Histochemical analysis of arthritc paw sections. Immunohistochemistry with the small immunoprotein L19 on arthritic paw sections reveals expression of extra domain B of fibronectin (EDB) in the tunica synovialis and inflammatory infiltrates (a) and (b). An immunofluorescent co-staining shows co-localisation of CD31 (green) and EDB (red) surrounding vascular structures (c)–(e). Scale bars = 100 μm.

Investigation of the selective accumulation of the small immunoproteins L19 and G11 in inflamed limbs of arthritic mice. Arthritic mice were injected with SIP(L19)–Alexa750 (Molecular Probes, Leiden, The Netherlands), SIP(G11)–Alexa750 or the control antibody SIP(HyHEL10)–Alexa750. Near-infrared fluorescence imaging analysis was performed 24 hours after injection of the fluorescence-labelled antibodies. Arrows indicate grade 2 swelling in the front paws of the mice. In a similar experiment, arthritic mice were injected with ¹²⁵I-labelled SIP(L19), SIP(G11) or SIP control. Uptake of radio-iodinated antibodies was analysed by phosphorimaging 24 hours after injection. The mouse injected with ¹²⁵I-labelled SIP(L19) had an arthritic score of 2 in the left paw, whereas the right paw was classified as grade 1 arthritis. In the mouse injected with ¹²⁵I-labelled SIP(G11) the left paw was classified as grade 1 arhritis, whereas the right paw was classified as grade 2 arthritis. In the mouse injected with ¹²⁵I-labelled SIP(F16) the left paw was classified as grade 2 arthritis, whereas the right paw as grade 1 arthritis.

Selective delivery of cytokines to arthritic lesions. Arthritic mice were given an intravenous (i.v.) injection in the lateral tail vein with saline (black circles), L19–IL-2 (black triangles; dashed line), L19–tumour necrosis factor (TNF) α (crosses; dashed line) or L19–IL-10 (open squares) diluted in a volume of 200 μl of saline. Injections were started at day 1 after arthritis onset and then repeated every second day for three injections per animal, as indicated by the arrows. The cumulative doses for the fusion proteins were 60 μg of L19–IL-2, 15 μg of L19–TNF and 450 μg of L19–IL-10 per mouse. The arthritic score was evaluated daily and expressed as the mean ± the standard error of the mean (SEM) (a). Paw swelling was measured every second day and paw thickness was expressed as the mean of all four paws of each animal. The results are displayed as the mean ± SEM for each group (b). Each group contained seven mice. Comparison of targeted delivery and systemic application of IL-10 to arthritic mice. Arthritic mice were given an i.v. injection in the lateral tail vein with saline (black circles), L19–IL-10 (open squares) or HyHel10–IL-10 (crosses; dashed line) diluted in a volume of 200 μl of saline. Injections were started at day 1 of arthritis onset and then repeated every second day for three injections per animal, as indicated by the arrows. The cumulative doses for the fusion proteins were 450 μg per mouse. The arthritic score was evaluated daily and expressed as the mean ± SEM (c). Paw swelling was measured every second day and paw thickness was expressed as the mean of all four paws for each animal. The results are displayed the mean ± SEM for each group (d). Each group contained six mice.

Cloning, expression and purification of L19–IL-10 and HyHel10–IL-10. (a) Schematic representation of single-chain variable fragment-IL-10 fusion proteins. (b) Schematic representation of a pcDNA3.1 vector (Invitrogen Basel, Switzerland) containing the essential elements of the L19–IL-10 or HyHEL10–IL-10 fusion proteins. The human IL-10 moiety was fused to the C-terminal of the single-chain Fv antibody fragment by the 15 amino acid linker (SSSSG)₃. The secretion sequence at the N-terminal is required for secretion of recombinant proteins and the His₆ tag at the C-terminal of human IL-10 was used for detection of the fusion proteins. (c) SDS-PAGE analysis of purified fusion proteins: lane 1, molecular-weight marker; lanes 2 and 3, L19–IL-10 under nonreducing and reducing conditions, respectively; lanes 4 and 5, HyHEL-IL-10 under nonreducing and reducing conditions, respectively. Monomeric fusion proteins are expected to have a molecular weight of 46 kDa. (d) The size-exclusion chromatography profile of purified L19–IL-10 (Superdex 200, GE Healthcare, Duebendorf, Switzerland). The peak eluting at a retention volume of 13 ml corresponds to the noncovalent homodimeric form of L19–IL-10, the smaller peak eluting at a retention volume of 16 ml corresponds to the monomeric fraction. (e) Biodistribution profile of L19–IL-10 in 129Sv mice grafted with a subcutaneous F9 tumour (n = 4). L19–IL-10 was labelled with ¹²⁵I and administered by intravenous (i.v.) injection into tumour-bearing mice (3 μg corresponding to 4 μCi L19–IL-10 per mouse). Mice were sacrificed 24 hours after injection and the tumours and organs were weighed and counted. Values are displayed as percent injected dose per gram (%ID/g); standard errors of the means (SEMs) are indicated.