Expression of single-domain antibody in different systems

Applied Microbiology and Biotechnology - Camelid single-domain antibodies (sdAbs, VHHs, or Nanobodies®) are types of antibody fragments that are composed of the heavy-chain variable domain...     

Development and Utilization of VHH Antibodies Derived from Camelus Dromedarius Against Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is an acute, highly contagious, and economically devastating viral disease of domestic and wildlife species. For effective implementation of FMD control program, there is an imperative need for developing a rapid, sensitive, and specific diagnostics which help in the identification of serotypes involved in the outbreaks. The humoral immune response of the Camelidae is unique since in these animals 75% of circulating antibodies are constituted by heavy-chain antibodies and 25% are conventional immunoglobulin with two identical heavy chains. In the present study, we developed and characterized FMD virus-specific single-domain heavy-chain antibodies (VHHs) against inactivated whole-virus antigens of FMDV serotypes O (INDR2/1975), A (IND40/2000), and Asia 1 (IND63/1972) vaccine strains. After six rounds of panning and enrichment, these VHHs were stably expressed in Escherichia coli cells. The VHHs directed against outer capsid proteins of FMD virus were successfully utilized as the capture antibody in liquid-phase blocking ELISA (LPBE) thus replacing rabbit coating antibodies. Our study demonstrated the utility of FMD virus-specific VHHs as potential candidates in FMD research and diagnostic application.     

Analysis of idiotope variability as a function of distance from the binding site for anti-streptococcal group A carbohydrate antibodies

We have extended our analysis of rat monoclonal anti-idiotopes (anti-Id) specific for previously mapped binding site-associated (distal) and less-or nonbinding site-associated (proximal) idiotopes on a murine monoclonal anti-streptococcal group A carbohydrate (GAC) antibody. By utilizing other monoclonal anti-GAC antibodies and anti-idiotypic antibodies as radiolabeled probands in both competitive and direct radioimmunoassays, we have detected previously unsuspected reactivities of some of the anti-Id. Although the anti-Id recognizing the most proximal idiotopes manifest relatively narrow ranges of binding strengths for anti-GAC antibodies, the anti-Id recognizing the most distal idiotopes display broader, more continuous distributions of binding strengths. These results suggest that mimicry of antigen structure by anti-Id might best be understood from a quantitative perspective, and that idiotopes intimately associated with binding sites display a broader range of variants than those not associated with binding sites. In addition, for one monoclonal anti-Id recognizing a distal determinant, changing the radiolabeled proband in inhibition radioimmunoassays results in dramatic changes in relative inhibitory efficacies for certain anti-GAC antibody inhibitors. This observation suggests the possibility that this anti-Id represents an example of a multispecific (polyfunctional) anti-idiotypic antibody.     

Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen

The importance of the lymphocyte source to generate hybridomas or to construct antibody gene libraries from which to identify potent monoclonal antibodies is understudied. However, the few comparative studies that exist seem to favor the lymph node tissue as a B-cell source. Here the peripheral blood and lymph node lymphocytes of a dromedary immunized with prostate-specific antigen (PSA) have been employed to clone two independent gene banks of the variable domains of heavy-chain antibodies (i.e. the VHHs). Several PSA-specific VHHs were retrieved after panning of these phage-displayed VHH libraries. Some of them were derived from the same B-cell lineage, possibly reflecting the restricted primary repertoire of heavy-chain antibodies. Other binders originated from different B-cell lineages and apparently converged toward a striking homologous amino acid sequence motif in their CDR3. This illustrates the strong somatic hypermutation and stringent antigen-driven selection ongoing in these animals. Although the various antigen binders exhibit a broad range of kinetic rate constants for their interaction with the PSA, leading to equilibrium constants from 70 pM to 100 nM, no significant difference existed between the binders from the two B-cell sources. The VHHs of both libraries were categorized in three groups based on nonoverlapping epitopes. Some of these VHHs could inhibit and others could enhance the proteolytic activity of the antigen. Remarkably, VHHs seem to sense or induce conformational changes on different PSA isoforms, a feature that might be exploited to study the PSA conformational flexibility and to discriminate the stages of prostate cancer.     

Identification and characterization of species-specific nanobodies for the detection of Listeria monocytogenes in milk

Listeria monocytogenes (LM), one of the eight species belonging to the genus Listeria, is pathogenic for both humans and animals. In this study, two novel LM-specific clones, designated L5-78 and L5-79, were isolated from a phage display antibody library that was derived from the variable domain of heavy-chain antibodies (VHHs) of non-immunized alpaca. These two clones were expressed, purified, and characterized. Results showed that both isolated VHHs recognize three serotypes (1/2a, 1/2b, and 4b), which are responsible for more than 95% of documented human listeriosis cases. The recombinant VHHs possess high thermal stability, pH tolerance, and urea resistance. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the VHH clone L5-79 and a monoclonal antibody was developed to detect LM in pasteurized milk, with a detection limit of 1 × 10⁴ colony-forming units (CFU)/ml. These findings indicated that the species-specific VHHs could be directly isolated from the non-immunized library with a properly designed panning strategy and VHH could be a new source for possible diagnosis/detection of foodborne pathogens in food because it was shown to be highly specific and stable.     

Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase

Camelidae single domain antibodies (VHHs) have structural and binding features that render them suitable alternatives to conventional IgG antibodies. VHHs are usually easier to produce as recombinant proteins than other antibody fragments. However, for some of the biotechnological applications for which they have been proposed, such as immunochromatography and assisted-crystallography, large amounts of purified antibodies are necessary, whereas some VHH-fusions with common tags such as GFP and SNAP are poorly expressed in the bacterial periplasm. Here we have shown that the co-expression of Erv1p sulfhydryl oxidase resulted in an astonishing yield increase of VHH-SNAP constructs expressed in the bacterial cytoplasm. The resulting recombinant antibodies were also more stable than the antibodies produced using the same plasmid, but in wild-type bacteria. Using this approach, it was possible to obtain tens of milligram of purified fusion antibodies using a basic flask fermentation protocol. Therefore, the described method represents a valid solution to produce inexpensively large amounts of single domain antibodies for in vitro applications and we expect it will be suitable for the production of other antibody fragments.     

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs.     

VH-related idiotopes detected by site-directed mutagenesis. A study induced by the failure to find CD4 anti-idiotypic antibodies mimicking the cellular receptor of HIV.

The function of the CD4 cell surface protein as coreceptor on T helper lymphocytes and as receptor for HIV makes this glycoprotein a prime target for an immune intervention with mAb. A detailed understanding of the structural determinants on the therapeutic CD4 mAb that are involved in Ag binding or are recognized by anti-idiotypic mAb (anti-Id) may be important for designing antibodies with optimal therapeutic efficacy. Seven anti-Id raised against the CD4 mAb M-T310 were selected from a large panel with the intention to obtain CD4 mimicking structures with specificity for HIV gp120. The selected anti-Id did not react with other CD4-specific mAb cross-blocking M-T310. Among these, mAb M-T404, although having the same L chain as M-T310 and a VH region sequence differing only at 14 amino acid positions, was not recognized by the anti-Id. M-T310 H chain complexed with the J558L L chain reacted with all anti-Id, thus demonstrating that the recognized idiotopes are located within the VH region. To identify the idiotopes of M-T310 seen by the anti-Id, variants of M-T404 containing one or more of the M-T310-derived substitutions were generated by oligonucleotide-directed mutagenesis. The reactivity pattern of the mutant proteins with the anti-Id demonstrated that the idiotopes reside within the complementarity determining region (CDR) 2 and CDR3 loops of the VH region. A major idiotope was defined by a single amino acid in CDR2 that was recognized by three anti-Id, whereas the four other anti-Id reacted with determinants of CDR3. Although the performed amino acid substitutions did influence the Id recognition, Ag binding was not significantly affected, suggesting that none of the anti-Id can be considered as a mimicry of the CD4 Ag.     

Lateral recognition of a dye hapten by a llama VHH domain

Camelids, camels and llamas, have a unique immune system able to produce heavy-chain only antibodies. Their VH domains (VHHs) are the smallest binding units produced by immune systems, and therefore suitable for biotechnological applications through heterologous expression. The recognition of protein antigens by these VHHs is rather well documented, while less is known about the VHH/hapten interactions. The recently reported X-ray structure of a VHH in complex with a copper-containing azo-dye settled the ability of VHH to recognize haptens by forming a cavity between the three complementarity-determining regions (CDR). Here we report the structures of a VHH (VHH A52) free or complexed with an azo-dye, RR1, without metal ion. The structure of the complex illustrates the involvement of CDR2, CDR3 and a framework residue in a lateral interaction with the hapten. Such a lateral combining site is comparable to that found in classical antibodies, although in the absence of the VL.     

Biological Mimicry of the Bluetongue Virus Core Protein VP7 by Rabbit Anti-Idiotype

A subpopulation of rabbit polyclonal anti-idiotypic antibody (anti-Id) was previously produced to a murine monoclonal antibody (mAb) (M1875) specific for the bluetongue virus core protein VP7. In this report, mimicry of VP7 by this anti-Id (designated RAb2-A) was functionally analyzed through immunization of Balb/c mice with RAb2-A or purified VP7. Animals immunized with RAb2-A were able to produce an M1875-like Ab3 antibody response with idiotype and epitope specificity resembling that of M1875 without subsequent exposure to the nominal antigen. This conclusion was supported by experiments showing that the RAb2-A-induced Ab3 antibodies (i) reacted specifically with the immunizing anti-Id; (ii) were capable of binding VP7; (iii) inhibited M1875 from binding to VP7; and (iv) inhibited M1875 from binding to RAb2-A. Similarly, mice immunized with purified VP7 also produced antibodies that exhibited characteristics such as idiotype and epitope specificity in common with M1875. No antibody response to VP7 was detected in control groups of mice immunized with either normal rabbit IgG or BHK-21 cell components. Therefore, it can be concluded that rabbit anti-Id RAb2-A mimics an M1875-defined VP7 epitope sufficiently to function as a surrogate antigen for inducing an anti-bluetongue virus response.     

