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Abstract: High affinity and discriminating specificity are important parameters for any successful antibody based targeting strategy. We herein describe a system for the construction and subsequent selection of affinity-optimised chelating recombinant antibodies (CRAbs) from a randomised filamentous phage-display inter-scFv linker library. Using a simple, robust and highly degenerate tandem scFv cloning strategy a phage-display library of CRAbs with varied inter-scFv linkers was constructed and characterised. The library consisted of two single-chain Fvs (scFvs) of well characterised anti-lysozyme antibodies D1.3 and HyHEL-10TF, specific for distinct non-overlapping epitopes, separated by flexible polypeptide linkers of varying lengths and sequences. The use of a stringent affinity-based selection strategy quickly led to the enrichment of CRAbs with a restricted set of linker lengths (16–21 amino acids) which agrees very closely with previously described crystal structure data, affinity measurements and mathematical modelling. This CRAb linker phage-display selection strategy is a widely applicable approach for the selection of very high affinity CRAbs for pairs of scFvs against potentially any target antigen, complementing the more arbitrary affinity maturation approaches based on random mutagenesis. [Copyright &y& Elsevier]

States that when glycine of Escherichia coli glutahione is replaced by a bulky tryptophan residue, the enzyme becomes highly cooperative for glutathione binding; Two principal models advanced to explain cooperative bahavior; Cooperativity for glutathione binding and loss of cooperativity when mutant subunit is hybridized with a wild-type subunit to create a heterodimer.

Antibody–Drug Conjugates (ADCs) have been through multiple cycles of technological innovation since the concept was first practically demonstrated ~40 years ago. Current technology is focusing on large, whole immunoglobulin formats (of which there are approaching 100 in clinical development), many with site-specifically conjugated payloads numbering 2 or 4. Despite the success of trastuzumab-emtansine in breast cancer, ADCs have generally failed to have an impact in solid tumours, leading many to explore alternative, smaller formats which have better penetrating properties as well as more rapid pharmacokinetics (PK). This review describes research and development progress over the last ~10 years obtained from the primary literature or conferences covering over a dozen different smaller format-drug conjugates from 80 kDa to around 1 kDa in total size. In general, these agents are potent in vitro, particularly more recent ones incorporating ultra-potent payloads such as auristatins or maytansinoids, but this potency profile changes when testing in vivo due to the more rapid clearance. Strategies to manipulate the PK properties, whilst retaining the more effective tumour penetrating properties could at last make small-format drug conjugates viable alternative therapeutics to the more established ADCs. [ABSTRACT FROM AUTHOR]

Background: A significant number of cancers are caused by defects in p21 causing functional defects in p21 or p53 tumour-suppressor proteins. This has led to many therapeutic approaches including restoration by gene therapy with wild-type p53 or p21 using viral or liposomal vectors, which have toxicity or side-effect limitations. We set out to develop a safer, novel fusion protein which has the ability to reconstitute cancer cell lines with active p21 by protein transduction. Methods: The fusion protein was produced from the cell-translocating peptide Antennapedia (Antp) and wild-type, full-length p21 (Antp-p21). This was expressed and refolded from E. coli and tested on a variety of cell lines and tumours (in a BALB/c nude xenograft model) with differing p21 or p53 status. Results: Antp-p21 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over expression of MDM2. Non-specific toxicity was excluded by showing that Antp-p21 penetrated but did not kill p53- or p21- wild-type cells. Antp-p21 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of Antp-p21. Antp-p21 was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with conventionally-used chemotherapeutic agents, which alone were unable to produce such an effect. Conclusions: Antp-p21 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of medical oncology. [ABSTRACT FROM AUTHOR]

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 (≤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. [ABSTRACT FROM AUTHOR]

Background: Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections. Methods: PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29. Results: Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm2) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT. Conclusion: This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon. [ABSTRACT FROM AUTHOR]

Review: 124 refs. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug) and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs), which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP) to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs. [ABSTRACT FROM AUTHOR]

Abstract: Tumour-associated splice variants of fibronectin are a major source of tumour-matrix associated targets and are proving very successful in the development of clinical agents to treat cancer. One of the first monoclonal antibodies to be produced to this target, murine BC-1, recognises a cryptic epitope in domain 7 of the B-form splice variant (EDB-FN). Antibody fragments based on this immunoglobulin (IgG) were unstable, but BC-1 humanisation provided an opportunity to produce a more stable single-chain Fv (scFv). The variable domains of the humanized BC-1 IgG were sub-cloned and constructed into a scFv (HuBC-1 scFv) which was successfully expressed in Escherichia coli. The scFv retained its conformationally-sensitive epitope recognition and demonstrated a good affinity to the target of around 50nM as measured by ELISA, Surface Plasmon Resonance and Flow Cytometry. Furthermore, the scFv was thermostable and stable in serum allowing substantial localisation to human tumours grown in mouse xenograft models. This scFv could form the basis of future tumour-specific biopharmaceuticals. [Copyright &y& Elsevier]

Abstract: An approach for enhancing antibody affinity is to engineer Chelating Recombinant Antibodies (CRAbs) which consist of two tandemly linked single-chain Fvs (scFvs) that bind to distinct non-overlapping epitopes on the antigen molecule leading to a synergistic decrease in K D. In order to develop this technology, the aim of this present study was to identify scFvs which can simultaneously bind to the tetanus toxin heavy chain C-terminal sub-domain (Hc), characterise their bio-physical properties and determine their functional efficacy. Over 50 antibodies specific for Hc were isolated from a human scFv phagemid library and found to bind specifically to the C-terminal sub-domain of Hc (HcC clones), the N-terminal sub-domain (HcN clones) or junctional epitopes on the whole Hc fragment only (HcJ clones). Fifteen clones were assayed in a pairwise competition binding study. The revealed, with few exceptions, that HcC clones were able to simultaneously bind to the toxin with HcN or HcJ clones. All other combinations competed for binding. Interestingly, we also observed cooperative binding with many non-competing scFv pairings which may impact upon the binding mechanism of CRAbs. We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity. This included clones that did not bind to the HcC sub-domain which is responsible for direct interaction with gangliosides on nerve cells. For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency. The diverse array of clones characterised in this paper can be used to construct CRAbs and will prove useful in further characterisation of toxin biology and in measuring the effects of polyclonal antibody therapy. [Copyright &y& Elsevier]

Natural killer (NK) cells discern the health of other cells by recognising the balance of activating and inhibitory ligands expressed by each target cell. However, how the integration of activating and inhibitory signals relates to formation of the NK cell immune synapse remains a central question in our understanding of NK cell recognition. Here we report that ligation of LFA-1 on NK cells induced asymmetrical cell spreading and migration. In contrast, ligation of the activating receptor NKG2D induced symmetrical spreading of ruffled lamellipodia encompassing a dynamic ring of f-actin, concurrent with polarization towards a target cell and a ''stop'' signal. Ligation of both LFA-1 and NKG2D together resulted in symmetrical spreading but co-ligation of inhibitory receptors reverted NK cells to an asymmetrical migratory configuration leading to inhibitory synapses being smaller and more rapidly disassembled. Using micropatterned activating and inhibitory ligands, signals were found to be continuously and locally integrated during spreading. Together, these data demonstrate that NK cells spread to form large, stable, symmetrical synapses if activating signals dominate, whereas asymmetrical migratory ''kinapses'' are favoured if inhibitory signals dominate. This clarifies how the integration of activating and inhibitory receptor signals is translated to an appropriate NK cell response. [ABSTRACT FROM AUTHOR]