Browsing by Subject "Antibody engineering"
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Item Advancing high-throughput antibody discovery and engineering(2014-05) Kluwe, Christien Alexandre; Ellington, Andrew D.; Georgiou, GeorgeThe development of hybridoma technology nearly forty years ago set the foundation for the use of antibodies in the life sciences. Subsequent advances in recombinant DNA technology have allowed us to adapt antibody genes to various screening systems, greatly increasing the throughput and specialized applications for which these complex biomolecules can be adapted. While selection systems are a powerful tool for discovery and evolution, they can be slow and prone to unintended biases. We see computational approaches as an efficient process for rapid discovery and engineering of antibodies. This is particularly relevant for biodefense and emerging infectious disease applications, for which time is a valuable commodity. In the first chapter of this work, we examine computational protocols for ‘supercharging’ proteins. This process resurfaces the target protein, adding charged moieties to impart specialized functions such as thermoresistance and cell penetration. Current algorithms for resurfacing proteins are static, treating each mutation as an event within a vacuum. The net result is that while several variants can be created, each must be tested experimentally to ensure the resultant protein is functional. In many cases, the designed proteins were severely impaired or incapable of folding. We hypothesize that a more dynamic approach, keeping an eye on energetics and the consequences of mutations will yield a more efficient and robust method for supercharging, successfully adding charges to proteins while minimizing deleterious effects. We continue on this theme applying the successful algorithm to supercharging antibodies for increased function. Utilizing the MS2 model biosensor system, we rationally engineer charges onto the surface of an antibody fragment, increasing thermoresistance, minimizing destabilizing effects, and in some cases actually increasing affinity. Finally, we apply next-generation sequencing approaches to the rapid discovery of antibodies directed against the Zaire Ebolavirus species. We utilize a local immunization strategy to generate a polarized antibody repertoire that is then sequenced to provide a database of antigen-specific variants. This repertoire is probed in silico and individual antibodies selected for analysis, bypassing time- and resource-consuming selection experiments.Item Engineering anti-infective antibodies(2009-12) Rani, Mridula; Iverson, Brent L.; Georgiou, George; Brown, Katherine A.; Maynard, Jennifer A.; Ren, PengyuIn the past 15-20 years, advances in antibody engineering have facilitated the generation and isolation of monoclonal antibodies (mAbs) to a wide array of antigens. Consequently, mAbs have become essential therapeutic tools and currently dominate the global protein therapeutics market. The engineering of anti-infective antibodies, however, has proven quite a challenge, despite the fact that antibodies were naturally evolved to fight infections. The identification of suitable antigens, the mode of administration and the high cost associated with the production of antibody therapeutics are some of the major hurdles for the progress of anti-infective antibodies. This dissertation addresses issues concerning the development of anti-infective antibodies against two different pathogens: SARS coronavirus (CoV) and two pathogenic species of Burkholderia bacteria. To investigate the role of affinity in viral neutralization and evolution of escape mutants, we first sought to isolate an antibody with high affinity towards the receptor binding domain (RBD) of SARS-CoV. Following high-throughput screening of a library of random mutants via the APEx display system, we isolated antibodies with affinities in the range of 0.8 nM - 0.1 nM. The affinity was further improved by additional mutagenesis and DNA shuffling, and a high affinity variant (45pM) with ~300-fold improvement over the parental antibody was isolated. Evaluation of these antibodies in an in vitro assay demonstrated that neutralization of wild-type Urbani strain of SARS-CoV correlates well with the affinity of the antibody, with higher affinity leading to greater neutralization. Moreover, the antibody exhibiting the highest affinity could neutralize SARS-CoV escape mutants that evaded neutralization by both parental and lower affinity antibodies. Another important aspect for the development of anti-infective antibodies concerns the identification of suitable antigen targets to be used in the isolation of antibodies. In an effort to develop a high-throughput screening method for the isolation of antibodies to a wide array of antigens, we used a synthetic antibody (Fab) library constructed by a minimalist approach and displayed on the surface of filamentous bacteriophage. The library was screened against antigens from Burkholderia pseudomallei and Burkholderia mallei. After only three rounds of selection and enrichment against five different antigens, we obtained Fabs specific to four of the antigens as confirmed by ELISA. These results not only demonstrate the use of a synthetic antibody library for the isolation of antibodies against infectious pathogens, but also its feasibility, and potential applicability as a high-throughput screen for a variety of antigens.Item Engineering antibody Fc domains for improved therapeutic function(2013-12) Kelton, William James; Georgiou, GeorgeTherapeutic antibodies have achieved exceptional clinical success in the treatment of cancer and other human diseases. Now, new approaches are required to enhance the potency of antibodies to further increase the number of patients responding to therapy. By engineering the antibody Fc domain through mutation of the amino acid sequence, binding affinity to activating or inhibitory Fc receptors on effector cells can be increased to modulate the cellular immune response. However, attaining selectivity for closely related Fc receptors has proved challenging and the technique has not been applied to access the function of antibody isotypes other than IgG. Here we present new methods for enhancing antibody potency using both hybrid IgA/G and aglycosylated Fc domains. In the first instance, a chimeric antibody Fc domain has been created by combining residues from IgA with those from IgG. The new variant, MutD, introduces binding to FcαRI while retaining affinity for certain members of the FcγR family. ADCC assays show MutD, when part of a full length trastuzumab antibody against Her2 antigen, can kill Her2-overexpressing tumor cell lines as effectively as IgA antibodies. Moreover, MutD shows improved assembly compared to IgA and thus provides access to potent FcαRI function while overcoming the expression and purification barriers that have limited the use of IgA as a therapeutic. Alternatively, aglycosylated antibodies may be engineered for exceptional effector function. Glycans anchored to residue N297 of the antibody IgG Fc domain are typically critical in mediating binding toward the FcγRs. Yet, using a full length bacterial IgG display system, we have isolated aglycosylated Fc1004 with mutations that confer a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous inhibitory receptor, FcγRIIb. Incorporation of this engineered Fc into trastuzumab resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with medium Her2-expressing cancer cells. In vivo testing of Fc1004 using NOD/SCID mouse model, reconstituted by adoptive transfer of leukocytes from FcγRIIa-R131 homozygous donors, showed a promising reduction in tumor burden in SkBr-3 Her2+ xenografts.