The growth of solid tumours is characterized not only by the uncontrolled proliferation of cancer cells but also by changes in the tumour environment that support the growth of the neoplastic mass and the metastatic spread of cancer cells to distant sites 1 . The formation of new blood vessels from pre-existing ones (angiogenesis) provides oxygen and nutrients to the tumour, which are essential for tumour growth 2 . A complex dynamic interplay exists between the expanding neoplastic mass and the tumour environment, which is affected by the metabolic requirements of cancer cells and by their products, such as secreted proteins (for example, extracellular matrix components and proteases) and metabolites.Upregulated glucose metabolism is a hallmark of invasive cancers 3 . In normal cells glucose is converted to glucose-6-phosphate and subsequently to pyruvate, which is then oxidized in the mitochondria to carbon dioxide and water; this releases 38 moles of ATP per mole of glucose 3 . However, inadequate oxygen delivery to some regions of tumours leads to hypoxia, which restricts oxidative phosphorylation. As a consequence, hypoxic tumour cells shift their metabolism towards glycolysis so that the pyruvate generated in the first step of the process is reduced to lactate, generating only 2 moles of ATP per mole of glucose. This is a less energy-efficient process but it does not depend on oxygen. Furthermore, glycolysis often persists even after reoxygenation because the obtained metabolic intermediates (that is, lactate and pyruvate) can be used for the biosynthesis of amino acids, nucleotides and lipids, thus providing a selective advantage to proliferating tumour cells. This explains Warburg's observation of high glucose consumption and high lactate production in tumour tissues 3-5 (known as the Warburg effect).Oncogenic metabolism also generates an excess of protons and carbon dioxide, which are kept in equilibrium with carbonic acid by the enzyme carbonic anhydrase 3-9 . Thus, increased glucose metabolism in tumour cells leads to enhanced acidification of the extracellular milieu, which is frequently accompanied by various levels of hypoxia. This phenotype confers a substantial Darwinian growth advantage to tumour cells over normal cells, which undergo apoptosis in response to such an acidic extracellular environment 3 .Tumour cells have evolved various mechanisms to cope with the acidic and hypoxic stress mentioned above. They eliminate acidic catabolites by ion transporters and pumps to preserve a slightly alkaline intracellular pH (pH i ), which is optimal for cell proliferation and tumour survival 3-10 . Acid export leads to a reduction in the extracellular pH (pH e ) to values as low as 6.0 (the usual pH e in tumours is in the range of 6.5-7.0) 3 , which is a salient feature of the tumour microenvironment 3-9 . As well as triggering the overexpression of many proteins involved in glucose metabolism -such as the glucose transporter GLUT1 (also known as SLC2A1) and pH-regulating proteins such as carbonic anhyd...

It is now accepted that the growth of solid tumours is dependent on their capacity to acquire a blood supply, and much effort has been directed towards the development of agents (known as anti-angiogenics) that disrupt this process. More recently, it has become apparent that targeted destruction of the established tumour vasculature is another avenue for exciting therapeutic opportunities. In this article, we present evidence that vascular targeting is an effective antitumour strategy in animal models, describe strategies for identifying putative tumour vascular targets and discuss future prospects for vascular targeting in the clinic.

Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.

A nuclear magnetic resonance (NMR) structure determination is reported for the polypeptide chain of a globular protein in strongly denaturing solution. Nuclear Overhauser effect (NOE) measurements with a 7 molar urea solution of the amino-terminal 63-residue domain of the 434-repressor and distance geometry calculations showed that the polypeptide segment 54 to 59 forms a hydrophobic cluster containing the side chains of Val54, Val56, Trp58, and Leu59. This residual structure in the urea-unfolded protein is related to the corresponding region of the native, folded protein by simple rearrangements of the residues 58 to 60. Based on these observations a model for the early phase of refolding of the 434-repressor(1-63) is proposed.

We recently demonstrated that a human recombinant scFv, L19, reacting with the ED-B domain of fibronectin, a marker of angiogenesis, selectively targets tumoral vasculature in vivo. Using the variable regions of L19, we constructed and expressed a human "small immunoprotein" (SIP) and a complete human IgG1 and performed biodistribution studies in tumor-bearing mice to compare the blood clearance rate, in vivo stability and performance in tumor targeting of the 3 L19 formats [dimeric scFv (scFv) 2 , SIP and IgG1]. The accumulation of the different antibody formats in the tumors studied was a consequence of the clearance rate and in vivo stability of the molecules. Using the SIP, the %ID/g in tumors was 2-5 times higher than that of the (scFv) 2 , reaching a maximum 4 -6 hr after injection. By contrast, the accumulation of IgG1 in tumors constantly rose during the experiments. However, due to its slow clearance, the tumor-blood ratio of the %ID/g after 144 hr was only about 3 compared to a ratio of 10 for the (scFv) 2 and 70 for the SIP after the same period of time. The different in vivo behavior of these 3 completely human L19 formats could be exploited for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease. Furthermore, the fact that ED-B is 100% homologous in human and mouse, which ensures that L19 reacts equally well with the human and the murine antigen, should expedite the transfer of these reagents to clinical trials. © 2002 Wiley-Liss, Inc. Key words: antibody formats; tumor vasculature; tumor targeting; clinical applications; cancer diagnosis and therapyDespite their enormous potential as therapeutic agents, monoclonal antibodies (mAbs) of nonhuman origin have not performed as well as expected in clinical trials as a result of their immunogenicity, 1,2 poor pharmacokinetic properties 3,4 and inefficiency in recruiting effector functions. 5,6 The recent prospect of isolating human antibody fragments from phage display libraries 7-10 transcends these problems, revitalizing studies and rekindling hopes of using these reagents to treat major diseases. Indeed, these molecules should serve as ideal building blocks for novel diagnostic and therapeutic tools. 11,12 Furthermore, these antibodies can be "matured" to reach affinities in the picomolar range, 13 desirable, if not necessary, for their clinical use. 14,15 Clinical applications of human antibody fragments for the selective delivery of diagnostic or therapeutic agents nonetheless require highly specific targets. In the case of tumors, the most popular targets are cell-surface antigens, which are usually neither abundant nor stable. On the other hand, during tumor progression the microenvironment surrounding tumor cells undergoes extensive modification that generates a "tumoral environment" that could ultimately represent a suitable target for antibody-based tumor therapy. 16 In fact, the concept that the altered tumor microenvironment is itself a carcinogen that can be targeted is increasingly gaining consensus. Mol...