Potent enzyme inhibitors derived from dromedary heavy-chain antibodies
1998; Springer Nature; Volume: 17; Issue: 13 Linguagem: Inglês
10.1093/emboj/17.13.3512
ISSN1460-2075
Autores Tópico(s)Transgenic Plants and Applications
ResumoArticle1 July 1998free access Potent enzyme inhibitors derived from dromedary heavy-chain antibodies Marc Lauwereys Corresponding Author Marc Lauwereys Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Mehdi Arbabi Ghahroudi Mehdi Arbabi Ghahroudi Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Aline Desmyter Aline Desmyter Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Jörg Kinne Jörg Kinne Central Veterinary Research Laboratories, PO Box 597, Dubai, United Arab Emirates Search for more papers by this author Wolfgang Hölzer Wolfgang Hölzer Central Veterinary Research Laboratories, PO Box 597, Dubai, United Arab Emirates Search for more papers by this author Erwin De Genst Erwin De Genst Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Lode Wyns Lode Wyns Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Serge Muyldermans Serge Muyldermans Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Marc Lauwereys Corresponding Author Marc Lauwereys Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Mehdi Arbabi Ghahroudi Mehdi Arbabi Ghahroudi Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Aline Desmyter Aline Desmyter Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Jörg Kinne Jörg Kinne Central Veterinary Research Laboratories, PO Box 597, Dubai, United Arab Emirates Search for more papers by this author Wolfgang Hölzer Wolfgang Hölzer Central Veterinary Research Laboratories, PO Box 597, Dubai, United Arab Emirates Search for more papers by this author Erwin De Genst Erwin De Genst Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Lode Wyns Lode Wyns Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Serge Muyldermans Serge Muyldermans Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium Search for more papers by this author Author Information Marc Lauwereys 1, Mehdi Arbabi Ghahroudi1, Aline Desmyter1, Jörg Kinne2, Wolfgang Hölzer2, Erwin De Genst1, Lode Wyns1 and Serge Muyldermans1 1Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium 2Central Veterinary Research Laboratories, PO Box 597, Dubai, United Arab Emirates *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:3512-3520https://doi.org/10.1093/emboj/17.13.3512 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic α-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors. Introduction Enzyme inhibitors, both low-molecular weight compounds and proteinaceous molecules, have emerged as important pharmaceutical agents. Recent advances in molecular biology and protein characterization, as well as the abundant information gathered from the genome sequencing projects, have led to the identification of a steadily growing number of new targets and created the need for the rapid development of specific inhibitors. Strategies to develop such inhibitors are often based on the synthesis of transition state analogues. In a number of cases molecules are identified after the screening of large numbers of chemical compounds or natural sources. The generation of antibody-based molecules forms an obvious alternative. It is widely recognized that the immune system is the preferred tool to generate specific binders or reporter molecules against virtually all agents (Paul, 1993). However, despite the omnipotence of the antibody repertoire, the number of conventional antibodies (i.e. heterotetramers of two light chains and two heavy chains) acting as competitive enzyme inhibitors remains disappointingly low. A satisfactory explanation for this scarce occurrence is given by the incompatible surface topography of the enzyme's active site and the antigen-binding site of conventional antibodies. From a recent survey of enzyme structures it appears that the active site is found almost exclusively in the largest cleft on the protein surface (Laskowski et al., 1996). Likewise, the antigen-binding surface of conventional antibodies forms either a cavity, a groove or flat surface depending on whether an interaction with haptens, oligopeptides or proteins is observed (Webster et al., 1994). It is striking that convex antigen-binding surfaces are not found in conventional antibodies. In this respect, we now demonstrate that functional heavy-chain antibodies from Camelidae behave quite differently in comparison with the conventional four-chain antibodies. More specifically, the