Synopsis The serine proteases of the trypsin-like (S1) family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Recent studies have identified members of this family which contain amino- or carboxy-terminal domains that serve to tether the serine protease catalytic domain directly at the plasma membrane. These membrane anchored serine proteases are proving to be key components of the cell machinery for activation of precursor molecules in the pericellular microenvironment, playing vital functions in the maintenance of homeostasis. Substrates activated by membrane anchored serine proteases include peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules and viral coat proteins. In addition, new insights into our understanding of the physiological functions of these proteases and their involvement in human pathology have come from animal models and patient studies. This review discusses emerging evidence for the diversity of this fascinating group of membrane serine proteases as potent modifiers of the pericellular microenvironment through proteolytic processing of diverse substrates. We also discuss the functional consequences of the activities of these proteases on mammalian physiology and disease.

Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2' þ '/ErbB3' þ ' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2' þ '/ErbB3' þ ' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.

Purpose: Successful radioimmunotherapy strategies depend on selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered antitumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that the C6.5 diabody, a noncovalent anti-HER2 single-chain Fv dimer, would be an ideal radioisotope carrier for the radioimmunotherapy of established tumors using the short-lived a-emitting radioisotope At-SAPS on the anti^Mu« llerian-inhibiting substance type II receptor diabody did not affect tumor growth rate, demonstrating specificity of the therapeutic effect. Conclusions: These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived a-emitting radioisotopes.Radioimmunotherapy exploits the highly specific antigen specificity of antitumor antibody molecules to selectively target and retain therapeutic radioisotopes in tumors. Radioimmunotherapy has been associated with significant clinical therapeutic outcomes in the treatment of hematologic (diffuse) malignant diseases, which has led to the approval of two anti-CD20 monoclonal antibodies (mAb) for radioimmunotherapy applications (1). However, similar clinical successes have yet to be achieved in the treatment of solid, established malignancies. This is thought to be due to a number of factors related to the size of the antibodies and the properties of commonly used therapeutic radioisotopes (2, 3). These include limited tumor penetration and prolonged circulation of intact mAb, which together impair the ability to treat a tumor and increases the bone marrow toxicity; the requirement for thousands of traversals of h-particles through a single tumor cell to mediate its death; and the disparate biological half-lives of intact mAbs and physical half-lives of the commonly used h-emitting radioisotopes such as iodine-131 ( 131 I) and yttrium-90 ( 90 Y). To address these issues, we have focused on developing smaller antibody-based molecules that are capable of greater tumortargeting specificity and pairing them based on their biological half-lives with high-energy therapeutic radioisotopes that have complementary physical half-lives.Noncovalent single-chain Fv (scFv) dimers, known as diabodies, can be formed by producing scFv molecules with short (5 aa) linkers between their variable light (V L ) and variable heavy (V H ) chains (4). This prevents the V H and V L chains from a single molecule from associating with each other Cancer Therapy: Preclinical

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