Antibody Core

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Abstract The core in charge of antibody engineering and production will serve projects #4, as well as Cores #1 and #3. Polyclonal as well as monoclonal antibodies will be produced and distributed. The flow of work will be organized along the lines of 4 specific tasks. Task 1: Produce and characterize anti-glycan polyclonal and monoclonal antibodies. Following immunization, we will use two main approaches to produce antibodies: standard fusion to an immortal partner to produce monoclonal antibodies the “classical” way, and expression of recombinant paired Heavy (H) and Light (L) chains in CHO cells after single B cell antibody chain sequencing (provided by Project #3). Polyclonal antibodies when needed in large quantities will be produced by immunizing large number of animals and regular bleeding. Monoclonal antibodies will be subcloned and isotyped. After purification, antibodies of interest will be characterized for binding to the target glycan by surface plasmon resonance on a Biacore T200. Recombinant IgA and IgM will be produced in insect cells. Task 2: Produce Fab fragments of antibodies for structural studies and others. Crystallization is greatly facilitated by the removal of the Fc fragment of antibodies and the production of Fab fragments. The production of these fragments will be accomplished by two routes. The first one is the proteolytic cleavage of the full-length antibodies with enzymes such as papain, the second one is the transient recombinant expression of paired truncated H - full length L chains in CHO cells. Task 3: Modifications of the Fc fragment to modify effector functions. The antibacterial functions of antibodies are highly dependent on the Fc fragment-associated functions of antibodies. Fc composition determines not only half-life but also complement binding and activation, as well as binding to some bacterial protein such as protein A. Both mouse and human Fc fragments will be grafted and/or mutated to modify the bioactivity of a particular antibody and expressed in a CHO cell recombinant expression system. Task 4: Quality control, storage, and distribution. This particular aim is essential to the success of a core within a large collaborative grant. Electronic archiving of all reagents will be centralized on a single computer and backed up on a separate hard drive as well as on the institutional backup system. For each antibody of interest, immunogen characterization, date of fusion, isotype, binding constants will be presented in a master spreadsheet. H and L sequences will be accessible for antibodies re-expressed from single cell sequencing. DNA and cells will be stored at -80ºC and LN2, respectively. The list of the fully described antibodies will be available on the Website of the PO1 and updated on a regular basis. Distribution within the group will be discussed at our monthly meetings. Outside distribution will follow the institutional rules of MTA agreement.