Alexander A. Kortt

A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn

Several cDNAs related to an ABA-induced cDNA from barley aleurone were isolated from barley and corn seedlings that were undergoing dehydration. Four different barley polypeptides with sizes of 22.6, 16.2, 14.4 and 14.2 kDa and a single corn polypeptide with a size of 17.0 kDa were predicted from the nucleotide sequences of the cDNAs. These dehydration-induced proteins (dehydrins) are very similar to each other and to a previously identified rice protein induced by ABA and salt, and have at least some similarity to a previously identified cotton embryo protein. Each dehydrin is extremely hydrophilic, glycine-rich, cysteine- and tryptophan-free and contains repeated units in a conserved linear order. A lysine-rich repeating unit occurs twice in each protein, once at the carboxy terminus and once partway through the polypeptide, adjacent to a succession of serines. This repeating unit and the adjacent flanking run of serines are conserved with minimal variation among all dehydrins. Another repeating unit is flanked by the two copies of the lysine-rich unit, and varies in number from one to five copies. This latter repeating unit is less conserved than the former, varying even within a singly dehydrin. The messenger RNAs corresponding to each cDNA are abundant in dehydrating, but not in well-watered seedlings. The amino acid sequence of tryptic peptides from purified dehydration-induced proteins of corn established that the corn cDNAs correspond to a protein that is produced in abundance during the response of corn seedlings to dehydration.

Design and application of diabodies, triabodies and tetrabodies for cancer targeting

Multivalent recombinant antibody fragments provide high binding avidity and unique specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies, triabodies and tetrabodies using examples of scFv molecules that target viruses (influenza neuraminidase) and cancer (Ep-CAM; epithelial cell adhesion molecule). We discuss the preferred choice of linker length between V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), each with size, flexibility and valency suited to different applications for in vivo imaging and therapy. The increased binding valency of these scFv multimers results in high avidity (low off-rates). A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared to the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv multimers that have recently successfully undergone pre-clinical trials for in vivo stability and efficacy. We also review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. These bi- and tri-specific multimers can be formed by association of different scFv molecules and, in the first examples, have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics).

High avidity scFv multimers; diabodies and triabodies.

Multivalent recombinant antibody fragments provide high binding avidity and unique specificity to a wide range of target antigens and haptens. This review describes how careful choice of linker length between V-domains creates new types of Fv modules with size, flexibility and valency suited to in vivo imaging and therapy. Further, we review the design of multi-specific Fv modules suited to cross-linking target antigens for cell-recruitment, viral delivery and immunodiagnostics. Single chain Fv antibody fragments (scFvs) are predominantly monomeric when the V(H) and V(L) domains are joined by polypeptide linkers of at least 12 residues. An scFv molecule with a linker of 3 to 12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approximately 60 kDa). Reducing the linker length below three residues can force scFv association into trimers (triabodies, approximately 90 kDa) or tetramers ( approximately 120 KDa) depending on linker length, composition and V-domain orientation. The increased binding valency in these scFv multimers results in high avidity (long off-rates). A particular advantage for tumor targeting is that molecules of approximately 60-100 kDa have increased tumor penetration and fast clearance rates compared to the parent Ig. A number of cancer-targeting scFv multimers have recently undergone pre-clinical evaluation for in vivo stability and efficacy. Bi- and tri-specific multimers can be formed by association of different scFv molecules and, in the first examples, have been designed as cross-linking reagents for T-cell recruitment into tumors (immunotherapy) and as red blood cell agglutination reagents (immunodiagnostics).

Dimeric and trimeric antibodies : high avidity scFvs for cancer targeting

Recombinant antibody fragments can be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies (dimers), triabodies (trimers) and tetrabodies (tetramers). In particular we discuss the role of linker length between V-domains and the orientation of the V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), and how the size, flexibility and valency of each molecules is suited to different applications for in vivo imaging and therapy. Single chain Fv antibody fragments joined by polypeptide linkers of at least 12 residues irrespective of V-domains orientation predominantly form monomers with varying amounts of dimer and higher molecular mass oligomers in equilibrium. A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approximately 60 kDa). Reducing the linker length below three residues can force scFv association into trimers (triabodies, approximately 90 kDa) or tetramers ( approximately 120 kDa) depending on linker length, composition and V-domain orientation. A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared with the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv diabodies that have undergone successful pre-clinical trials for in vivo stability and efficacy. We also briefly review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. Bi-specific diabodies formed by association of different scFv molecules have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics). The more challenging trispecific multimers (triabodies) remain to be described.

Triabodies: single chain Fv fragments without a linker form trivalent trimers

A single chain Fv fragment (scFv) of the murine monoclonal antibody 11‐1G10 was constructed by directly joining the C‐terminal residue of the VH domain to the N‐terminal residue of VL. 11‐1G10 is an anti‐idiotype and competes with the antigen, influenza virus neuraminidase (NA), for binding to the NC41 antibody. The scFv formed stable trimers with three active antigen combining sites for NC41 Fab fragments. We propose that trimeric scFvs may be the preferred conformation for directly linked VH‐VL molecules, which contrasts the formation of scFv dimers (diabodies) when the VH and VL domains are joined by short flexible linkers of between 5–10 residues. BIAcore biosensor binding experiments showed that the trimeric scFv showed an expected increase in binding affinity, due to avidity, compared to the monomeric 15‐residue linked scFv. The increase in avidity of scFv trimers offers advantages for imaging and immunotherapy.