heavy-chain antibodies acquired the potential to recognize protein cavities and as such the ability to inhibit enzymes. Camelidae produce an important fraction of their functional immunoglobulins as homodimers of only heavy chains, devoid of light chains (Hamers-Casterman et al., 1993). Specific heavy-chain antibodies can be raised in a dromedary or llama with a variety of protein antigens. The N-terminal variable region of these heavy-chain antibodies (referred to as VHH) contains a minimum-sized antigen-binding domain (Sheriff and Constantine, 1996). The structure of a dromedary VHH in complex with lysozyme revealed the unusual surface topography of the antigen-combining site of this single-domain antibody fragment (Desmyter et al., 1996) and the importance of the CDR3 loop for the binding interaction. The N-terminal part of the 24 amino acid-long CDR3 loop protrudes from the antigen-binding surface and penetrates deeply into the active site of lysozyme. However, this single observation does not allow the claim that the on average long CDR3 loop found in dromedary heavy-chain antibodies (Muyldermans et al., 1994) systematically prefers to interact with antigen clefts and automatically generates competitive inhibitors. In this study we report the immunization of a dromedary with additional enzymes and demonstrate that a substantial proportion of the polyclonal heavy-chain antibodies binds into the active site of the enzymes. The antigen-specific VHHs, binding with nanomolar affinity are easily cloned from the peripheral lymphocytes. We consider that cloning and expression in Escherichia coli of recombinant dromedary VHH antibody fragments is a general and powerful strategy to obtain a new type of potent and specific enzyme inhibitor in a short period of time. Results Two enzymes, porcine pancreatic α-amylase and bovine erythrocyte carbonic anhydrase, were selected to test the generality of producing enzyme-inhibiting dromedary heavy-chain antibodies. Both enzymes are readily available, while inhibitors of small molecular weight and proteinaceous nature are known for each (Vértesy et al., 1984; Alkazaz et al., 1996; Wuebbens et al., 1997); in addition, simple enzymatic activity assays have been described (Pocker and Stone, 1968; Winn-Deen et al., 1988). Specific antibodies in the heavy-chain IgG subclasses Injection of a dromedary with 1 mg of both immunogens every 7 days during a 2-month period resulted in successful immunization as indicated by ELISA (Figure 1A). A sharp increase in titre was observed after three to four injections for both antigens. Compared with the response of porcine α-amylase, a 1-week delay in response was observed for bovine carbonic anhydrase; maximal titre was obtained after 5 weeks and remained constant thereafter for at least an additional 3 weeks. Figure 1.Analysis of antigen-specific antibodies. (A) Presence of antigen-specific antibodies in total serum as a function of time. Antigens were immobilized in microtitre plates and incubated with total serum (at 8000-fold dilution) from different blood samples (days 0,7,14,21,28,35,42,54). α-Amylase- (▪) and carbonic anhydrase- (▴) specific immunoglobulins were subsequently detected with a rabbit anti-dromedary IgG antiserum and anti-rabbit IgG-alkaline phosphatase conjugate. Optical densities were measured after 10 min. (B, C) ELISA experiment with individual immunoglobulin subclasses isolated from serum collected at day 54. Bound IgG1 (▪), IgG2a (▴), IgG2b (▾) and IgG3 (●) to solid-phase coated α-amylase (B) and carbonic anhydrase (C) was detected as described above. (D) Analysis of amylase-binding compounds in serum collected at days 0, 28 and 54. Serum samples (200 μl) were incubated with immobilized α-amylase (50 μl wet gel). After washing, beads were resuspended in 100 μl non-reducing (lanes 1–3) or reducing (lanes 4 and 5) sample buffer and 10 μl aliquots were loaded on 10% SDS–PAGE. Lane 1, day 0; lanes 2 and 4, day 28; lanes 3 and 5, day 54. SDS–PAGE was stained with Coomassie Blue. Download figure Download PowerPoint Four IgG subclasses can be purified from the dromedary serum by differential absorption on Protein A and Protein G. The IgG1 subclass contains the conventional heterotetrameric antibodies composed of two light and two heavy chains, whereas IgG2a, IgG2b and IgG3 are the homodimeric heavy-chain antibodies, devoid of light chains (Hamers-Casterman et al., 1993). Two experiments proved that all dromedary IgG subclasses recognized the antigens. In the first