Llama DNA Immunization and Isolation of Functional Single-Domain Antibody Binders.

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.

Biparatopic single-domain antibodies against Axl achieve ultra-high affinity through intramolecular engagement.

Overexpression of Axl, a TAM-family receptor tyrosine kinase, plays key roles in the formation, growth, and spread of tumors as well as resistance to targeted therapies and chemotherapies. We identified novel llama VHHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity VHHs. The VHHs targeted multiple sites in Ig-like domains 1 and 2 of the Axl extracellular domain, including an immunodominant epitope overlapping the site of Gas6 interaction and two additional non-Gas6 competitive epitopes recognized by murine monoclonal antibodies. Only a subset of VHHs cross-reacted with cynomolgus monkey Axl and none recognized mouse Axl. As fusions to human IgG1 Fc, VHH-Fcs bound Axl+ tumor cell lines and mertansine-loaded VHH-Fcs were cytotoxic in vitro against Axl+ cells in proportion to their binding affinities. Engineered biparatopic VHH-VHH heterodimers bound Axl avidly, and a subset of molecules showed dramatically enhanced association rates indicative of intramolecular binding. These VHHs may have applications as modular elements of biologic drugs such as antibody-drug conjugates.

Incorporation of a Novel CD16-Specific Single-Domain Antibody into Multispecific Natural Killer Cell Engagers With Potent ADCC.

Multispecific antibodies that bridge immune effector and tumor cells have shown promising preclinical and clinical efficacies. Here, we isolated and characterized novel llama single-domain antibodies (sdAbs) against CD16. One sdAb, NRC-sdAb048, bound recombinant human and cynomolgus monkey CD16 ectodomains with equivalent affinity (KD: 1 nM) but did not recognize murine CD16. Binding was similar for human CD16a expressed on NK cells and CD16b (NA2) expressed on neutrophils but dramatically weaker (KD: ∼6 µM) for the CD16b (NA1) allotype. The sdAb stained primary human peripheral blood NK cells. Irrespective of fusion orientation and linker length, bispecific sdAb-sdAb and sdAb-scFv dimers (anti-CD16/EGFR, anti-CD16/HER2, and anti-CD16/CD19) retained full binding affinity for each target, coengaged both antigens simultaneously, elicited ADCC against target antigen-expressing tumor cells in a reporter bioassay, and triggered target-specific activation and degranulation of primary NK cells as measured via interferon-γ and CD107a expression. These molecules may have applications in cancer immunotherapy.

Serum albumin-binding VH Hs with variable pH sensitivities enable tailored half-life extension of biologics.

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VH Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VH H (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VH Hs maintained in vivo half-life extension capabilities. These VH Hs represent a new set of tools for extending protein therapeutic half-life and one (M75) demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life.

Antibody Binding to the O-Specific Antigen of Pseudomonas aeruginosa O6 Inhibits Cell Growth.

Pseudomonas aeruginosa is an opportunistic pathogen that is inherently resistant to many antibiotics and represents an increasing threat due to the emergence of drug-resistant strains. There is a pressing need to develop innovative antimicrobials against this pathogen. In this study, we identified the O-specific antigen (OSA) of P. aeruginosa serotype O6 as a novel target for therapeutic intervention. Binding of monoclonal antibodies and antigen-binding fragments therefrom to O6 OSA leads to rapid outer membrane destabilization and inhibition of cell growth. The antimicrobial effect correlated directly with antibody affinity. Antibody binding to the O antigen of a second lipopolysaccharide (LPS) type present in P. aeruginosa or to the LPS core did not affect cell viability. Atomic force microscopy showed that antibody binding to OSA resulted in early flagellum loss, formation of membrane blebs, and eventually complete outer membrane loss. We hypothesize that antibody binding to OSA disrupts a key interaction in the P. aeruginosa outer membrane.

Role of the non-hypervariable FR3 D-E loop in single-domain antibody recognition of haptens and carbohydrates.

Single-domain antibodies (sdAbs), the variable domains of camelid heavy chain-only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti-methotrexate, anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop, formed between ß-strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15-acetyl-deoxynivalenol (15-AcDON), and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb, NAT-267, was extended and randomized. From this library, we identified one sdAb, MA-232, bearing a 14-residue insertion in CDR4 and showing improved binding to 15-AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage-displayed libraries in which the CDR4 and other regions of three hapten- or carbohydrate-binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan-binding specificities, we panned the library against four tumor-associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten-specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired VL domain.

Llama peripheral B-cell populations producing conventional and heavy chain-only IgG subtypes are phenotypically indistinguishable but immunogenetically distinct.

