The Application of Monoclonal Therapies and Therapeutics to Asthma and Allergy
Designer Drug Design: How are Monoclonal Antibodies Made in 2008?
Shyam S. Mohapatra, PhD FAAAI
Mabel & Ellsworth Simmons Professor of Allergy and Immunology
VA Hosp & University of South Florida College of Medicine
Tampa, FL, United States
Since Paul Ehrlich, who first presented the concept that antibodies could be exploited in therapy, it took four decades before technological advances allowed the exploration of the potential of antibodies for immunotherapeutic applications. In the late 70s, Nobel laureates Kohler and Milstein introduced the monoclonal antibody technology, which was further revolutionized by advances in DNA technology, that led to the ability to tailor and manipulate the immunoglobulin molecule for specific functions and in vivo properties.
The ‘state of the art technology’ includes combinatorial chemistry, DNA mutagenesis, and the ability to fuse “display and selection” systems in the same setting; which allow physical linking of the mutated gene with its encoded proteins, making easier the recovery of the antibody with the desired properties and specificities. They enable conversion of a mousederived monoclonal complete antibody (Fc-[Fab]2) to different combinations or designs of “humanized” versions. These tools allow construction of antibodies comprising just the portion of the antibody with the antigen-binding property made up of both a heavy and light chain (Fv, single chain Fv – scFv) to larger engineered arrangements resulting from the multimerization of these Fv or scFv formats (diabodies, triabodies, tetrabodies). These same tools permit the construction of antibodies or antibody fragments having multiple specificity, which potentially favour the recognition of more than one antigen, the increase in the avidity of the antibody, the cross-activation of immune cells, or bridging an immune cell with its target. The engineering of these formats increase their affinity, stability and clearance time, reduce complications associated to undesirable immune reactions and make the new antibody versions carry drugs to their intended targets. Furthermore, current in vitro antibody generating systems utilize ribosomal display, phage display, yeast surface display, and mammalian cell display, which are capable of generating large and diverse libraries of clones (1010) expressing several combinations of Fv encoding genes. Depending on the display system, robotics or flow cytometry have facilitated the handling of many different clones to select those expressing the Fv with promising affinity properties. The Fv-encoding genes are mutated using directed mutagenesis approaches – CDR walking, windows mutagenesis, site-directed mutagenesis, hotspots CDR, and site saturation mutagenesis – or random mutagenesis approaches – error prone PCR, EvoGene, and DNA shuffling – or a combination of both. Moreover, a plethora of in vivo technologies allow antibody engineering at the level of single cells such as Escherichia coli, mammalian cells or whole animals. The success of an application of these technologies is evident from the recent approval by FDA of the Panitumumab, a fully human antibody directed against the epidermal growth factor receptor, which is obtained from transgenic mice expressing human antibody repertoires. These technological advances combined with the FDA fast-tracking policy are expected to expand the application of these new formats both in diagnostic kits for disease biomarkers and in therapeutic scenarios.