Mahendra Deonarain

Ligand-targeted receptor-mediated vectors for gene delivery

Gene therapy promises to cure human genetic diseases. One of the main obstacles to fulfilling this promise is in the ability to target a gene to a significant population of cells and express it at adequate levels for a long enough period of time. Viral methods for gene delivery have been studied for a number of years and are effective vectors for gene transfer. The great majority of gene therapy clinical trials currently in progress use retroviruses or adenoviruses. However, there are concerns for their clinical use because of possible risks of mutagenesis, immunogenic side-effects and toxicity. In addition to this, there are other limitations, including the size of gene that can be transferred. Over the last ten years, a new approach has emerged that has increasingly gathered speed thanks to advances in receptor cell biology and antibody production. This method involves ligand-targeted receptor mediated endocytosis (RME) of ‘polyplexes’. Here, synthetic complexes are composed of a cell-specific targeting...

Antibodies targeting cancer stem cells: A new paradigm in immunotherapy?

Antibody targeting of cancer is showing clinical and commercial success after much intense research and development over the last 30 years. They still have the potential to delivery long-term cures but a shift in thinking towards a cancer stem cell (CSC) model for tumour development is certain to impact on how antibodies are selected and developed, the targets they bind to and the drugs used in combination with them. CSCs have been identified from many human tumours and share many of the characteristics of normal stem cells. The ability to renew, metabolically or physically protect themselves from xenobiotics and DNA damage and the range of locomotory-related receptors expressed could explain the observations of drug resistance and radiation insensitivity leading to metastasis and patient relapse. Targeting CSCs could be a strategy to improve the outcome of cancer therapy but this is not as simple as it seems. Targets such as CD133 and EpCAM/ESA could mark out CSCs from normal cells enabling specific intervention but indirect strategies such as interfering with the establishment of a supportive niche through anti-angiogenic or anti-stroma therapy could be more effective. This review will outline the recent discoveries for CSCs across the major tumour types highlighting the possible molecules for intervention. Examples of antibody-directed CSC therapies will be given and the outlook for the future development of this emerging area will be given.

Natural Killer Cell Signal Integration Balances Synapse Symmetry and Migration

Imaging immune surveillance by natural killer (NK) cells has revealed that integration of activating and inhibitory signals determines whether or not NK cells stop to kill the target cell or retain a migratory configuration.

Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments

Current photodynamic therapy (PDT) of cancer is limited by inefficiencies involved in specifically targeting photosensitizers to tumors. Although antibodies are being explored as targeting vehicles, they present significant challenges, particularly in terms of pharmacokinetics and drug‐coupling. We describe here a novel and effective system to covalently attach multiple photosensitizer molecules (both preclinical, pyropheophorbide‐a and clinically approved, verteporfin photosensitizers) to single‐chain Fvs. Further, we demonstrate that not only do the resulting photoimmunoconjugates retain photophysical functionality, they are more potent than either free photosensitizer, effectively killing tumor cells in vitro and in vivo. For example, treatment of human breast cancer xenografts with a photoimmunoconjugate comprising an anti‐HER‐2 scFv linked to 8–10 molecules of pyropheophorbide‐a leads to significant tumor regression. These results give an insight into the important features that make scFvs good carriers for PDT drugs and provide proof of concept of our unique approach to targeted photodynamic therapy (tPDT). This promises to significantly improve on current photodynamic therapies for the treatment of cancer.

Modulation of Antibody Pharmacokinetics by Chemical Polysialylation

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.

Site-Specific Polysialylation of an Antitumor Single-Chain Fv Fragment

Protein pharmacokinetic modulation is becoming an important tool in the development of biotherapeutics. Proteins can be chemically or recombinantly modified to alter their half-lives and bioavailability to suit particular applications as well as improve side effect profiles. The most successful and clinically used approach to date is chemical conjugation with poly(ethylene glycol) polymers (PEGylation). Here, therapeutic protein half-life can be increased significantly while retaining biological function, reducing immunogenicity and cross-reaction. Naturally occurring alternatives to such synthetic polymers could have major advantages such as lower side effects due to biodegradability and metabolism. Polysialic acid (PSA) has been investigated as a pharmacokinetic modulatory biopolymer with many successful examples in preclinical and clinical development. Single-chain Fvs (scFvs) are a choice antibody format for human therapeutic antibody discovery. Because of their small size, they are rapidly eliminated from the circulation and often are rebuilt into larger proteins for drug development and a longer half-life. Here we show that chemical polysialylation can increase the half-life of an antiplacental alkaline (PLAP) and anticarcinoembryonic antigen (CEA) scFv (F1 and MFE-23, respectively) 3.4-4.9-fold, resulting in a 10.6-15.2-fold increase in blood exposure. Amine-directed coupling of the MFE-23 scFv reduced its immunoreactivity 20-fold which was resolved by site-specific polysialylation through an engineered C-terminal thiol residue. The site-specifically polysialylated MFE-23 scFv demonstrated up to 30-fold improved tumor uptake while displaying favorable tumor:normal tissue specificity. This suggests that engineering antibody fragments for site-specific polysialylation could be a useful approach to increase the half-life for a variety of therapeutic applications.