Camelid ungulates produce homodimeric heavy chain-only antibodies (HCAbs) in addition to conventional antibodies consisting of paired heavy and light chains. In the llama, HCAbs are made up by at least two subclasses (long-hinge IgG2b and short-hinge IgG2c HCAbs vs. conventional heterotetrameric IgG1s). Here, we generated murine monoclonal antibodies (mAbs) specific for the hinge-CH2 boundary of llama IgG2b (mAb 1C10) and the Fc of llama IgG2c HCAbs (mAb 5E4). Flow cytometric analysis of llama peripheral blood lymphocytes revealed that IgG1+, IgG2b+ and IgG2c+ B cells could be distinguished using mAbs 1C10/5E4 but had equivalent expression of three other cell-surface markers. MiSeq sequencing of the peripheral B cell repertoires of three llamas showed that (i) IgG2b and IgG2c HCAbs were present in similar proportions in the repertoire, (ii) a subset of IgG2b and IgG2c HCAbs, but not IgG1s, entirely lacked a hinge exon and showed direct VHH-CH2 splicing; these "hingeless" HCAbs were clonally expanded, somatically mutated and derived from hinged HCAb precursors, (iii) substantial repertoire overlap existed between IgG subclasses, especially between IgG2b and IgG2c HCAbs, (iv) the complementarity-determining region (CDR)-H3 length distributions of IgG2b and IgG2c HCAbs were broader and biased towards longer lengths compared with IgG1s due to increased N-nucleotide addition, (v) IgG2b and IgG2c HCAbs used a more restricted set of IGHV genes compared with IgG1s, and (vi) IgG2b and IgG2c HCAbs had elevated somatic mutations rates of both CDRs and framework regions (FRs) compared with IgG1s, especially of CDR-H1 and FR3. The distinct molecular features of llama IgG1, IgG2b and IgG2c antibodies imply that these subclasses may have divergent immunological functions and suggest that specific mechanisms operate to diversify HCAb repertoires in the absence of a light chain.

Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling.

Up-regulation of epidermal growth factor receptor (EGFR) is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here, we report the isolation and functional characterization of novel camelid single-domain antibodies (sdAbs or VHHs) directed against human EGFR. The source of these VHHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost), which is notable in that genetic immunization of large, outbred animals is generally poorly effective. The VHHs targeted multiple sites on the receptor's surface with high affinity (KD range: 1-40 nM), including one epitope overlapping that of cetuximab, several epitopes conserved in the cynomolgus EGFR orthologue, and at least one epitope conserved in the mouse EGFR orthologue. Interestingly, despite their generation against human EGFR expressed from cDNA by llama cells in vivo (presumably in native conformation), the VHHs exhibited wide and epitope-dependent variation in their apparent affinities for native EGFR displayed on tumor cell lines. As fusions to human IgG1 Fc, one of the VHH-Fcs inhibited EGFR signaling induced by EGF binding with a potency similar to that of cetuximab (IC50: ∼30 nM). Thus, DNA immunization elicited high-affinity, functional sdAbs that were vastly superior to those previously isolated by our group through protein immunization.

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.

Neutralization of Clostridium difficile toxin B with VHH-Fc fusions targeting the delivery and CROPs domains.

An increasing number of antibody-based therapies are being considered for controlling bacterial infections, including Clostridium difficile by targeting toxins A and B. In an effort to develop novel C. difficile immunotherapeutics, we previously isolated several single-domain antibodies (VHHs) capable of toxin A neutralization through recognition of the extreme C-terminal combined repetitive oligopeptides (CROPs) domain, but failed at identifying neutralizing VHHs that bound a similar region on toxin B. Here we report the isolation of a panel of 29 VHHs targeting at least seven unique epitopes on a toxin B immunogen composed of a portion of the central delivery domain and the entire CROPs domain. Despite monovalent affinities as high as KD = 70 pM, none of the VHHs tested were capable of toxin B neutralization; however, modest toxin B inhibition was observed with VHH-VHH dimers and to a much greater extent with VHH-Fc fusions, reaching the neutralizing potency of the recently approved anti-toxin B monoclonal antibody bezlotoxumab in in vitro assays. Epitope binning revealed that several VHH-Fcs bound toxin B at sites distinct from the region recognized by bezlotoxumab, while other VHH-Fcs partially competed with bezlotoxumab for toxin binding. Therefore, the VHHs described here are effective at toxin B neutralization when formatted as bivalent VHH-Fc fusions by targeting toxin B at regions both similar and distinct from the bezlotoxumab binding site.

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

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.

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display.

ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.

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 5 nM to 12 µM (average: 2-3 µM), but had highly variable expression yields (range: 0.1-19 mg/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.

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.

Isolation of TGF-ß-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing.

The epitope specificity of therapeutic antibodies is often critical to their efficacy and mode of action. Here, we report the isolation of single-domain antibodies (sdAbs) against a pre-specified epitope of TGF-ß3: namely, the site of interaction between the cytokine and its cell-surface type II receptor. By panning a phage-displayed immune llama VhH library against TGF-ß3 using competitive elution with soluble dimeric type II receptor ectodomain in tandem with next-generation DNA sequencing, we identified several sdAbs that competed with the receptor for TGF-ß3 binding and neutralized TGF-ß3 in in vitro cellular assays. In contrast, all other sdAbs identified using conventional panning approaches (i.e., without regard to epitope specificity) did not target the site of receptor:cytokine interaction. We expect this strategy to be generally applicable for identifying epitope-specific sdAbs when binding reagents directed against the epitope of interest are available. The sdAbs identified here are of potential interest as cancer immunotherapeutics.

Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.

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 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.

In situ proteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B.

The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a VHH). The VHH R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface-exposed and is required for virulence, hence a VHH targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X-ray diffraction of R303 are reported. Crystals of R303 were obtained following in situ proteolysis with trypsin. Gel filtration and SDS-PAGE revealed that trypsin removed the C-terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a=46.4, b=31.2, c=74.8 Å, ß=93.8°. The crystals exhibited a Matthews coefficient of 1.95 Å3 Da(-1) with two molecules in the asymmetric unit.