Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Multiple sclerosis is a neurodegenerative disorder affecting the central nervous system (CNS). Currently, the disease is incurable and immunomodulating drugs are the only option to control the disease. CD49d is an adhesion receptor expressed on most immune cells. Antibodies that bind to CD49d and block immune cells from trafficking toward the CNS are being pursued as one class of therapeutics. In this work, by combining recombinant antibody and phage display technologies we isolated 10 anti-CD49d single domain antibodies from a synthetic antibody light chain variable domain (VL) phage display library. Isolated VLs (~ 12 kDa) were expressed in Escherichia coli, purified and analysed for biophysical characteristics. The majority were expressed in good yields and were non-aggregating. All 10 VLs bound recombinant CD49d by ELISA, and 7 bound to CD49d-expressing cells in flow cytometry experiments. To empower the VLs for better therapeutic efficacy (thru increasing avidity and half-life), three of the lead VLs were re-engineered as fusions to fragment crystallisable (Fc) of human immunoglobulin gamma (IgG). The engineered hFc-VL fragments (~ 70 – 90 kDa) retained their specificity for CD49d by flow cytometry. With (i) being less immunogenic due to their human nature, (ii) their efficient access to cryptic epitopes (iii) having half-lives comparable to IgGs’ and (iv) being more cost effective compared to IgGs, these novel antibody fragments (monovalent VLs and bivalent hFc-VLs) provide a promising therapeutic platform against multiple sclerosis.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

In recent years a number of therapeutically useful immunotoxins have been produced using recombinant gene technology. In general, this involves fusion of a toxin gene with sequence encoding a variety of clinically relevant proteins or peptides. Using these techniques a recombinant immunotoxin has been engineered by fusing the genes encoding an antibody fragment with the sequence of a small cytolytic peptide, melittin. The antibody fragment consists of the antigen binding site derived from a murine monoclonal antibody K- 1-21, which binds to human free kappa light chains and recognises a specific epitope (KMA) expressed on the surface of human myeloma and lymphoma cells. The toxic portion of the molecule is melittin, a 26 amino acid, membrane lytic peptide which is a major component of bee venom. Using PCR a single chain Fv (scFv) was constructed by linking VH and VL genes with an oligonucleotide encoding a flexible, hydrophilic peptide. The melittin gene was synthesised as an oligonucleotide and extended by PCR. Nucleotide sequence encoding a linker peptide was added to the 5' end and a primer encoding a FLAG peptide was used to extend the 3' end. This gene construct was then ligated into the recombinant expression vector, pPOW scFv, to create the fusion gene encoding the recombinant immunotoxin. The gene construct was expressed in the periplasm of E.coli (TOPP2) using the secretion signal pelB . Expression of the foreign protein was monitored by western blot using a monoclonal antibody which recognises the FLAG peptide encoded at the carboxy terminal region of the gene construct. Expression of the recombinant immunotoxin was optimised and the resulting protein was purified using anti-FLAG M2 affinity chromatography. Antigen binding activity was assessed by ELISA and flow cytometry using a human myeloma cell line, HMy2, which expresses the KMA antigen.Binding of the immunotoxin to a control human cell line, K562, which does not express KMA on the cell surface was also assessed. The results indicated that the recombinant immunotoxin retained antigen binding specificity and it was cytotoxic towards the target cell line (HMy2).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Dynamics of antigens & antibodies. Includes bibliographical references (214-230). Also available online through Digital Dissertations.

Afhandling--Copenhagen. / Summary in Danish.

Thesis (Ph. D.)--University of Wisconsin, 1953. / Typescript (carbon copy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [38]-40).

Afhandling--Copenhagen. / Summary in Danish.