CCR7-guided neutrophil redirection to skin-draining lymph nodes regulates cutaneous inflammation and infection.

Neutrophils are the first nonresident effector immune cells that migrate to a site of infection or inflammation; however, improper control of neutrophil responses can cause considerable tissue damage. Here, we found that neutrophil responses in inflamed or infected skin were regulated by CCR7-dependent migration and phagocytosis of neutrophils in draining lymph nodes (dLNs). In mouse models of Toll-like receptor-induced skin inflammation and cutaneous Staphylococcus aureus infection, neutrophils migrated from the skin to the dLNs via lymphatic vessels in a CCR7-mediated manner. In the dLNs, these neutrophils were phagocytosed by lymph node-resident type 1 and type 2 conventional dendritic cells. CCR7 up-regulation on neutrophils was a conserved mechanism across different tissues and was induced by a broad range of microbial stimuli. In the context of cutaneous immune responses, disruption of CCR7 interactions by selective CCR7 deficiency of neutrophils resulted in increased antistaphylococcal immunity and aggravated skin inflammation. Thus, neutrophil homing to and clearance in skin-dLNs affects cutaneous immunity versus pathology.

Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature.

Interleukin-12 (IL-12) is a heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. Its clinical applications are limited, however, by severe side-effects. Here we report that an IL-12 fusion protein, consisting of IL-12 fused to a human antibody fragment specific to the oncofetal ED-B domain of fibronectin, markedly enhances the antitumor activity of this cytokine, as demonstrated in a mouse lung-metastasis model and in two models of mice bearing different aggressive murine tumors. The residual small tumor masses seen in the treated mice were infiltrated with lymphocytes, macrophages, and natural killer cells and had elevated interferon gamma (IFN-gamma). These results are of therapeutic relevance as the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is not detectable in normal vessels and tissues.

Antibody-based targeting of angiogenesis.

The selective targeting of neovasculature opens new avenues for the diagnosis and therapy of angiogenesis-related diseases such as cancer, blinding ocular disorders, and rheumatoid arthritis. Here we review recent advances in the identification of markers of angiogenesis as well as in the isolation and use of antibodies (and their derivatives) for the in vivo targeting of both tumoral and nontumoral neovasculature.

Antibody-Based targeting of angiogenesis.

Angiogenesis-the sprouting of new blood vessels from pre-existing ones-is a characteristic feature of relevant diseases such as cancer, some blinding ocular disorders, rheumatoid arthritis, and psoriasis. This article reviews recent progress in the generation of monoclonal antibodies, which recognize and target new blood vessels in vivo, while sparing mature vessels and healthy tissues. The use of such antibodies as selective "molecular vehicles" is likely to open new important diagnostic and therapeutic opportunities.

Elastase substrate specificity tailored through substrate-assisted catalysis and phage display.

The catalytic histidine of human neutrophil elastase was replaced with alanine (H57A) to determine if a substrate histidine could substitute for the missing catalytic group-'substrate-assisted catalysis'. H57A and wild-type elastase were recovered directly from Pichia pastoris following expression from a synthetic gene lacking the elastase pro sequence, thereby obviating the need for zymogen activation. Potential histidine-containing substrates for H57A elastase were identified from a phage library of randomized sequences. One such sequence, REHVVY, was cleaved by H57A elastase with a catalytic efficiency, k(cat)/K(M), of 2800 s(-1) M(-1), that is within 160-fold of wild-type elastase. In contrast, wild-type but not H57A elastase cleaved the related non-histidine containing sequence, REAVVY. Ten different histidine-containing linkers were cleaved by H57A elastase. In addition to the requirement for a P2 histidine, significant preferences were observed at other subsites including valine or threonine at P1, and methionine or arginine at P4. A designed sequence, MEHVVY, containing the preferred residues identified at each subsite proved to be a more favorable substrate than any of the phage-derived sequences. Extension of substrate-assisted catalysis to elastase suggests that this engineering strategy may be widely applicable to other serine proteases thereby creating a family of highly specific histidine-dependant proteases.

Infrared photodetection for the in vivo localisation of phage-derived antibodies directed against angiogenic markers.

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a characteristic process which underlies many diseases, including cancer, rheumatoid arthritis and blinding ocular disorders. Antibodies capable of selective targeting and occlusion of neovasculature would open diagnostic and therapeutic opportunities. We have recently demonstrated that phage-derived human antibody fragments with high affinity for the extra-domain B (ED-B) of fibronectin, a marker of angiogenesis, selectively localise in new-forming blood vessels upon intravenous injection. Here, we show that infrared fluorescence methodologies nicely complement radioactive techniques for the study of the antibody-mediated targeting of angiogenesis in a variety of animal models. Methods are presented for the construction and use of infrared fluorescence imagers, as well as for the production and characterisation of recombinant antibodies labeled with infrared fluorophores.

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.