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.     

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.     

Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.     

Suppression of murine lupus nephritis by administration of an anti-idiotypic antibody to anti-DNA

The suppression of pathogenic antibodies to DNA in NZB/NZW f1 female mice was achieved by repeated inoculation of the mice with a monoclonal anti-idiotypic antibody (anti-Id). The anti-Id, an IgG1, kappa, was directed against a major cross-reactive idiotype (Id) on NZB/NZW IgG antibodies to DNA. One hundred micrograms of the anti-Id were inoculated i.p. every 2 wk, beginning at 6 wk of age (nondiseased mice--no circulating anti-DNA or proteinuria) or 20 wk of age (diseased mice--all with circulating anti-DNA, one-third with proteinuria). As controls, littermates received an IgG, kappa non-DNA-binding myeloma or no treatment. In the young mice, nephritis and anti-DNA antibodies appeared at the same time in all groups, and their circulating antibodies to DNA did not bear the target Id. In the older (20-wk-old) mice, survival was significantly prolonged because of delay in the onset of nephritis; the total quantities of antibodies to DNA were diminished, and the target Id, initially present on circulating IgG, was deleted. These benefits were transient; the suppression of antibodies was followed by the appearance of large quantities of anti-DNA that did not bear the major Id. Therefore, although administration of anti-Id was effective in reducing an undesirable antibody response after the target Id was present on circulating antibodies, the benefits were limited, probably by Id "switch" or by increased synthesis of pathogenic antibodies bearing a minor Id.     

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange–Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.     

Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins

Antibodies and antibody derived fragments are excellent tools for the detection and purification of proteins. However, only few antibodies targeting Arabidopsis seed proteins are currently available. Here, we evaluate the process to make antibody libraries against crude protein extracts and more particularly to generate a VHH phage library against native Arabidopsis thaliana seed proteins. After immunising a dromedary with a crude Arabidopsis seed extract, we cloned the single-domain antigen-binding fragments from their heavy-chain only antibodies in a phage display vector and selected nanobodies (VHHs) against native Arabidopsis seed proteins. For 16 VHHs, the corresponding antigens were identified by affinity purification and MS/MS analysis. They were shown to bind the major Arabidopsis seed storage proteins albumin and globulin (14 to albumin and 2 to globulin). All 16 VHHs were suitable primary reagents for the detection of the Arabidopsis seed storage proteins by ELISA. Furthermore, several of the anti-albumin VHHs were used successfully for storage protein localisation via electron microscopy. The easy cloning, selection and production, together with the demonstrated functionality and applicability, strongly suggest that the VHH antibody format will play a more prominent role in future protein research, in particular for the study of native proteins.     

A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops

BACKGROUND: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor. RESULTS: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies. CONCLUSIONS: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.     

Tumor idiotype vaccines. VII. Analysis and correlation of structural, idiotypic, and biologic properties of protective and nonprotective Ab2

We have previously generated and used anti-Id mAb (Ab2) to induce protective immunity against the L1210 DBA/2 tumor and for immunotherapy of established tumors. Among various anti-Id that were typed serologically as internal image Ab2 of the mouse mammary tumor virus tumor-associated Ag gp52, only one induced protective immunity and was effective in immunotherapy. In this study we compared the structural, idiotypic, and network properties of the protective and nonprotective antiidiotypic antibodies. The DNA sequence of the variable regions of six anti-Id was determined. The VH sequence of four Ab2, including the protective Ab2, are highly homologous, whereas the VL sequences differ and were assigned to different Vk families. In addition, the DH sequence region of the same four Ab2 are identical, whereas one is highly homologous and another one without homology. Search for amino acid sequence homologies between the Ab2 and gp52 showed the strongest similarities in the CDR2 of the L chain from the protective Ab2. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Id inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there is evidence for differences among the Ab1-Ab2-Ab3 cascade induced by protective and nonprotective anti-Id.     

Carcinoembryonic antigen (CEA) mimicry by an anti-idiotypic scFv isolated from anti-Id 6.C4 hybridoma

Since carcinoembryonic antigen (CEA) is expressed during embryonic life, it is not immunogenic in humans. The use of anti-idiotypic (Id) antibodies as a surrogate of antigen in the immunization has been considered a promising strategy for breaking tolerance to some tumor associated antigens. We have described an anti-Id monoclonal antibody (MAb), designated 6.C4, which is able to mimic CEA functionally. The anti-Id MAb 6.C4 was shown to elicit antibodies that recognized CEA in vitro and in vivo. In the present study, we sought to verify whether a single chain (scFv) antibody obtained, the scFv 6.C4, would retain the ability to mimic CEA. Two scFv containing the variable heavy and light chain domains of 6.C4 were constructed with a 15-amino acid linker: one with and another without signal peptide. DNA immunization of mice with both forms of scFv individually elicited antibodies able to recognize CEA.