experiment we purified the individual IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) from the sera and tested them individually in a solid-phase ELISA (Figure 1B and C). From the binding curves it was clear that antibodies with specificity for α-amylase or carbonic anhydrase were present in all the IgG subclasses. The prevalence of specific heavy-chain antibodies was confirmed following a separate approach in which total serum was incubated with the native enzymes immobilized on Sepharose. The captured proteins were thereafter analysed on reducing and non-reducing SDS–PAGE (Figure 1D). In the serum collected at days 28 and 54, in addition to the conventional IgG1 antibodies of Mr 160 000 Da, huge amounts of heavy-chain antibodies with Mr of ∼95 000 Da were retained on the beads with immobilized α-amylase (Figure 1D, lanes 2 and 3). As shown in lanes 4 and 5 of Figure 1D, the same samples contain a mixture of two bands (Mr 45 000 and 42 000 Da) under reducing conditions. These are the monomeric heavy chains of the dromedary IgG2 and IgG3, respectively (Hamers-Casterman et al., 1993). The controls in which equivalent amounts of pre-immune serum (day 0) were used, revealed that only minor amounts of proteins were adsorbed onto the beads (Figure 1D, lane 1). Similar results were obtained with carbonic anhydrase (data not shown). Presence of inhibitory antibodies in the heavy-chain subclasses Two experiments were performed to demonstrate that a substantial proportion of the polyclonal heavy-chain antibodies interact specifically with the active site of the enzymes. In a first set of experiments the individual isolated subclasses were tested in a competitive ELISA. It was demonstrated that an important fraction of the antibodies of IgG2a, IgG2b and IgG3 subclasses were prevented from binding to α-amylase in the presence of acarbose, a pseudohexasaccharide competitive inhibitor (Figure 2A). Apparently, ∼50% of the heavy-chain antibodies are displaced upon addition of acarbose, which is known to bind into the active site of α-amylase (Gilles et al., 1996). In contrast, no significant difference in signal is observed with the conventional antibody IgG1 subclass. A similar observation was made for carbonic anhydrase with dorzolamide as competitive inhibitor. Here, the drop in signal is most pronounced in the IgG3 subclass (Figure 2B). Figure 2.Competitive ELISA with small molecular weight inhibitors and enzyme inhibition of α-amylase by dromedary VHH fragments. (A, B) ELISA signals obtained after binding of IgG1, IgG2a, IgG2b and IgG3, isolated from serum collected at day 54, and at two different protein concentrations (0.6 and 0.3 μg/ml), to amylase (A) and carbonic anhydrase (B) in the absence (filled bars) or presence (open bars) of inhibitor. Detection of bound IgG was determined as described in the legend of Figure 1. (C) Inhibition of α-amylase by VHH fragments isolated from serum (collected at day 54). VHH fragments were prepared by limited proteolysis of the IgG3 fraction. Varying amounts of this pool of antibody fragments (range 0–300 μg/ml) were preincubated for 2 h with α-amylase at a concentration of 1.5 μg/ml in a final volume of 50 μl. The residual enzymatic activity (increase OD405/min) was measured after addition of 950 μl 0.2 mM 2-chloro-4-nitrophenyl maltotrioside. Download figure Download PowerPoint In the second approach we tested the enzyme-inhibiting capacity of the heavy-chain antibodies. To avoid immunoprecipitation, we first prepared pure VHHs starting from IgG3 heavy-chain antibodies. IgG3 was chosen as it constitutes the most abundant heavy-chain subclass in dromedaries and llamas (Hamers-Casterman et al., 1993). These VHHs are readily obtained by the limited proteolytic digestion of IgG3 with endo-Glu V8 protease. This enzyme apparently cleaves in the short hinge region between the VHH and the CH2 domain (R.Hamers, in preparation). Undigested IgG3 and the Fc-part were retained on a Protein A column. The flow-through, containing the VHHs, was dialysed and subsequently tested in enzymatic assays. No inhibition could be demonstrated for carbonic anhydrase due to the insensitivity of the colorimetric assay (p-nitrophenylacetate hydrolysis). On the other hand, the cleavage reaction of 2-chloro 4-nitrophenyl maltotriose by porcine α-amylase is a much more sensitive assay. Here, preincubation of the enzyme with the purified polyclonal VHH fraction resulted in a substantial drop of the initial cleavage rate (Figure 2C). A VHH pool prepared