The pharmacokinetics, tissue distribution, and efficacy of a systemic gene transfer method were examined in male BALB/c mice (6-8 weeks old) using 33P-labeled plasmid DNA for luciferase. The DNA was delivered via tail vein injection in saline ([33P]DNA) or in a cationic lipid formulation ([33P]DNA/lipid). One group of mice received approximately equal to 1-3 microCi (45 micrograms of DNA) of either formulation, and mice were euthanized at 2 and 20 min, and 1 and 24 h postdose (2 mice/time point). Blood and plasma radioactivity were quantified, and whole body autoradiographic (WBAR) images were obtained from 20-microns whole body sections. A tissue distribution (TD) study was conducted in a second group of mice, which received approximately equal to 4-6 microCi (45-60 micrograms of DNA) of [33P]DNA/lipid. Mice were euthanized at 1.5 h (1 mouse; [33P]DNA/lipid) or 24 h (2 mice/ group), and organ radioactivity and luciferase expression were measured in lung, liver, kidney, spleen thymus, and parotid salivary gland by direct quantitation methods. Microautoradiography (MAR) was performed on a third group of mice (n = 2), which received 3 microCi (45 micrograms of DNA) of [33P]DNA/lipid and were euthanized at 24 h postdose. For WBAR, the [33P]DNA/lipid tissue distribution (% dose equiv/g) at 2 min was lung >> liver > spleen (red pulp) > kidney (cortex); at 24 h the ranking was spleen (red pulp) > liver > lung, kidney (cortex). The [33P]DNA organ distribution observed at 2 min was liver >> spleen (red pulp) > lung, blood > kidney (cortex); at 24 h the ranking was liver, spleen (red pulp) > kidney (cortex) > lung, blood. High levels of radioactivity in bone (cortical, marrow, growth plate) in both groups may represent uptake of the 33P-labeled test articles by the cellular component of the bone marrow, particularly macrophages, as well as deposition of [33P]phosphate in the bone matrix following metabolism of the [33P]DNA. In the luciferase component of the study, no expression was observed in the [33P]DNA group at 24 h. The [33P]- DNA/lip group exhibited expression as early as 1.5 h in the lung; at 24 h, expression was seen in all the organs examined. Microautoradiography of 24-h tissue samples revealed radioactivity in hepatic Kupffer cells, reticuloendothelial system cells in the marginal zone of the spleen, and diffusely along alveolar septae with scattered accumulations in alveolar macrophages. The results of the WBAR, TD, MAR, and luciferase assay show that the use of cationic lipids significantly altered the biodistribution and resulting expression of the DNA plasmid. Further, 33P (0.25 MeV beta, half-life = 25 days) was shown to be an excellent radionuclide for quantitative WBA and MAR, providing sharp images with less personal hazard and greater ease of handling than 32P (1.71 MeV beta, half-life = 14.3 days).

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Tricalcium phosphate (TCP) was combined with amylopectin to form a deliverable carrier paste for recombinant human transforming growth factor beta 1 (rhTGF-beta 1) intended for bone repair applications. Approximately 80% of rhTGF-beta 1 was released from the carrier within 24 h following in vitro incubation in serum. Full biological activity was maintained, suggesting the growth factor was stable in this formulation before and after in vitro release. In vivo efficacy also was assessed, in comparison to a sham control group and a placebo-treated group, using a rabbit unilateral segmental defect model (1 cm). Radiographs of defect sites taken at scheduled intervals and the mechanical testing of treated limbs at 56 days demonstrated a higher incidence of radiographic bone union, in concert with a stronger torque strength, in the rhTGF-beta 1-treated group compared to the placebo group. The short duration of the study and the fact that the model used was not a critical defect may account for the lack of superiority of the rhTGF-beta 1-treated group over the healing of the sham control. The in vivo pharmacokinetics of the growth factor evaluated in the same rabbit model suggested that rhTGF-beta 1 persisted intact at the defect site for more than 21 days. Gamma imaging and radioactivity recovery at defects administered to [131I]- and [125I]-labeled rhTGF-beta 1, respectively, estimated the half-life of rhTGF-beta 1 eliminated from the applied site to be 4-6 days. The present report substantiates the potential of rhTGF-beta 1 and its carrier for treatment of bone defects.

We developed a novel bivalent antibody fragment, the linear (L-) F(ab')2, comprising tandem repeats of a heavy chain fragment VH-CH1-VH-CH1 cosecreted with a light chain. Functional humanized L-F(ab')2 directed against p185HER2 was secreted from Escherichia coli at high titer (> or = 100 mg/l) and purified to homogeneity. The L-F(ab')2 binds two equivalents of antigen with an apparent affinity (Kd = 0.46 nM) that is within 3-fold of the corresponding thioether-linked F(ab')2 fragment. The N-terminal site binds antigen with an affinity (Kd = 1.2 nM) that is approximately 4-fold greater than that for the C-terminal site, as shown by the comparison of L-F(ab')2 variants containing a single functional binding site. L-F(ab')2 has greater antiproliferative activity than the thioether-linked F(ab')2 against the p185HER2-overexpressing tumor cell line BT474. Linear and thioether-linked F(ab')2 have very similar pharmacokinetic properties in normal mice, and their serum permanence times are respectively 7- and 8-fold longer than the corresponding Fab fragment. L-F(ab')2 offers a facile route to bivalent antibody fragments that are potentially suitable for clinical applications, and that may have improved biological activity compared with thioether-linked F(ab')2 fragments.

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