Modulating antibody pharmacokinetics using hydrophilic polymers

Introduction: 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. Areas covered: This report reviews the state of the art for antibody PEGylation in terms of approaches and effects. Additionally, non-biological (such as N-(2-hydroxypropyl)methacrylamide) and potentially superior biological alternatives (such as polysialylation) are described, ending with recombinant approaches (such as hydrophilic peptides and glyco-engineering), which promise to circumvent the need for chemical modification altogether. Expert opinion: The emergence of many small, antibody fragment-like mimics will drive the need for such technologies, and PEGylation is still the choice polymer due to its established use and track record. However, there will be a place for many alternative technologies if they can match the pharmacokinetics of PEG-conjugates and bring addition beneficial features such as easier production.

Modulating the pharmacokinetics of therapeutic antibodies

With the advent of antibody fragments and alternative binding scaffolds, that are devoid of Fc-regions, strategies to increase the half-life of small proteins are becoming increasingly important. Currently, the established method is chemical PEGylation, but more elaborate approaches are being described such as polysialylation, amino acid polymers and albumin-binding derivatives. This article reviews the main strategies for pharmacokinetic enhancement, primarily chemical conjugates and recombinant fusions that increase apparent molecular weight or hydrodynamic radius or interact with serum albumin which itself has a long plasma half-life. We highlight the key chemical linkage methods that preserve antibody function and retain stability and look forward to the next generation of technologies which promise to make better quality pharmaceuticals with lower side effects. Although restricted to antibodies, all of the approaches covered can be applied to other biotherapeutics.

Fluorescence characterisation of multiply-loaded anti-HER2 single chain Fv-photosesitizer conjugates suitable for photodynamic therapy

We report the synthesis, spectroscopic properties and intracellular imaging of recombinant antibody single chain fragment (scFv) conjugates with photosensitizers used for photodynamic therapy of cancer (PDT). Two widely-studied photosensitizers have been selected: preclinical pyropheophorbide-a (PPa) and verteporfin (VP), which has been clinically approved for the treatment of acute macular degeneration (Visudyne). Pyropheophorbide-a and verteporfin have been conjugated to an anti-HER2 scFv containing on average ten photosensitizer molecules per scFv with a small contribution (<or=20%) from non-covalently bound molecules. Confocal fluorescence microscopy demonstrates good cellular uptake of PPa conjugate with the HER2-positive cell line, SKOV-3, while negligible cell uptake is demonstrated for the HER2-negative cell line, KB. For the VP conjugate, increased rate of cellular uptake and prolonged retention in SKOV-3 cells is observed compared to free photosensitizer. In clinical applications this could provide increased potency and desired selectivity towards malignant tissue, leaving surrounding healthy tissue unharmed and reducing skin photosensitivity. The present study highlights the usefulness of photosensitizer immunoconjugates with scFvs for targeted PDT.

Recombinant antibodies for cancer therapy

Background: Recombinant antibodies have evolved into successful therapeutics with 10 approved for cancer and more in the pipeline. Four of the top ten cancer therapy drugs are recombinant antibodies. Objectives: To survey the current state-of-the-art highlighting the reasons for this success and looking ahead to the next generation of antibody therapy. Methods: An analysis was carried out to identify preclinical and clinical examples and the underlying concepts and mechanisms that have shown how to design better therapies. Results/conclusions: Greater understanding of the molecular basis of cancer has led to improved antibodies and a greater selection of targets. Fine tuning of successful antibodies through modification of glycosylation, affinity, size and other parameters are paying dividends. Fc-engineering is likely to be predominant in the near future but conjugates, fragments and fusion proteins will continue to be developed and find their place in the arsenal of antibody therapeutics.