrometry. These antibodies are designed for flow cytometry, immunohistochemistry, immunoprecipitation—in low or non-denaturing conditions—as well as in vivo imaging and therapeutics. For a successful project using this approach, a high throughput screening assay is essential.
Vesicle preparations can also be used when the protein targets are known but native protein folding is essential (in vivo/therapeutic targets). In these cases, the target protein can be overexpressed in the vesicles for more specific antibody production.
Production of Human Monoclonal Antibodies
To circumvent the anti-species response, technology for the direct production of human monoclonal antibodies is being developed. Depending upon the immune status of the donor (patient or subject), antibody secreting B cells can be isolated through a number of methods that are currently available in our facility.
Human monoclonal antibodies are being produced by multiple methods including in vitro immortalization of memory B cell and V gene cloning from antigen specific B cells single cell sorted on a FACS sorter. We use molecular technology to generate V gene clones from the isolated monoclonal antibody producing B cells for the purposes of generating recombinant versions of the antibodies for production, characterization, genetic manipulation, and DNA storage. Sophisticated analysis of antibody specificity and binding is being performed (surface plasmon resonance, etc.) to maintain binding affinity and specificity of cloned molecules.