Figure 3

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.