In 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. / text

1 / Yue Sun

Therapeutic 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. / text

The development of safer and more accessible contraceptive options is necessary to reduce the high number of unintended pregnancies worldwide. As monoclonal antibody engineering continues to revolutionize drug development, a variety of strategies are being harnessed to establish antibody-based contraceptives. Human Contraception Antibody (HCA), an immunoglobulin G1 (IgG1) monoclonal antibody that potently agglutinates human sperm, is a promising candidate for nonhormonal immunocontraception in women. Our group recently established the safety and efficacy of a topical IgG1 HCA-formulated dissolvable vaginal film. Though successful, we are currently working to further optimize and improve the HCA product. In this study, we characterized engineered variants of HCA. Bioactivities, specifically agglutination and effector functions, of multimeric and fragment crystallizable (Fc)-mutated variants were compared and inform further engineering of an optimal clinical profile. We then established an atomized mRNA mechanism for delivery of HCA to the female reproductive tract (FRT). The use of mRNA could provide several advantages including: efficiency, reversibility, safety, durability, and cost-effectiveness. mRNA-encoded HCAs were expressed in several models of the FRT and were functional, sperm-specific, and safe. We also analyzed Fc N-glycans at the conserved glycosylation site on IgGs that regulate effector functions and compared the site-specific glycosylation on antibodies generated by two HCA expression platforms of interest, namely Nicotiana benthamiana and mRNA-transfected vaginal cells. Disparities in glycan site occupancy and glycoform populations between the two platforms were observed. Platform-specific HCA glycans resulted in differing levels of sperm phagocytosis, an Fc function. In summary, these studies provide a clearer understanding of engineered variants and delivery platforms to further advance the development of HCA as a novel, antibody-based female contraceptive.

The 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. / text

Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups. / <p>QC 20170828</p>

In the medical and veterinary diagnostic fields there is an ongoing need for stable and specific antibodies. There is also a requirement for simple, robust and cost-effective diagnostic assays to be used in the developing world. Recombinant antibodies from phage displayed libraries are economical to produce and can often be engineered to improve affinity, avidity and stability. While recombinant antibody fragments are useful in immunoassays, they are not strictly comparable to normal immunoglobulins and may under-perform in certain assays. Converting monovalent single-chain antibody fragments (scFvs) to bivalent immunoglobulin-like formats could conceivably provide a more suitable molecular scaffold for use in immunoassays. Two scFvs that recognised the 65 kDa heat-shock protein (HSP65) of Mycobacterium bovis were used in this study. They were originally derived from the Nkuku® repertoire, a phage displayed antibody library based on the immune repertoire of the chicken, Gallus gallus. The genes coding for these scFvs were subcloned in expression vectors containing chicken IgY constant-heavy domains, to create bivalent constructs which were designated ‘gallibodies’. Expression of these constructs was attempted in three heterologous systems. While they were successfully produced in adherent mammalian cell cultures, the growth requirements of these cultures complicated subsequent purification. Bacteria and yeasts were investigated as alternative expression systems, but antibodies were not produced in either system. The gallibodies were compared to their monovalent scFv counterparts for stability as well as their applicability in ELISAs and gold-conjugated immunochromatographic lateral-flow assays. As gallibodies, both retained their functionality after exposure to different conditions and they were capable of immunocapture in ELISA. This was in contrast to their performance as scFvs. Furthermore, these antibody-like molecules could be stably conjugated to colloidal gold and used in lateral flow tests where positive and specific signals were obtained. This confirmed that recombinant single-chain monomeric antibody fragments could be reconstituted as bivalent immunoglobulin-like molecules and that they are a potentially useful platform for developing practical, robust immunodiagnostic reagents. It appeared from these experiments that the antibodies could act as a pair in which one captures, and the other detects HSP65. To find out whether they recognised discrete regions on the protein, their epitopes were mapped using a phage displayed peptide library in combination with computer-based algorithms. The presumptive epitope of one was mapped to residues 350 to 370 on HSP65 of M. bovis. The sequences selected from the peptide library by the other corresponded to three separate regions on the target protein. These recombinant antibody recognition sites are analogous to some of those that have been mapped by others using traditional monoclonal antibodies. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

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京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24304号 / 生博第487号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 渡邊 直樹, 教授 見学 美根子, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.