C. Roger MacKenzie

Transgenic Tobacco Plants Expressing a Dimeric Single-chain Variable Fragment (scFv) Antibody Against Salmonella enterica Serotype Paratyphi B

Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T0) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7xa0μg of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T1 plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T0 plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T1 generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.

Isolation and Characterization of Clostridium difficile Toxin-Specific Single-Domain Antibodies

Camelidae single-domain antibodies (VHHs) are a unique class of small binding proteins that are promising inhibitors of targets relevant to infection and immunity. With VHH selection from hyperimmunized phage display libraries now routine and the fact that VHHs possess long, extended complementarity-determining region (CDR3) loop structures that can access traditionally immunosilent epitopes, VHH-based inhibition of targets such as bacterial toxins are being explored. Toxin A and toxin B are high molecular weight exotoxins (308 kDa and 269 kDa, respectively) secreted by Clostridium difficile that are the causative agents of C. difficile-associated diseases in humans and in animals. Here, we provide protocols for the rapid generation of C. difficile toxin A- and toxin B-specific VHHs by llama immunization and recombinant antibody/phage display technology approaches and for further characterization of the VHHs with respect to toxin-binding affinity and specificity and the conformational nature of their epitopes.

Intracellular Expression of a Single Domain Antibody Reduces Cytotoxicity of 15-Acetyldeoxynivalenol in Yeast

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (VHH) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and VHH immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific VHH within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 μg·ml−1. We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 μg·ml−1. The proof of concept established within this eukaryotic system suggests that in planta VHH expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.

Characterization of single-domain antibodies with an engineered disulfide bond.

Camelidae single-domain antibodies (VHHs) represent a unique class of emerging therapeutics. Similar to other recombinant antibody fragments (e.g., Fabs, scFvs), VHHs are amenable to library screening and selection, but benefit from superior intrinsic biophysical properties such as high refolding efficiency, high solubility, no tendency for aggregation, resistance to proteases and chemical denaturants, and high expression, making them ideal agents for antibody-based drug design. Despite these favorable biophysical characteristics, further improvements to VHH stability are desirable when considering applications in adverse environments like high heat, low humidity, pH extremes, and the acidic, protease-rich gastrointestinal tract. Recently, the introduction of a disulfide bond into the hydrophobic core of camelid VHHs increased antibody thermal and conformational stability. Here, we present additional protocols for characterizing the effects of the introduced disulfide bond on a panel of llama VHHs. Specifically, we employ mass spectrometry fingerprinting analysis of VHH peptides to confirm the presence of the introduced disulfide bond, size exclusion chromatography, and surface plasmon resonance to examine the effects on aggregation state and target affinity, and circular dichroism spectroscopy and protease digestion assays to assess the effects on thermal and proteolytic stability. The disulfide bond stabilization strategy can be incorporated into antibody library design and should lead to hyperstabilized single-domain antibodies (VHHs, VHs), and possibly Fabs and scFvs, if selection pressures such as denaturants or proteases are introduced during antibody selection.

A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies.

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.

Semiautomated panning of naive camelidae libraries and selection of single-domain antibodies against peptide antigens.

With the identification of vast numbers of novel proteins through genomic and proteomic initiatives, the need for efficient processes to characterize and target them has increased. Antibodies are naturally designed molecules that can fulfill this need, and in vitro methodologies for isolating them from either immune or naïve sources have been extensively developed. However, access to pure protein antigens for screening purposes is a major hurdle due to the limitations associated with recombinant production of eukaryotic proteins. Consequently, rational peptide design based on proteomic methodologies such as protein modeling, secondary sequence prediction, and hydrophobicity/hydrophilicity prediction, in combination with other bioinformatics data, is being explored as a viable solution to isolate specific antibodies against difficult antigens. Single-domain antibodies are becoming the ideal antibody format due to their structural advantages and ease of production compared to conventional antibodies and antibody fragments derived from conventional antibodies. For screening purposes, phage display technology is a well-established technique. With this technique, a repertoire of antibody fragments can be displayed on the surface of filamentous phages (f1, fd, M13) followed by screening against various antigenic targets. Furthermore, the technique can be expanded to a high-throughput scale using a magnetic-based, in-solution panning protocol which allows for the screening of multiple target antigens simultaneously. In this chapter, we describe a semiautomated panning method to screen a naïve Camelidae library against rationally designed peptide antigens, followed by preliminary characterization of isolated binders.

Multivalent Display of Single-Domain Antibodies

Antigen-binding fragments, such as single-domain antibodies (sdAbs), can now be readily isolated by in vitro technologies. Antibody fragment libraries derived from immune or nonimmune sources are presented in a molecular display format, typically phage display, and binders to individual antigens are selected from the libraries by a so-called panning process. Nonimmune libraries can serve as sources of binders to a wide range of targets but yield antigen-binding fragments that generally have much lower affinities than those obtained from immune sources. Here we describe a strategy for constructing pentameric sdAbs termed pentabodies. Pentamerization introduces avidity which can greatly enhance the binding of low affinity sdAbs to antigens presented on surfaces.

High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires

The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historically used in detection platforms due to their antigen specificity and robust physicochemical properties. In this study, we report the isolation and characterization of antibody fragments from the heavy chain antibody repertoire (VHH) of Camelidae which bind with specificity and high affinity to the Listeria monocytogenes invasin, Internalin B (InlB). To the best of our knowledge, this is the first report of anti-InlB VHHs from camelids. These anti-InlB VHHs were not cross-reactive to the structurally related Listeria invasin Internalin A (InlA) and are potential reagents to be used in the development of detection and medical technologies.

Antigen recognition by single-domain antibodies: structural latitudes and constraints

ABSTRACT Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional VH:VL antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.

Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries

Human autonomous VH/VL single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from in vitro display libraries constructed via synthetic randomization of rearranged VH/VL domains. Here, we describe the design and characterization of three novel human VH/VL sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential VH/VL sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) in vitro randomization of the CDRs of three VH/VL sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three VH/VL libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 VHs and 7 VLs in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5u2009nM to 12u2009µM (average: 2–3u2009µM), but had highly variable expression yields (range: 0.1–19u2009mg/L). Despite our efforts to identify the most stable VH/VL scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing VH/VL sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some VH/VL sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence clearly dramatically impacts the stability of human autonomous VH/VL immunoglobulin domain folds, and sequence-stability tradeoffs must be taken into account during the design of such libraries.