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

Targeting surface-layer proteins with single-domain antibodies : a potential therapeutic approach against Clostridium difficile-associated disease

Clostridium difficile is a leading cause of death from gastrointestinal infections in North America. Antibiotic therapy is effective, but the high incidence of relapse and the rise in hypervirulent strains warrant the search for novel treatments. Surface layer proteins (SLPs) cover the entire C. difficile bacterial surface, are composed of high-molecular-weight (HMW) and low-molecular-weight (LMW) subunits, and mediate adherence to host cells. Passive and active immunization against SLPs has enhanced hamster survival, suggesting that antibody-mediated neutralization may be an effective therapeutic strategy. Here, we isolated a panel of SLP-specific single-domain antibodies (VHHs) using an immune llama phage display library and SLPs isolated from C. difficile hypervirulent strain QCD-32g58 (027 ribotype) as a target antigen. Binding studies revealed a number of VHHs that bound QCD-32g58 SLPs with high affinity (K D\u2009=\u20093\u20136 nM) and targeted epitopes located on the LMW subunit of the SLP. The VHHs demonstrated melting temperatures as high as 75 \ub0C, and a few were resistant to the gastrointestinal protease pepsin at physiologically relevant concentrations. In addition, we demonstrated the binding specificity of the VHHs to the major C. difficile ribotypes by whole cell ELISA, where all VHHs were found to bind 001 and 027 ribotypes, and a subset of antibodies were found to be broadly cross-reactive in binding cells representative of 012, 017, 023, and 078 ribotypes. Finally, we showed that several of the VHHs inhibited C. difficile QCD-32g58 motility in vitro. Targeting SLPs with VHHs may be a viable therapeutic approach against C. difficile-associated disease.Peer reviewed: YesNRC publication: Ye

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 β-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

Pentavalent single-domain antibodies reduce Campylobacter jejuni motility and colonization in chickens.

Campylobacter jejuni is the leading cause of bacterial foodborne illness in the world, with symptoms ranging from acute diarrhea to severe neurological disorders. Contaminated poultry meat is a major source of C. jejuni infection, and therefore, strategies to reduce this organism in poultry, are expected to reduce the incidence of Campylobacter-associated diseases. We have investigated whether oral administration of C. jejuni-specific single-domain antibodies would reduce bacterial colonization levels in chickens. Llama single-domain antibodies specific for C. jejuni were isolated from a phage display library generated from the heavy chain IgG variable domain repertoire of a llama immunized with C. jejuni flagella. Two flagella-specific single-domain antibodies were pentamerized to yield high avidity antibodies capable of multivalent binding to the target antigen. When administered orally to C. jejuni-infected two-day old chicks, the pentabodies significantly reduced C. jejuni colonization in the ceca. In vitro, the motility of the bacteria was also reduced in the presence of the flagella-specific pentabodies, suggesting the mechanism of action is through either direct interference with flagellar motility or antibody-mediated aggregation. Fluorescent microscopy and Western blot analyses revealed specific binding of the anti-flagella pentabodies to the C. jejuni flagellin

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 conformati

Binding of anti-flagella pentabodies to intact flagellar filament.

<p>Fluorescence microscopy showing FlagV1P (A) and FlagV6P (B) pentabody binding to <i>C. jejuni</i> flagella. Fluorescently labelled FlagV1P and FlagV6P were hybridized with either <i>C. jejuni</i> strain 81–176, 81–176 <i>flaA⁻flaB⁻</i>, or <i>C. jejuni</i> strain 11168. (C) Fluorescence microscopy showing polyclonal anti-81–176 flagella binding to both strains, 81–176 and 11168. Representative fields of view are shown for all images at the same magnification, as indicated by the 5 µm bar.</p

The effect of administration of flagella-specific antibodies on chicken body weight.

<p>Chicken were weighed one day and four days after challenging with <i>C. jejuni</i> alone or <i>C. jejuni</i> followed by pentabody administration. PBS was used as a control and body weights (in grams) were measured at day one and day four. The average body weight ± standard deviation of the values obtained from 28 replicates is shown for each group. No significant differences were found between the groups.</p

Motility assay of <i>C. jejuni</i> strain 81–176 in the absence or presence of pentabodies.

<p><i>C. jejuni</i> 81–176 was incubated for 30 min with (A) buffer, (B) control unrelated pentabody at 0.5 µg/µl, (C) FlagV1P at 0.25 µg/µl, and (D) FlagV1P at 0.5 µg/µl. Images were taken after 53 h of incubation.</p

Motility assays show the cross-reactivity of the pentabodies FlagV1P and FlagV6P with <i>C. coli VC167</i>.

<p>Significant reduction in motility of <i>C. coli</i> was noticed with FlagV6P pentabody. The values were subjected to the Student's <i>t</i>-test for statistical analysis.</p><p>*p value <0.05.</p><p>**p value <0.005.</p