Characterisation of the wheat Mr 15000 “grain-softness protein” and analysis of the relationship between its accumulation in the whole seed and grain softness

The Mr 15000 protein associated with water-washed wheat starch granules from soft wheats was shown to be heterogeneous: it could be divided into a fraction containing one or moreα-amylase inhibitor subunits and a fraction largely composed of a previously uncharacterised polypeptide(s) referred to as the “grainsoftness protein” (GSP). The major N-terminal sequence and sequences of peptides derived from protease digests of GSP are reported. An antiserum specific for GSP was used to show that GSP accumulated in both hard and soft wheat grains, but the GSP in soft grains associated more strongly with starch granules than the GSP in hard grains. A positive correlation between grain softness and accumulation of GSP in the seed was demonstrated for a range of cultivars. This differs from the qualitative relationship, based on the isolated starch fraction, between GSP and grain softness that has already been reported. Analysis of wholemeal extracts with the antiserum demonstrated that the accumulation of GSP in the seed was dependent on the short arm of chromosome 5D, which also encodes theHa locus. In addition, examination of near-isogenic lines differing in hardness indicated that the gene(s) controlling GSP was (were) linked with theHa locus. The findings indicate that GSP may be the product of theHa locus and thus be the major factor that determines the milling characteristics of bread wheats.

Affinity maturation of recombinant antibodies using E. coli mutator cells.

BACKGROUND Phage libraries can display repertoires of antibodies which are greater in number than the mammalian immune response. However, the selected antibodies often have low binding affinity to their target antigen or hapten (KD below 10(-6) M), which is characteristic of the primary immune repertoire. There is a need for procedures to mimic somatic hypermutation through antigen driven affinity maturation, thereby increasing the affinity of selected immunoglobulins. OBJECTIVE To investigate the effectiveness of mutation and affinity selection of recombinant antibody genes with mutator E. coli cells, incorporating phage-display strategies. STUDY DESIGN Unique human scFvs were selected from a naive Fd-phage library. These genes were mutated by propagation in mutD5 mutator E. coli cells (mutD5-FIT) which were competent for Fd (M13) based phagemid transfections and generated point mutations (transversions and transitions) in the scFv genes. Individual phage-displayed scFvs were affinity selected from the mutation library and were assayed as soluble scFvs by ELISA and BIAcore for binding to antigen. RESULTS The in vivo mutation of phage-displayed scFvs in E. coli mutD5-FIT, combined with affinity selection against antigen, produced scFv molecules with improved binding activity. The point mutations which resulted in single amino acid substitutions frequently produced ten fold increases in apparent binding affinity. Structural comparisons revealed that these point mutations were in framework regions (adjacent to the CDRs) and within the CDRs. In one case the apparent affinity of an anti-glycophorin scFv after mutation in the VL framework region close to CDR3 increased by 10(3). However, this increase in apparent affinity was accompanied by an increased propensity to dimerise and form aggregates. CONCLUSIONS A strategy for the rapid affinity maturation of scFv and Fab antibody fragments has been developed which utilises mutator strains of E. coli and incorporates phage display of antibody repertoires (libraries).

A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis

The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysine‐specific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (K d=1.31±0.26×10−7 M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody‐like molecule.

Isolation and characterization of the trypsin inhibitors from winged bean seed (Psophocarpus tetragonolobus (L) Dc.)

The trypsin inhibitors from winged bean seed were isolated by affinity chromatography on trypsin-Sepharose 4B and the components fractionated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The major components, inhibitors 2 and 3 were found to be homogeneous proteins with molecular weights of about 20,000. The inhibitors stoichiometrically inhibited bovine trypsin in the molar ratio of 1 : 1 whereas the inhibition of bovine alpha-chymotrypsin was weak and non-stoichiometric. Amino acid analysis indicated that both the inhibitors contain four cysteine residues and are rich in aspartic acid, glutamic acid, glycine, valine and leucine; however, inhibitor 3 lacks histidine and methionine while inhibitor 2 contains one histidine and three methionines. A minor trypsin inhibitor fraction was also isolated which contained at least three proteins with a molecular weight of about 10,000 and a high content of half-cystine.

Cytotoxic properties of immunoconjugates containing melittin-like peptide 101 against prostate cancer: in vitro and in vivo studies

Background: Monoclonal antibodies (MAbs) can target therapy to tumours while minimising normal tissue exposure. Efficacy of immunoconjugates containing peptide 101, designed around the first 22 amino acids of bee venom, melittin, to maintain the amphipathic helix, to enhance water solubility, and to increase hemolytic activity, was assessed in nude mice bearing subcutaneous human prostate cancer xenografts. Methods: Mouse MAbs, J591 and BLCA-38, which recognise human prostate cancer cells, were cross-linked to peptide 101 using SPDP. Tumour-bearing mice were used to compare biodistributions of radiolabeled immunoconjugates and MAb, or received multiple sequential injections of immunoconjugates. Therapeutic efficacy was assessed by delay in tumour growth and increased mouse survival. Results: Radiolabeled immunoconjugates and antibodies showed similar xenograft tropism. Systemic or intratumoural injection of immunoconjugates inhibited tumour growth in mice relative to carrier alone, unconjugated antibody and nonspecific antibody-peptide conjugates and improved survival for treated mice. Conclusions: Immunoconjugates deliver beneficial effects; further peptide modifications may increase cytotoxicity.