A llama single domain antibody (dAb) library designed and constructed to contain only heavy chain antibody variable domains (VHHs) also contained a substantial number of typical conventional antibody heavy chain variable sequences (VHs). Panning the library against two carbohydrate-specific antibodies yielded anti-idiotypic dAbs and enriched solely for sequences from the VH subpopulation of the library. The conventional antibody origin of these VHs was confirmed by using oligonucleotide probes, specific for the enriched VHs, to identify the parental sequences in the message employed in library construction. Surprisingly, these VH dAbs, which are produced in high yield in Escherichia coli, are highly soluble, have excellent temperature stability profiles and do not display any aggregation tendencies. The very close similarity of these molecules to human VHs makes them potentially very useful as therapeutic dAbs.

We have constructed a human VHlibrary based on a camelized VH sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate VH sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the1H-15N HSQC spectra, indicated that the VH sequence for library construction was a good choice. These results should be useful in the design of VHlibraries with optimal features.

The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.

The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 --> Gly, Gly-235 --> Ser, Leu-266 --> Met, and Gly-268 - Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase, To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time, A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the k(cat) being 5-fold higher for the A donor, Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 --> Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the k(cat) and increased specificity, The increases observed in k(cat) are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (k ≈ 0.25 s). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 × 105M s. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the on-rate was the same as that obtained with Fab and scFv monomer.

The application of phage display to the screening of antibody V-gene libraries promises to replace animal immunization and hybridoma technology as a means of antibody generation (,). Synthetic libraries offer the potential for expanding the immune system repertoire and designing libraries for particular sets of antigens. We have developed a method for the generation of synthetic antibody gene libraries by which spiked oligonucleotides are assembled by a ligase chain reaction protocol (). This is a synthetic adaptation of a reaction that was previously used as a diagnostic tool (,).

A 1034-bp synthetic gene encoding the human blood group B glycosyltransferase, which catalyzes the transfer of galactose from UDP-Gal to Fuc alpha(1-2)Gal beta-OR to give the blood group B determinant Gal alpha(1-3)[Fuc alpha(1-2)]Gal beta-OR (where R is a glycoprotein or glycolipid), has been expressed in Escherichia coli by replacing its membrane-anchoring domain with an ompA bacterial secretory signal. The active enzyme was purified from the periplasm using UDP-hexanolamine affinity chromatography and used in the synthesis of preparative amounts of the human blood group B trisaccharide antigen. The substrate specificity and kinetics of the recombinant enzyme were comparable to the enzyme from human sera. Thus we have achieved the construction of a completely synthetic glycosyltransferase gene and its successful expression.

A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the salt-bridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp, The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics, Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy, The T-m was raised from 60 degrees C to 69 degrees C, The ds-scFv form thus combines the stable monomeric form of the disulphide form with the expression advantages of the scFv.

The combination of an antibody fragment with a lanthanide chelating protein has desirable characteristics for fluorescence-based immunoassays and tumor radioimmunotherapy. As a model for this design, a fusion protein consisting of a single-chain antibody linked to an engineered version of oncomodulin, a protein with two Ca2+-binding motifs (the CD and EF loops), was produced by secretion from Escherichia coli in good yield. The single-chain antibody was specific for a Salmonella O-polysaccharide. The CD loop of oncomodulin had been redesigned to bind lanthanide ions with high affinity. The fusion protein was shown to have antigen-binding activity that was comparable to that of the unfused single-chain antibody, to bind Tb3+ with very high affinity and to give strong, sensitized Tb3+ luminescence via excitation of the tryptophan residue in the CD loop. A second fusion protein containing a 30-residue helix-loop-helix motif as the lanthanide-binding component was also prepared, but showed considerably lower solubility, Competition for Tb3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb3+ were approximately 10(11) M(-1) and 10(7) M(-1) respectively. Time-resolved lanthanide luminescence photography of electrophoresis gels demonstrated that the helix-loop-helix Ca2+-binding could be used to specifically visualize the scFv fragment.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

We describe here the 1.7-A resolution structure of a single-chain antibody variable domain (scFv) molecule, based on the carbohydrate-binding antibody Se155-4, complexed with the trisaccharide ligand alpha-D-Gal(1-->2)[alpha-D-Abe(1-->3)]alpha-D-Manp1-->OMe, where Abe is abequose. The scFv expressed in Escherichia coli has the variable region light chain to heavy chain polarity with the domains connected by a 19-residue linker. Although the linker is partially disordered in the crystal, the packing of the molecules suggests a monomeric state of the scFv. The carbohydrate adopts a different conformation about the Man-Gal linkage than was observed previously in the Fab-trisaccharide complex. Instead of a direct hydrogen bond between O2Abe and O2Gal, these two atoms are bridged by a water molecule in the present complex.

