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Genetic immunization is a simple, cost-effective, and powerful tool for inducing innate and adaptive immune responses to combat infectious diseases and difficult-to-treat illnesses. DNA immunization is increasingly used in the generation of monoclonal antibodies against targets for which pure proteins are unavailable or are difficult to express and purify (e.g., ion channels and receptors, transmembrane proteins, and emerging infectious pathogens). Genetic immunization has been successfully utilized in small inbred laboratory animals (mostly rodents); however, low immunogenicity of DNA/RNA injected into large mammals, including humans, is still a major challenge. Here, we provide a method for the genetic immunization of llamas, using a combination of biolistic transfection with a gene gun and intradermal injection with a DERMOJET® device, to elicit heavy-chain IgG responses against epidermal growth factor receptor (EGFR). We show the technique can be used to generate single-domain antibodies (VHHs) with nanomolar affinities to EGFR. We provide methods for gene gun bullet preparation, llama immunization, serology, phage-display library construction and panning, and VHH characterization., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.

High avidity, pentameric, single-domain antibodies, oligomerized through the B subunit of verotoxin, are excellent immunohistochemical reagents. The resulting molecules are termed pentabodies. Here, we describe the immunostaining of tissue sections with ES1, a pentabody recognizing CEACAM6 which is overexpressed in several cancers. The advantages of pentabodies, compared to conventional antibody reagents, in immunohistochemical studies are highlighted.

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.

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.

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.

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.