Saran A. Narang

Selection of phage-displayed llama single-domain antibodies that transmigrate across human blood-brain barrier endothelium

Delivery to the brain of drugs, peptides, and genes depends on the availability of brain‐specific delivery vectors. We used a phage‐displayed library of llama single‐domain antibodies (sdAbs) to enrich for species that selectively bind to and are internalized by human cerebromicrovascular endothelial cells (HCEC). Two sdAbs (FC5 and FC44) were selected, sequenced, subcloned, and expressed as fusion proteins with c‐Myc‐His5 tags. Similar to phage‐displayed sdAbs, soluble FC5 and FC44 were shown to selectively bind HCEC and to transmigrate across an in vitro human blood‐brain barrier (BBB) model. Both FC5 and FC44, in contrast to an unrelated llama sdAb, were also detected in the brain after i.v. injection into mice. These small (~14 kDa) antibodies have characteristics essential for a carrier‐vector and can be used to facilitate drug transport across the BBB.

Selection by phage display of llama conventional VH fragments with heavy chain antibody VHH properties

A llama single domain antibody (dAb) library designed and constructed to contain only heavy chain antibody variable domains (V(H)Hs) also contained a substantial number of typical conventional antibody heavy chain variable sequences (V(H)s). Panning the library against two carbohydrate-specific antibodies yielded anti-idiotypic dAbs and enriched solely for sequences from the V(H) subpopulation of the library. The conventional antibody origin of these V(H)s was confirmed by using oligonucleotide probes, specific for the enriched V(H)s, to identify the parental sequences in the message employed in library construction. Surprisingly, these V(H) 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 V(H)s makes them potentially very useful as therapeutic dAbs.

[6] Improved phosphotriester method for the synthesis of gene fragments

Publisher Summary This chapter discusses an experimental study focusing on the improved phosphotriester method for the synthesis of gene fragments. During studies with a modified triester approach, it was observed that the pure, fully protected product was difficult to separate quantitatively by the conventional silica gel chromatography from the crude reaction mixture, especially from the components containing several guanine bases. It was found that thin-layer chromatography (TLC) on silanized silica gel (RP-2) and KC 18 (RP-18) plates in an acetone–water solvent gave excellent separation of the components containing trityl and hydroxyl groups and also of those differing in sizes. The polar component (containing 3′ phosphodiester) generally moved to the solvent front, and the fully protected component (containing the trityl group) was the slowest. The mobility of a component containing a 5′-hydroxyl group was between that of the polar and nonpolar compounds. The purification of GGCA could not be achieved on silica gel as determined after complete deblocking and TLC on a polyethyleneimine (PEI), whereas purification by TLC on reverse-phase (RP-2) plates gave pure GGCA as analyzed on a PEI plate. These results indicated that reverse-phase chromatography of a triester oligonucleotide could yield a pure product at each step of synthesis.

Analysis by Surface Plasmon Resonance of the Influence of Valence on the Ligand Binding Affinity and Kinetics of an Anti-carbohydrate Antibody

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.

Optimal Design Features of Camelized Human Single-domain Antibody Libraries

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.

Sequential Interchange of Four Amino Acids from Blood Group B to Blood Group A Glycosyltransferase Boosts Catalytic Activity and Progressively Modifies Substrate Recognition in Human Recombinant Enzymes

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 thek 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.

Thermal stabilization of a single-chain Fv antibody fragment by introduction of a disulphide bond

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 saltbridge 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 6°C to 69°C. The ds‐scFv form thus combines the stable monomeric form of the disulphide form with the expression advantages of the scFv.

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.

A Modified Triester Method for the Synthesis of Deoxyribopolynucleotides

A modified triester method has been developed for the synthesis of oligonucleotides starting from mononucleosides as more economical starting materials. A pentadecanucleotide has been successfully prepared.

Synthesis of a human insulin gene V. Enzymatic assembly, cloning and characterization of the human proinsulin DNA

To form a 258-bp sequence coding for human proinsulin, 41 synthetic deoxyribo-oligonucleotide fragments of 11 to 15 nucleotides in length were assembled by enzymatic methods. The coding sequence is preceded by ATG and following by TGA for translation start and stop signals, and terminated in an EcoRI and a BamHI recognition sequence. The complete synthetic sequence was ligated to a plasmid and cloned in Escherichia coli. The cloned DNA was shown to have the correct human proinsulin coding sequence.