Formatting antibody fragments to mediate specific therapeutic functions.

Monoclonal antibodies are increasingly being used as therapeutic agents in a wide range of indications, including oncology, inflammation and infectious disease. In most cases the basis of the therapeutic function is the high degree of specificity and affinity the antibody-based drug has for its target antigen. However, the mechanism of action (MOA), the way the drug takes advantage of this specificity to mediate a therapeutic effect, varies considerably from drug to drug. Three basic potential categories of MOAs exist: antagonists, agonists and specific delivery mechanisms to target an active function to a particular cell type. The latter functions include selective cell killing, based on Fc-mediated events, recruitment of effector cells, and drug or radioisotope delivery. The majority of these mechanisms are not necessarily optimally mediated by an IgG structure; clearly, in the case of antibody-dependent cellular cytotoxicity or complement-mediated lysis, Fc is required. However, Fab fragments (the fragment comprising one antigen-binding arm of the Y-shaped IgG molecule) can be formatted to mediate most mechanisms and have the advantage that valency and half-life can be controlled to simplify the drug and address only the mechanism required. Moreover, Fab fragments can be produced in microbial expression systems which address manufacturing issues such as scale of supply and cost of goods.

Prolonged in vivo residence times of antibody fragments associated with albumin.

Antibody fragments can be expressed at a high level in microbial systems, but they may have limited therapeutic value because they are rapidly eliminated from the body. We demonstrate here that site-specific conjugation or binding of bacterially derived Fab' to the long-lived protein serum albumin allows full retention of the antibody's binding characteristics while imparting the albumin's longevity in vivo. In rats the area under the curve for Fab' conjugated to rat serum albumin was 17-fold greater than for the control of Fab' conjugated to cysteine. Again, a bispecific F(ab')(2) with specificity for rat serum albumin showed an area under the curve about 8-fold greater than did a F(ab')(2) without specificity to albumin. Genetic fusions of scFv to albumin were similarly long-lived and could be expressed in yeast to provide the basis of a cost-effective production system.

High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence.

We investigated the ability of signal peptides of eukaryotic origin (human, mouse, and yeast) to efficiently direct model proteins to the Escherichia coli periplasm. These were compared against a well-characterized prokaryotic signal peptide-OmpA. Surprisingly, eukaryotic signal peptides can work very efficiently in E. coli, but require optimization of codon usage by codon-based mutagenesis of the signal peptide coding region. Analysis of the 5' of periplasmic and cytoplasmic E. coli genes shows some codon usage differences.

Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site.

The peptide sequence (N)DKTH(C) was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized gamma1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu(2+), such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni(2+). Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.

Predictive accuracy of population viability analysis in conservation biology.

Population viability analysis (PVA) is widely applied in conservation biology to predict extinction risks for threatened species and to compare alternative options for their management. It can also be used as a basis for listing species as endangered under World Conservation Union criteria. However, there is considerable scepticism regarding the predictive accuracy of PVA, mainly because of a lack of validation in real systems. Here we conducted a retrospective test of PVA based on 21 long-term ecological studies--the first comprehensive and replicated evaluation of the predictive powers of PVA. Parameters were estimated from the first half of each data set and the second half was used to evaluate the performance of the model. Contrary to recent criticisms, we found that PVA predictions were surprisingly accurate. The risk of population decline closely matched observed outcomes, there was no significant bias, and population size projections did not differ significantly from reality. Furthermore, the predictions of the five PVA software packages were highly concordant. We conclude that PVA is a valid and sufficiently accurate tool for categorizing and managing endangered species.

Therapeutic antibody fragments with prolonged in vivo half-lives.

Antibody fragments can be isolated rapidly using techniques such as phage display and can be expressed to high levels in microbial systems. However, to date such antibody fragments have been of limited use for many therapeutic applications because they are rapidly cleared from the body. We present a strategy for the site-specific chemical modification of antibody fragments with polyethylene glycol, which results in the production of antibody fragments with long in vivo half-lives and full retention of antigen-binding properties. This technology should allow more rapid and economical production of therapeutic antibodies for chronic disease therapy.

F(ab')2 molecules made from Escherichia coli produced Fab' with hinge sequences conferring increased serum survival in an animal model.

Fab's with hinges based on the human gamma1 sequence containing 1, 2, or 4 cysteines have been produced by high level Escherichia coli periplasmic secretion, and coupled in vitro by reduction/oxidation to form F(ab')2. We find that the F(ab')2 made with hinges containing 2 or 4 cysteines have a high level (approximately 70%) of multiple disulphide bonds. These F(ab')2 molecules have an increased pharmacokinetic stability as measured by area under the curve compared to those made by direct coupling through a single disulphide bond. One particular molecule containing 4 hinge cysteines has a greater pharmacokinetic stability than a F(ab')2 formed by chemical cross-linking. F(ab')2 made from the Fab' with 4 hinge cysteines is also relatively resistant to chemical reduction in vitro allowing partial reduction to expose reactive hinge thiols. These hinge sequences provide a simple method for producing robust F(ab')2 in vitro, obviating the need to use chemical cross-linkers, and provide a route to hinge specific chemical modification with thiol-reactive conjugates.

Formation of dimeric Fabs in Escherichia coli: effect of hinge size and isotype, presence of interchain disulphide bond, Fab' expression levels, tail piece sequences and growth conditions.

We have made hinge variants of two human Fab's in order to investigate the factors involved in the formation of dimeric Fab's in the periplasm of E. coli. Hinges containing one or more copies of the IgG1 hinge with various numbers of spacing residues were tested. Fab's with hinges based on the gamma2, gamma3 and gamma4 isotypes were also tested. We find that the IgG1 hinge sequence can form approximately 35% F(ab')2 in vivo in shake flask experiments, but that only (approximately) 5% F(ab')2 can be produced during fermentation. IgM and IgA tail-pieces added to Fab's did not effect their multimerisation. The possible role of growth conditions upon F(ab')2 formation in vivo is discussed.

Multiple ubiquitin conjugates are present in rat brain synaptic membranes and postsynaptic densities.

The pattern of ubiquitin-protein conjugates present in a range of adult rat forebrain subcellular fractions has been investigated by immunoblotting with a monoclonal antibody specific for ubiquitin and its conjugates. Each fraction contains a complex and characteristic pattern of ubiquitin conjugates. Many integral synaptic membrane proteins are ubiquitinated, including a subset of high M(r) (> 120 kD) concanavalin A-binding glycoproteins. Postsynaptic densities are also enriched in ubiquitin conjugates, the profile being distinct from that of synaptic membranes. These results suggest that many plasma membrane and synaptic proteins are ubiquitinated.