from IgG3 of a non-immunized dromedary did not interfere with the enzyme activity. Thus, the observed inhibition is not due to trace amounts of co-purified contaminants or residual protease. In summary, the first experiment revealed the presence of heavy-chain antibodies interacting with the active site of the enzymes, while the second experiment showed that the VHHs of the IgG3 subclass contained enzyme-inhibiting binders. Taken together, these observations strongly suggest that at least part of the VHHs of the IgG3 population are competitive enzyme inhibitors. Isolation of individual VHH binders The VHHs of 107 peripheral blood lymphocytes were cloned in the pHEN4 vector after RT–PCR amplification (Ghahroudi et al., 1997). A VHH library of 5×106 individual clones was obtained and panned for the presence of carbonic anhydrase or α-amylase binders. After the third round of panning, individual colonies were randomly selected and the VHHs produced in their phage-attached form or as soluble protein in a TG1 E.coli strain. For carbonic anhydrase, the detection of the binders in an ELISA with the anti-M13 or with the anti-decapeptide monoclonal antibody revealed that 23 out of the 24 clones were positive. The binding characteristics of these clones, produced as soluble periplasmic protein in TG1 cells, were examined in further detail in a competitive ELISA. In this assay the immobilized enzyme was preincubated with the inhibitor, dorzolamide, before adding the soluble VHH. From the optical reading, it appeared that VHH binding to carbonic anhydrase was inhibited by the presence of this low-molecular weight inhibitor in 14 of the 24 clones tested. For α-amylase, 20 individual clones randomly selected after three rounds of panning with biotinylated α-amylase, were tested in solid-phase ELISA either in their phage-attached form or as soluble protein. The ELISA indicated 17 positive clones out of 20 tested. In order to identify putative active site binders a direct enzyme inhibition assay was used. In this assay an equal volume of a 10-fold diluted periplasmic fraction was preincubated with the enzyme for 30 min. Upon adding the chromogenic substrate, the residual enzymatic activity was determined spectrophotometrically. This allowed us to identify seven out of 20 clones as α-amylase inhibitors. Sequence alignment Sequence analysis of the 23 clones with specificity for carbonic anhydrase revealed that four different VHH fragments were selected, the deduced amino acid sequences of which are compiled in Figure 3. The clones encoding the enzyme-inhibiting camel single-domain antibody (cAb) fragments cAb-CA04, cAb-CA06 and the non-inhibiting cAb-CA05, cAb-CA10 occurred 12, four, four and three times respectively among the 23 selected clones. Figure 3.Amino acid sequence of enzyme binders. Amino acid sequence alignment of α-amylase (cAb-AMxx), carbonic anhydrase (cAb-CAxx), lysozyme (cAb-Lys3) binders along with human POT VH. Numbering and CDRs are according to Kabat et al. (1991). Gaps ‘–’ were inserted in order to have maximal sequence overlap. Download figure Download PowerPoint For the α-amylase binders, the sequence results revealed that only two different inhibitory clones were present. These were referred to as cAb-AMD7 and cAb-AMD9 and occurred four and three times, respectively. Among the non-inhibitory VHH fragments, four different clones were present. The amino acid sequences of the four different carbonic anhydrase binders (two enzyme inhibitors and two non-inhibitors) and the six α-amylase binders (two inhibitors and four non-inhibitors) are aligned along with the human POT VH (Fan et al., 1992) and the dromedary cAb-Lys3 (Figure 3). The cAb-Lys3 is an inhibitor for hen egg-white lysozyme which was identified from a previous immunization experiment (Desmyter et al., 1996; Ghahroudi et al., 1997). It is clear that all isolated single-domain binders are derived from heavy-chain antibodies: they are VHHs and not VHs. Indeed, the substitution of Leu11, Val37, Gly44, Leu45 and Trp47 by Ser11, Phe or Tyr37, Glu44, Arg45 (two clones with Cys45) and mostly Gly47 supports this statement (Muyldermans et al., 1994). It is anticipated that these VHHs are generated by a recombination mechanism from a VHH germline gene (Nguyen et al., 1998). The divergence of the CDR1 and CDR2 region indicates that all binders are derived from different VHH germline genes. This underlines the extended repertoire present within the dromedary heavy-chain antibodies. The average length of the