Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine in the specialty of Virology, June 2018 / The discovery of broad and potent neutralizing HIV antibodies (bNAbs) has opened up new opportunities of passive immunization for HIV-1 prevention. In this study, we have engineered CAP256-VRC26.25, a V1V2 bNAb that neutralizes 70% of clade C viruses, as a single domain antibody (sdAb). These small antigen binding entities are derived from naturally occurring heavy chain only antibodies present in members of the dromedary families, and are characterized by the absence of a light chain, long complementarity-determining regions (CDR) heavy (H) chain 3 and high stability. Since CAP256.25 contains a highly charged and protruding CDR-H3 that binds mainly through its heavy chain, we hypothesized that it may function well as an sdAb. Multiple camelization approaches to engineer CAP256.25 as a sdAb were tested in silico utilising structural modelling software. Parameters such as germline sequence homology, hydrophobicity and solubility, folding energy, torsion angles and native conformation of CAP256.25 in complex with its binding epitope were major factors considered during the modelling process. Four CAP256.25 sdAb derivatives were generated from parental antibody, the mut_0 or a wild type (WT), which was used as a base line for downstream optimization. CAP256.25 mut_4 in which residues involved in LC interactions were replaced with residues strongly conserved in camel sdAbs, which minimize hydrophobic interface of the sdAb. Mut_8 variant, which included four additional substitutions to increase solubility and mut_9 contained a single additional mutation at the base of CDR-H3 to improve the energetic landscape of sdAb. All genes were synthesized and sub-cloned into a mammalian expression vector and recombinant proteins expressed in HEK293T cell line, and purified by Immobilized Metal Ion Affinity Chromatography (IMAC) and Fast Protein Liquid Chromatography (FPLC). CAP256.25_mut0 expression was below the detectable level and whilst mut_4 expressed at low levels, it showed no neutralization activity. CAP256.25 sdAb mut_8 and mut_9 expressed at significantly lower levels compared to m36, a previously described sdAb used a positive control. Nevertheless CAP256.25mut_8 sdAb showed neutralization capability although it lost significant potency in comparison to the parental antibody, yet still within the therapeutic window of the VRC01 bNAb. Importantly, CAP256.25 sdAb was unable to neutralize the K169E mutant confirming that it retained specificity for the V2 epitope. These data suggest that camelization of human antibodies is possible although further engineering is required to increase expression and improve stability. As such, sdAb engineering could be an encouraging step for the generation of small antigen binding fragments for future therapeutic purposes including topical delivery at mucosal surfaces, to interrupt or block sexual transmission of HIV. / XL2018

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.

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.

A ferromagnetic particle with single domain, at some kinds of applied field (at some angle or strangth), the particle's free energy would be two state model. The rate of barrier crossing could be solve by Fokker-Planck equation .And use master equation to find out the Total rate between two potential well. In this thysis, we use the upper method to simulate particle's magnetic moment under time varying magnetic field at fixed angle or fixed magnetic applied rotate the particle. In numerical method, we use the back Euler method to prevent the divergence of the calculation.