fibs-enabled-noninvasive-metabolic-profiling

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Imperial College London

Chemical coupling of a variety of polymers to therapeutic proteins has been studied as a way of improving their pharmacokinetics and pharmacodynamics in vivo. Conjugates have been shown to possess greater stability, lower immunogenicity, and a longer blood circulation time due to the chemicophysical properties of these hydrophilic long chain molecules. Naturally occurring colominic acid (polysialic acid, PSA) has been investigated as an alternative to synthetic polymers such as poly(ethylene glycol) (PEG) due to its lower toxicity and natural metabolism. Antibodies and their fragments are a good example of the types of proteins which benefit from pharmacokinetic engineering. Here, we chemically attached differing amounts and differing lengths of short (11 kDa) and longer (22 kDa) chain colominic acid molecules to the antitumor monoclonal antibody H17E2 Fab fragment. Different coupling ratios and lengths were seen to alter the electrophoretic mobility of the Fab fragment but have a minor effect on the antibody immunoreactivity toward the placental alkaline phosphatase (PLAP) antigen. Polysialylation generally increased Fab fragment blood half-life resulting in higher tumor uptake in a KB human tumor xenograft mouse model. One H17E2 Fab-PSA conjugate had over a 5-fold increase in blood exposure and over a 3-fold higher tumor uptake with only a marginal decrease in tumor/blood selectivity ratio compared to the unconjugated Fab. This conjugate also had a blood bioavailability approaching that of a whole immunoglobulin.

Modulation of antibody pharmacokinetics by chemical polysialylation.

The use of hydrophilic polymers as a substitute for the Fc-domain in immuno- or non-immuno-based binding proteins is accelerating. Chemical PEGylation has led the way and is still the most advanced and clinically-approved approach. Hydrophilic polymers act by maintaining a flexible conformation and hydrogen bonding to a network of water molecules to acquire a larger hydrodynamic volume and apparent mass than their actual molecular mass suggest. The benefits are increased blood half-life and bioavailability, stability and reduced immunogenicity. In the case of PEG, there is also evidence of enhanced targeting and reduced side effects, but drawbacks include the fact that PEG is non-biodegradable.

Modulating antibody pharmacokinetics using hydrophilic polymers.

The potential for protein-engineered biotherapeutics is enormous, but pharmacokinetic modulation is a major challenge. Manipulating pharmacokinetics, biodistribution, and bioavailability of small peptide/protein units such as antibody fragments is a major pharmaceutical ambition, illustrated by the many chemical conjugation and recombinant fusion approaches being developed. We describe a recombinant approach that leads to successful incorporation of polysialic acid, PSA for the first time, onto a therapeutically valuable protein. This was achieved by protein engineering of the PSA carrier domain of NCAM onto single-chain Fv antibody fragments (one directed against noninternalizing carcinoembryonic antigen-CEA and one against internalizing human epidermal growth factor receptor-2-HER2). This created novel polysialylated antibody fragments with desired pharmacokinetics. Production was achieved in human embryonic kidney cells engineered to express human polysialyltransferase, and the recombinant, glycosylated product was successfully fractionated by ion-exchange chromatography. Polysialylation was verified by glycosidase digestion and mass spectrometry, which showed the correct glycan structures and PSA chain length similar to that of native NCAM. Binding was demonstrated by ELISA and surface plasmon resonance and on live cells by flow cytometry and confocal immunofluorescence. Unexpectedly, polysialylation inhibited receptor-mediated endocytosis of the anti-HER2 scFv. Recombinant polysialylation led to an estimated 3-fold increase in hydrodynamic radius, comparable to PEGylation, leading to an almost 30-fold increase in blood half-life and a similar increase in blood exposure. This increase in bioavailability led to a 12-fold increase in tumor uptake by 24 h. In summary, recombinant polysialylation of antibody fragments in our system is a novel and feasible approach applicable for pharmacokinetic modulation, and may have wider applications.

Glycoengineering approach to half-life extension of recombinant biotherapeutics.

Bioprocess monitoring is used to track the progress of a cell culture and ensure that the product quality is maintained. Current schemes for monitoring metabolism rely on offline measurements of samples of the extracellular medium. However, in the era of synthetic biology, it is now possible to design and implement biosensors that consist of biological macromolecules and are able to report on the intracellular environment of cells. The use of fluorescent reporter signals allows non-invasive, non-destructive and online monitoring of the culture, which reduces the delay between measurement and any necessary intervention. The present mini-review focuses on protein-based biosensors that utilize FRET as the signal transduction mechanism. The mechanism of FRET, which utilizes the ratio of emission intensity at two wavelengths, has an inherent advantage of being ratiometric, meaning that small differences in the experimental set-up or biosensor expression level can be normalized away. This allows for more reliable quantitative estimation of the concentration of the target molecule. Existing FRET biosensors that are of potential interest to bioprocess monitoring include those developed for primary metabolites, redox potential, pH and product formation. For target molecules where a biosensor has not yet been developed, some candidate binding domains can be identified from the existing biological databases. However, the remaining challenge is to make the process of developing a FRET biosensor faster and more efficient.

Article	Year
Modulation of antibody pharmacokinetics by chemical polysialylation. Bioconjugate chemistry\| PubMed ID: 18307285	2008
Modulating antibody pharmacokinetics using hydrophilic polymers. Expert opinion on drug delivery\| PubMed ID: 21854300	2011
Glycoengineering approach to half-life extension of recombinant biotherapeutics. Bioconjugate chemistry\| PubMed ID: 22681552	2012
Opportunities for bioprocess monitoring using FRET biosensors. Biochemical Society transactions\| PubMed ID: 24059500	2013

Antony Constantinou

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