CDR3 of these new binders is 15.1 amino acids. With the notable exception of cAb-AMD9, all the binders contain an additional pair of cysteines (one in the CDR3 and one in either the CDR1 or at position 45). We could not yet allocate the D germline minigenes. Neither did sequence comparison of the dromedary VHH inhibitors with naturally occurring proteinaceous carbonic anhydrase inhibitors or tendamistat reveal any homology. Nor could we detect any specific feature (sequence, CDR length, position of Cys in CDR3) to discriminate the inhibitors from the non-inhibitors. VHH purification and affinity measurements The four carbonic anhydrase and four α-amylase binders (cAb-AMB7, cAb-AMB10, cAb-AMD7 and cAb-AMD9) were selected for detailed characterization. These single-domain binders were expressed in the periplasm of E.coli WK6 as a fusion protein with a C-terminal (His)6 purification tag. The proteins were purified to >95% homogeneity, as judged by SDS–PAGE, by two successive chromatographic steps. All purified proteins remained soluble and were present only in their monomeric form. For all VHH fragments, 1–3 mg pure protein was obtained from a 3 l shakeflask culture. Real-time binding was monitored with an IAsys Biosensor instrument. As the α-amylase lost all of its enzymatic activity upon immobilization to the cuvette, we carried out all binding experiments by immobilizing the purified antibody fragments to the dextran layer. The equilibrium dissociation constant KD for the individual binders was derived from the ratio of the kinetic rate constants (Table I). As shown, the on-rates range from 2.5×104 up to 2.36×105 M−1s−1, whereas dissociation rates as low as 0.0008 s−1 for cAb-AM-D9 were obtained. Combined, this resulted in equilibrium dissociation constants of between 3.5 and 70 nM for all the binders with the exception of cAb-AMD7. Table 1. Kinetic rate constants and equilibrium dissociation constant of α-amylase- (AM) and carbonic anhydrase- (CA) specific antibody fragments as determined with IAsys Biosensor Antibody fragment kon (105 M−1s−1) koff (s−1) KD (nM) AM-B7 1.49 ± 0.19 0.0348 ± 0.0027 232 ± 47 AM-B10 0.48 ± 0.03 0.0011 ± 0.0001 24 ± 4 AM-D7 1.62 ± 0.10 0.0024 ± 0.0003 15 ± 3 AM-D9 2.36 ± 0.38 0.0008 ± 0.0002 3.5 ± 1.4 CA-04 1.32 ± 0.13 0.0039 ± 0.0008 29 ± 9 CA-05 0.64 ± 0.05 0.0050 ± 0.0004 72 ± 11 CA-06 0.72 ± 0.02 0.0014 ± 0.0002 20 ± 3 CA-10 0.25 ± 0.01 0.0011 ± 0.0001 42 ± 6 Enzyme inhibition All carbonic anhydrase-specific VHH fragments were tested for their inhibitory potency in an esterolytic assay. Dorzolamide was developed as an inhibitor for human carbonic anhydrase. We have evidence that the bovine carbonic anhydrase is also inhibited by dorzolamide when tested under identical conditions (IC50 = 3 μM). From our observation that this drug prevented the binding of two VHH fragments (cAb-CA04 and cAb-CA06) in an ELISA assay, we anticipated that both VHH fragments would inhibit the enzymatic activity of carbonic anhydrase. As shown in Figure 4A, cAb-CA04 and cAb-CA06 clearly inhibit the esterolytic activity of bovine erythrocyte carbonic anhydrase. The lower IC50 for cAb-CA06 (1.5 μM) versus cAb-CA04 (2 μM) is in agreement with its higher affinity as determined by IAsys. No inhibition of p-nitrophenylacetate hydrolysis was observed for cAb-CA05 or cAb-CA10, even when tested at concentrations as high as 10 μM. Figure 4.Inhibition of enzyme by recombinant antibody fragments. (A) The bovine erythrocyte carbonic anhydrase was preincubated for 30 min at a fixed concentration of 2.3 μM with varying amounts of cAb-CA04 (▪) or cAb-CA06 (▴) in 60 μl PBS. The residual enzymatic activity (increase OD405/min) was measured after addition of p-nitrophenylacetate. (B) The α-amylase was preincubated for 30 min at a fixed concentration (1.5 μg/ml) of enzyme with varying amounts of cAb-AMD09 (▪) or cAM-D07 (▾) in 150 mM NaCl, 2 mM CaCl2, 50 mM Tris pH 7.4. The residual enzymatic activity (increase OD405/min) was measured after addition of 950 μl 0.2 mM substrate and plotted relative to the velocity measured in the absence of antibody. Download figure Download PowerPoint The crude periplasmic proteins from two out of four selected α-amylase-specific clones inhibited the enzymatic activity of the porcine pancreatic α-amylase. This observation was confirmed with the purified proteins (Figure 4B). Both cAb-AMD9 and cAb-AMD7 appeared to be potent inhibitors (IC50 of 10 and 25 nM, respectively). The order of potency is in agreement with the KD values determined