Abstract: Using genetic engineering a humanized Fab fragment with specificity for CD19 was fused to a disulfide-stabilized single-chain antibody (dsFv) recognizing CD5. This format should show reduced immunogenicity and improved tissue penetration. The specificity of bsAb FabCD19xdsFvCD5 binding to target cells was verified by flow cytometry on B and T lymphoma cell lines. Binding affinities of both arms were compared with the bivalent parental antibodies against CD19 and CD5 by binding competition assay. Redirected lysis of B lymphoma cells by preactivated PBMC from healthy donors was demonstrated in a chromium-release assay. A clear dose-response relationship could be established in the range from 1 ng/mL to 10 mg/mL bsAb. To evaluate the in vivo efficacy of bsAb FabCD19xdsFvCD5, NOD/SCID mice were intravenously injected with luciferase transfected Raji lymphoma cells together with pre-activated PBMC. Mice received five injections of therapeutic bsAb or control antibodies. While in the control groups all mice died within 40 to 50 days, 40% of bsAb treated animals survived longer than 60 days.

Abstract: We previously demonstrated that the Fc receptor γ-chain Y⁵⁸(C-terminal tyrosine) is highly susceptible to dephosphorylation; a mechanism that controls the extent of Syk activation and the downstream signaling in mast cells. Here, we explored the importance of the γ-chain Y⁴⁷ (N-terminal tyrosine) in mast cell signaling. We generated a highly sensitive and versatile phospho-specific antibody that recognized the phosphorylated Y⁴⁷ in various species. Using this antibody, we found that mutation of the FcεRIβ Y²¹⁹ to phenylalanine caused a loss in the phosphorylation of the γ-chain Y⁴⁷, consistent with the previously described role of Y²¹⁹ in Lyn association with FcεRIβ and subsequent FcεRIγ phosphorylation. These conditions also diminished the tyrosine phosphorylation of Syk and LAT1 but, surprisingly, not the phosphorylation of Akt at T³⁰⁸. Mutation of Y⁴⁷ or Y⁵⁸ of the γ-chain also caused a marked inhibition of Syk and LAT1 phosphorylation, but only the latter mutant showed a reduction in Akt phosphorylation. These findings show that the full phosphorylation of Syk and LAT1 requires the FcεRIβ Y²¹⁹ and both Y⁴⁷ and Y⁵⁸ of the γ-chain. However, T³⁰⁸ phosphorylation of Akt is largely independent of FcεRIγ Y⁴⁷ phosphorylation and of the Lyn-binding site (Y²¹⁹) on the FcεRIβ.

Abstract: Many human diseases are caused by mutant or abnormal protein functions that are largely confined to the inside of cells, rather than being displayed on the abnormal cell surface. Furthermore, many of the functional consequences of aberrant proteins, such as in cancer cells, are due to protein–protein interactions (PPIs). Developing reagents that can specifically interfere with PPI is an important goal for both therapeutic use and as reagents to interrogate the functional importance of PPI. Antibody fragments can be used for inhibiting PPI. Our recent technology development has provided a set of simple protocols that allow development of single antibody variable (V) region domains that can function inside the reducing environment of the cell. The heavy chain variable region (VH) segments mainly used in this technology are based on a designer framework that folds inside cells without the need for the intra-chain disulphide bond and can be used as drug surrogates to determine on-target effects (target validation) and as templates for small molecule drug development. In this review, we discuss our work on single domains as intracellular antibodies and where this work might in the future.

Abstract: 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: Ricin is a type II ribosome inactivating protein (RIP) isolated from castor beans. Its high toxicity classifies it as a possible biological weapon. On the other hand, ricin linked to specific monoclonal antibodies or used in other conjugates has powerful medical applications. Ricin consists of an A-chain (RTA) that damages ribosomes and inhibits protein synthesis, and a B-chain that plays a role in binding and cellular uptake. A number of recent studies have demonstrated that ricin-induced inhibition of protein synthesis is not the only mechanism responsible for cell death. It turns out that ricin is able to induce apoptosis in different cell lines and multiple organs in animals. However, the molecular link between protein synthesis inhibition and ricin-dependent triggering of apoptotic cell death is unclear. This review describes the intracellular transport of ricin and ricin-based immunotoxins and their mechanism of action in different non-malignant and cancer cell lines. Moreover, various ricin-containing immunotoxins, their composition, medical applications and side-effects will be described and discussed. Understanding the mechanism of action of ricin-based immunotoxins will facilitate construction of effectively acting immunotoxins that can be used in the clinic for cancer treatment.

Abstract: Membranes are vital barriers by which cells control the flux of molecules and energy between their exterior and interior and also between their various intracellular compartments. While numerous transport systems exist for ions and small molecules, the cytosolic uptake of larger biological molecules and in particular antibody-targeted drugs, is a big challenge. Inducing leakage of the plasma membrane is unfavorable since the target cell specificity mediated by the antibody would likely be lost in this case. After binding and internalization, the antibody drug conjugates reach the endosomes. Thus, enforcing the endosomal escape of anti-tumor toxins without affecting the integrity of other cellular membranes is of paramount importance. Different strategies have been developed in the last decades to overcome endosomal accumulation and subsequent lysosomal degradation of targeted protein-based drugs. In this review we summarize the various efforts made to establish efficient techniques to disrupt the endosomal membrane barrier including the use of molecular ferries such as cell penetrating peptides or viral membrane fusion proteins, endosomal leakage inducing molecules such as saponins or monensin and physicochemical methods as represented by photochemical internalization.