We have used a strategy of hybrid gene synthesis and constant domain shuffling to construct and functionally express in Escherichia coli genes encoding two anti-carbohydrate Fabs, one specific for a Brucella cell-surface polysaccharide and the second for the human blood group A determinant. Very similar VL amino acid sequences made possible the simultaneous synthesis of the two corresponding genes. A class switching approach was used in Fd and light chain gene assembly. The two independently synthesized VH genes were fused to a previously made sequence encoding the C(gamma 1)1 domain as an alternative to synthesis of the natural C gamma 2b 1 and C mu 1 sequences. The VL genes were initially coupled to a synthetic C kappa gene. When these light chain and the above Fd genes, each preceded by the ompA signal sequence, were expressed from two-cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Replacement of the C kappa domains with a C lambda 1 domain resulted in a significant increase in the amount of soluble periplasmic light chain and functional Fab for both the Brucella and blood group A antibodies. The C kappa and C lambda 1 forms of each of the Brucella and blood group A Fabs, with His5 fusions at the C-termini of the Fd chains, were purified by immobilized metal affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, rive of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77 --> Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties.

The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.

a thermostable ligase to ligate two oligonucleotides which are hybridized to a targeted DNA sequence, provided the adjoining ends are perfectly base-paired to the target. Using eight oligonucleotides, instead of two or four as used in diagnostic applications, we have constructed a library of sequences encoding the variable domain (VH) of an antibody heavy chain in which the three complementarity-determining regions (CDRs) have been randomly and simultaneously mutated by spiking the synthetic fragments (Figure 1). Only oligonucleotides of the correct size and base-pairing at adjoining ends are incorporated into the LCR product. The method provides a highly efficient way of assembling randomized CDR sequences. Introduction of diversity is an essential feature of antibody redesign and in vitro affinity maturation by the phage display technology. Semisynthetic approaches to this problem have thus far been limited to a single CDR (3, 4). In this work, the wild-type gene encoding a singlechain antibody specific for Salmonella serogroup B 0polysaccharide (5) was randomized in all of the heavy chain CDR regions. Oligodeoxynucleotides (56mer 85mer) were synthesized using an ABI 384 DNA synthesizer such that the central 15-24 bases encoding the CDRs were randomized by equimolar spiking with A, G, C and T at a level of 10%. The LCR mixture contained 8 ,tl oligonucleotides (4 pmol each of non-phosphosphorylated

The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.

A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se 155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 μg/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.

Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and V(L)-V(H) orientation on expression, secretion, and binding to a Salmonella O-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS), sequence. V(L) and V(H) domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were approximately 50 mg/liter, chiefly in the form of inclusion bodies. With V(L) in the NH2-terminal position, the amount of secreted product obtained was 2.4 mg/liter, but when V(H) occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG.

A new hypothesis on carcinogenesis is set forth on the basis of the neighborhood coherence principle (NCP). NCP constitutes a general rule of pattern formation and maintenance. According to this principle, a system of interacting cells can produce and maintain a spatial organization by virtue of cell-cell communication. This hypothesis suggests that this homeostasis primarily results from a NCP-like process implying cell-cell communication. Each cell is constrained by its neighbors to maintain the mature phenotype despite its inherent individual variability. If the cell-cell mature communication happens to be impaired, tissue homeostasis is disrupted and a proliferative state can be initiated. A further potential effect may result from the establishment of NCP-like communication specific for proliferative cells allied to paracrine and outocrine factors which can lock the cells into the proliferative mode. Most mechanisms implied in this hypothesis have already been investigated. There is a large body of experimental results supporting the role of junctional communication in cooperative metabolism, growth, differentiation and tumour-related events. This new hypothesis provides a framework within which these known facts may be put in a theoretical perspective; it might well constitute the unifying theory--as yet missing--in carcinogenesis.