by IAsys biosensor measurements. In contrast, neither cAb-AMB7 nor cAb-AMB10, when tested at micromolar concentration, had any effect on the catalytic efficiency of the enzyme. This proves that the observed inhibition was due to the VHH and not as a result of a co-purified contaminant. For comparison, the IC50 for the α-amylase with the non-antibody inhibitor acarbose was found to be in the order of 5 μM when measured under similar conditions. Discussion In conventional antibodies the antigen-binding site is formed by combining the variable regions of light and heavy chains. Residues present in all six hypervariable regions (three in each domain) may be actively involved in the molecular interaction with the antigen (Padlan, 1994, 1996). They create a sufficiently large surface area which is essentially flat for protein antigens (Webster et al., 1994; Padlan, 1996) (see Figure 5 for an example of a mouse anti-lysozyme binder). Large protruding flexible loops are not encountered frequently at the antigen-binding surface (Wu et al., 1993; Padlan, 1996). These would be immobilized upon antigen interaction and would have a negative influence on the binding energy. Figure 5.Crystallographic model of mouse D1.3 and dromedary cAb-Lys3 antibodies with lysozyme. Ribbon representation of crystal structures of lysozyme (blue) in complex with the mouse D1.3 VH-VL fragments (grey, left of lysozyme) and the dromedary single-domain cAb-Lys3 (grey, below lysozyme). The CDR1, CDR2 and CDR3 of D1.3 VH and VL, shown in red, orange and yellow, respectively form the flat antigen-binding surface characteristic for most protein binders. The lower complexity of the cAb-Lys3 compared with the mouse D1.3 is evident, as well as the large protruding CDR3 loop which penetrates into the active site of lysozyme. The catalytic residue Asp35 of lysozyme is shown in ball-and-stick representation for reference. The pdb file 1VFB of D1.3 (Bhat et al., 1994) and pdb file 1MEL of cAb-Lys3 (Desmyter et al., 1996) in complex with lysozyme were used to generate this figure. The lysozyme of both complexes were superimposed on each other. Both antibodies bind to their original epitope as determined by crystallography. Download figure Download PowerPoint The catalytic amino acids are mostly buried inside a cleft on the enzyme's surface (Laskowski et al., 1996). This part of the molecule is regarded to be of low immunogenicity (Novotny, 1991; Sheriff and Constantine, 1996), which is easy to conceive in view of the failure of conventional antibodies to generate convex antigen-binding sites. Occasionally, conventional antibodies are able to inhibit the enzymatic activity (Bibi and Laskov, 1990); however, these are more the exception than the rule. In contrast, we infer that the situation might be quite different for camelid heavy-chain antibodies. The crystallographic structure of a recombinant dromedary heavy-chain antibody fragment in complex with lysozyme illustrates how the N-terminal part of the long CDR3-loop protrudes from the remaining antigen-binding site (Figure 5; Desmyter et al., 1996). These amino acids are primarily involved in antigen recognition and form an internal image of the lysozyme active site cavity. Biochemical analysis further demonstrated that the antibody fragment inhibits the enzymatic activity of lysozyme in a competitive manner (T.Transue, in preparation). This came as no surprise as seven consecutive amino acids of the CDR3 loop form a structural mimic of the natural carbohydrate substrate of the enzyme. Therefore, the exposed CDR3 loop of dromedary VHHs might be a good candidate to serve as a lead compound for new drugs (Sheriff and Constantine, 1996). The lower complexity of the antigen-binding site in dromedary VHHs, being composed of only three loops versus six loops in Fvs, reduces the problem of choosing the optimal amino acid sequence to derive small molecular recognition units (Sheriff and Constantine, 1996). The generality of a protruding CDR3 loop in camelid heavy-chain antibodies might be questioned upon examination of the structure of a llama VHH fragment (Spinelli et al., 1996). This antibody fragment has a moderate affinity (KD = 300 nM) for the α-subunit of human chorionic gonadotrophin hormone. Here, the first hypervariable loop protrudes due to the presence of amino acid substitutions at key positions for the loop configuration. Hence, the CDR1 loops of VHHs might adopt many as yet unknown exposed con
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