There are great demands on the stability, expression yield and resistance to aggregation of antibody fragments. To untangle intrinsic domain effects from domain interactions, we present first a systematic evaluation of the isolated human immunoglobulin variable heavy (V(H)) and light (V(L)) germline family consensus domains and then a systematic series of V(H)-V(L) combinations in the scFv format. The constructs were evaluated in terms of their expression behavior, oligomeric state in solution and denaturant-induced unfolding equilibria under non-reducing conditions. The seven V(H) and seven V(L) domains represent the consensus sequences of the major human germline subclasses, derived from the Human Combinatorial Antibody Library (HuCAL). The isolated V(H) and V(L) domains with the highest thermodynamic stability and yield of soluble protein were V(H)3 and V(kappa)3, respectively. Similar measurements on all domain combinations in scFv fragments allowed the scFv fragments to be classified according to thermodynamic stability and in vivo folding yield. The scFv fragments containing the variable domain combinations H3kappa3, H1bkappa3, H5kappa3 and H3kappa1 show superior properties concerning yield and stability. Domain interactions diminish the intrinsic differences of the domains. ScFv fragments containing V(lambda) domains show high levels of stability, even though V(lambda) domains are surprisingly unstable by themselves. This is due to a strong interaction with the V(H) domain and depends on the amino acid sequence of the CDR-L3. On the basis of these analyses and model structures, we suggest possibilities for further improvement of the biophysical properties of individual frameworks and give recommendations for library design.
In a systematic study of V gene families carried out with consensus V(H) and V(L) domains alone and in combinations in the scFv format, we found comparatively low expression yields and lower cooperativity in equilibrium unfolding in antibody fragments containing V(H) domains of human germline families 2, 4, and 6. From an analysis of the packing of the hydrophobic core, the completeness of charge clusters, the occurrence of unsatisfied hydrogen bonds, and residues with low beta-sheet propensities, positive Phi angles, and exposed hydrophobic side chains, we pinpointed residues potentially responsible for the unsatisfactory properties of these germline-encoded sequences. Several of those are in common between the domains of the even-numbered subgroups, but do not occur in the odd-numbered ones. In this study, we have systematically exchanged those residues alone and in combination in two different scFvs using the V(H)6 framework, and we describe their effect on equilibrium stability and folding yield. We improved the stability by 20.9 kJ/mol and the expression yield by a factor of 4 and can now use these data to rationally engineer antibodies derived from this and similar germline families for better biophysical properties. Furthermore, we provide an improved design for libraries exploiting the significant additional diversity provided by these frameworks. Both antibodies studied here completely retain their binding affinity, demonstrating that the CDR conformations were not affected.
Kaufmann, M., Lindner, P., Honegger, A., Blank, K., Tschopp, M., Capitani, G., Plückthun, A., Grütter, M. G.
The crystal structure of a mutant form of the single-chain fragment (scFv), derived from the monoclonal anti-His tag antibody 3D5, in complex with a hexahistidine peptide has been determined at 2.7 A° resolution. The peptide binds to a deep pocket formed at the interface of the variable domains of the light and the heavy chain, mainly through hydrophobic interaction to aromatic residues and hydrogen bonds to acidic residues. The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole sidechains. The crystals have a solvent content of 77% (v/v) and form 70 A° - wide channels that would allow the diffusion of peptides or even small proteins. The anti-His scFv crystals could thus act as a framework for the crystallization of His-tagged target proteins. Designed mutations in framework regions of the scFv lead to high-level expression of soluble protein in the periplasm of Escherichia coli. The recombinant anti-His scFv is a convenient detection tool when fused to alkaline phosphatase. When immobilized on a matrix, the antibody can be used for affinity purification of recombinant proteins carrying a very short tag of just three histidine residues, suitable for crystallization. The experimental structure is now the basis for the design of antibodies with even higher stability and affinity.
Auf der Maur, A., Zahnd, C., Fischer, F., Spinelli, S., Honegger, A., Cambillau, C., Escher, D., Plückthun, A., Barberis, A.
Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.
Immunoglobulin VH domain frameworks can be grouped into four distinct types, depending on the main-chain conformation of Framework 1. Based on the analysis of over 200 X-ray structures representing more than 100 non-redundant VH domain sequences, we have come to the conclusion that the marked structural variability of the VH Framework 1 region is caused by three residues: the buried side chain of H6, which can be either a glutamate or a glutamine residue, the residue in position H7, which may only be a proline if H6 is a glutamine, and by H9 (H10 according to a new consensus nomenclature, Honegger & Plückthun, J. Mol. Biol. 2000)), which has to be either a glycine or a proline if H6 is a glutamate. In natural antibodies, these three residues are encoded in combinations which are compatible with each other and with the rest of the structure and therefore will yield functional molecules. However, the degenerate primer mixtures commonly used for PCR cloning of antibody fragments can and frequently do introduce out-of-context mutations to combinations which can lead to severe reduction of stability, production yield and antigen affinity.
A common residue numbering scheme for all immunoglobulin variable domains (immunoglobulin light chain lambda (Vl) and kappa (Vk) variable domains, heavy chain variable domains (VH) and T-cell receptor alpha (Va), beta (Vb), gamma (Vg) and delta (Vd) variable domains) has been devised. Based on the spatial alignment of known three-dimensional structures of immunoglobulin domains, it places the alignment gaps in a way which minimizes the average deviation from the averaged structure of the aligned domains. This residue numbering scheme was applied to the immunoglobulin variable domain structures in the PDB database to automate the extraction of information on structural variations in homologous positions of the different molecules. A number of methods are presented which allow the automated projection of information derived from individual structures or from the comparison of multi-structure alignments onto a graphical representation of the sequence alignment.
Burmester, J., Spinelli, S., Pugliese, L., Krebber, A., Honegger, A., Jung, S., Schimmele, B., Cambilleau, C. and Plückthun, A.
Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an ampicillin-bovine serum albumin conjugate. Several antibodies with specificity for intact ampicillin were selected by phage display and characterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyzed ampicillin. We determined the X-ray structures of two crystal forms of w.t. aL2, which mainly differ in the side-chain conformation of Trp H109 according to a new consensus nomenclature (Honegger & Plückthun, J. Mol. Biol. 2000; Kabat residue number H95) in the extremely short (3 residues) CDR H3 and the presence or absence of a well-resolved molecule of 2-methyl-pentane-2,4-diol (MPD) in the bottom of the binding pocket. Attempts to co-crystallize aL2 with its antigen or to diffuse ampicillin into the wild-type aL2 crystals were unsuccessful, since crystal contacts obstruct the binding pocket. However, a mutant with two point mutations near the N-terminus (Gln H6 replaced by Glu and Ala H10 (Kabat H9) replaced by Gly) crystallized in a form compatible with antigen binding. Although the mutations affect the conformation of framework I, the conformations of the binding pocket of the uncomplexed w.t. aL2 and of the mutant complex were almost identical. The structure explains the specificity of the antibody for intact ampicillin and the degree of cross-reactivity of aL2 with a wide variety of ampicillin analogs. This antibody system will be very useful as a diagnostic reagent for antibiotics use and abuse, as a model for the effect of expression of antibiotic binding molecules in E. coli, and for directed evolution towards high antibiotic resistance.
Jung, S., Spinelli, S., Schimmele, B., Honegger, A., Pugliese, L., Cambilleau, C. and Plückthun, A.
The N-terminal segment (FR-H1) of the heavy chain (VH) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to important biophysical properties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (Kabat H9). For the aL2 wildtype (w.t.) with the sequence 6Q7P10A and for two of the mutants, the X-ray structures have been solveddetermined. The structure of the triple mutant aL2-6E7S10G shows the FR-H1 backbone conformations predicted for this amino acid combination, which is distinctly different from the structure of the w.t, thus proving supporting our hypothesis that these residues determine the conformation of this segment. The mutant aL2-6E7P10G represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique structure in the first b-strand of VH, not observed in natural Fv fragments and forms a novel type of diabody. Two VH domains of the mutant associate by swapping the first b-strand. Concentration dependent changes in Trp fluorescence indicate that this dimerization is not just an artefact of crystallization, but occurs also under normal experimental conditions. The mutations in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization behavior of the scFv and its thermodynamic stability. All the observations reported here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR grafting or synthetic libraries.
We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (k(off) at 4 degrees C of 10(-4) s(-1)) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible.
Schaer, D. J., Boretti, F. S., Honegger, A., Poehler, D., Linnscheid, P., Staege, H., Muller, C., Schoedon, G., Schaffner, A.
Schwesinger, F., Ros, R., Strunz, T., Anselmetti, D., Guntherodt, H. J., Honegger, A., Jermutus, L., Tiefenauer, L. and Plückthun, A.
Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.
Wörn, A., Auf der Maur, A., Escher, D., Honegger, A., Barberis, A. and Plückthun, A.
A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.
Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Plückthun, A. and Virnekas, B.
By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.
Langedijk, A. C., Spinelli, S., Anguille, C., Hermans, P., Nederlof, J., Butenandt, J., Honegger, A., Cambillau, C. and Plückthun, A.
Monoclonal antibodies were elicited against the small hydrophobic hapten traseolide, a commercially available musk fragrance. Antibody variable region sequences were found to belong to different sequence groups, and the binding characteristics of the corresponding antibody fragments were investigated. The antibodies M02/01/01 and M02/05/01 are highly homologous and differ in the binding pocket only at position H93. M02/05/01 (H93 Val) binds the hapten traseolide about 75-fold better than M02/01/01 (H93 Ala). A traseolide analog, missing only one methyl group, does not have the characteristic musk odorant fragrance. The antibody M02/05/01 binds this hapten analog about tenfold less tightly than the original traseolide hapten, and mimics the odorant receptor in this respect, while the antibody M02/01/01 does not distinguish between the analog and traseolide. To elucidate the structural basis for the fine specificity of binding, we determined the crystal structure of the Fab fragment of M02/05/01 complexed with the hapten at 2.6 A resolution. The crystal structure showed that only van der Waals interactions are involved in binding. The somatic Ala H93 Val mutation in M02/05/01 fills up an empty cavity in the binding pocket. This leads to an increase in binding energy and to the ability to discriminate between the hapten traseolide and its derivatives. The structural understanding of odorant specificity in an antibody gives insight in the physical principles on how specificity for such hydrophobic molecules may be achieved.
A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display.
Willuda, J., Honegger, A., Waibel, R., Schubiger, P. A., Stahel, R., Zangemeister-Wittke, U. and Plückthun, A.
The epithelial glycoprotein-2 is abundantly expressed on many solid tumors and is a suitable target for antibody-based therapy. In the present study, an antiepithelial glycoprotein-2 single-chain Fv (scFv) was derived from the hybridoma MOC31 by phage display. Despite its high affinity (KD = 3.9 x 10(-9) M), however, this antibody fragment failed to significantly enrich at lung tumor xenografts in mice, mostly because of its insufficient thermal stability. To overcome this limitation, the antigen-binding residues of the MOC31 scFv fragment were grafted onto the framework of the highly stable and well-folding anti-c-erbB2 scFv 4D5. Further modification of the resulting 4D5 MOC-A, which was performed by transferring eight additional residues of the heavy chain variable domain core of the parent MOC31 antibody, produced 4D5 MOC-B, resulting in increased serum stability at 37 degrees C and also significantly improved expression behavior while retaining the antigen specificity and affinity of the parent MOC31 scFv. In mice, the scFv 4D5 MOC-B, which was radiolabeled with 99mtechnetium using a new histidine-tag specific labeling method (Waibel et al., Nature Biotechnol., 17: 897-901, 1999), showed favorable blood clearance and efficient enriches at lung tumor xenografts, with a tumor:blood ratio of 5.25 and a total dose of 1.47% injected dose per gram after 24 h. Biophysical properties such as high thermal stability are thus decisive for whether these molecules are useful in vivo, and our approach may provide a general strategy to solve this problem. This is also the first report of using a humanized anti-EGP-2 scFv in vivo for targeting solid tumors, which is a promising targeting moiety for the diagnostics and therapy of EGP-2-positive tumors in patients.
Spada, S., Honegger, A. and Plückthun, A.
The beta-sandwich structure of immunoglobulin variable domains is characterized by a typical kink in the first strand, which allows the first part of the strand to hydrogen bond to the outer beta-sheet (away from the VH-VL interface) and the second part to the inner beta-sheet. This kink differs in length and sequence between the Vkappa, Vlambda and VH domains and yet is involved in several almost perfectly conserved interactions with framework residues. We have used the selectively infective phage (SIP) system to select the optimal kink region from several defined libraries, using an anti-hemagglutinin single-chain Fv (scFv) fragment as a model system. Both for the kink with the Vkappa domain length and that with the Vlambda length, a sequence distribution was selected that coincides remarkably well with the sequence distribution of natural antibodies. The selected scFv fragments were purified and characterized, and thermodynamic stability was found to be the prime factor responsible for selection. These data show that the SIP technology can be used for optimizing protein structural features by evolutionary approaches.
We generated stable and functional cysteine-free antibody single-chain fragments (scFv) lacking the conserved disulfide bonds in both VH and VL. This was achieved by molecular evolution, starting from the scFv fragment of the levan binding antibody ABPC48, which is naturally missing one of the conserved cysteine residues, by using DNA shuffling and phage display. Several of the selected sequences were expressed and the resulting scFv proteins characterized by equilibrium urea denaturation. Three of the characterized proteins exhibit thermodynamic stability similar to the wild-type protein, and these cysteine-free mutant proteins can now be expressed in functional form in the Escherichia coli cytoplasm. We believe that such molecules are of great utility for use as intrabodies, can be produced by simpler expression strategies and may give further insight into the folding and stability of the immunoglobulin fold.
Langedijk, A. C., Honegger, A., Maat, J., Planta, R. J., van Schaik, R. C. and Plückthun, A.
Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln), which is poised to interact with Ser H92 in the recombinant scFv fragment obtained from this antibody. In the scFv fragment derived from this antibody, the stabilizing effect of Gln H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have analyzed the conformation and hydrogen bond pattern of Gln H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to VH domain stability. We also show that the unpaired cysteine H22 is buried, and conforms to the expected VH structure. The antibody appears to have acquired two somatic mutations (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on VH stability. The overall domain stability is the decisive factor for generating functional, disulfide-free antibody domains, and several key residues play dominant roles.
Lindner, P., Bauer, K., Krebber, A., Nieba, L., Kremmer, E., Krebber, C., Honegger, A., Klinger, B., Mocikat, R. and Plückthun, A.
Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore method, Western blots and enzyme-linked immunosorbent assay (ELISA). Western blots and ELISAs can be developed directly by using crude extracts of E.coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent. Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose. The dissociation constants, KD of the peptide KGGHHHHH (KD = 4 x 10(-7) M) and of imidazole (KD = 4 x 10(-4) M) were determined. Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.
While the disulfide bridge is highly conserved within the immunoglobulin fold, a few antibody variable domains lack one of the essential cysteine residues. In the levan binding antibody ABPC48 one of the essential cysteine residues (Cys H92) of the heavy chain variable domain is replaced by tyrosine. We expressed scFv fragments with the ABPC48 sequence and a mutant in which the VH disulfide bond has been restored in Escherichia coli, purified both proteins by antigen affinity chromatography and characterized them by equilibrium denaturation. While the ABPC48 protein was found to be significantly less stable than an average scFv molecule, the restored disulfide increased its stability above that of other, unrelated scFv fragments, explaining why it tolerates the disulfide loss. Surprisingly, we observed that under some refolding conditions, the unpaired cysteine residue of functional scFv of ABPC48 is derivatized by glutathione. It is easily accessible to other reagents and thus appears to be solvent-exposed, in contrast to the deeply buried disulfide of ordinary variable domains. This implies a very unusual conformation of stand b containing the unpaired Cys H22, which might be stabilized by interactions with the tyrosine residue in position H92.
Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R. and Plückthun, A.
A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.
Nieba, L., Honegger, A., Krebber, C. and Plückthun, A.
By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface. We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out. The replacement of one of the residues (V84D in VH) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20. With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased. Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein. Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domains.
Pedrazzi, G., Schwesinger, F., Honegger, A., Krebber, C. and Plückthun, A.
We investigated which molecules are selected from a model library by the selectively infective phage (SIP) methodology. As a model system, we used the fluorescein binding single-chain Fv fragment FITC-E2, and from a 3D-model, we identified 11 residues potentially involved in hapten binding and mutated them individually to alanines. The binding constant of each mutant was determined by fluorescence titration, and each mutant was tested individually as well as in competitive SIP experiments for infectivity. After three rounds of SIP, only molecules with KD values within a factor of 2 of the tightest binder remain, and among those, a mutant no longer carrying an unnecessary exposed tryptophan residue is preferentially selected. SIP is shown to select for the best overall properties of the displayed molecules, including folding behavior, stability and affinity.
Bruggeman, Y. E., Honegger, A., Kreuwel, H., Visser, A. J., Laane, C., Schots, A. and Hilhorst, R.
Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Fl[ox]) and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Fl[red])] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These 'flavobodies' are characterized by time-resolved and steady-state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen-binding site), and by molecular modelling. The three-dimensional models of the antigen-binding sites are consistent with the experimental results. Binding of anti-Fl(red) 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti-Fl(red) 5 shows an 'oxidase-like' redox-potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti-Fl(ox), which do not resemble a natural flavoprotein, show that when the pyrimidine-like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin-binding protein.
Freund, C., Honegger, A., Hunziker, P., Holak, T. A. and Plückthun, A.
Last Modified by A.Honegger