Doubly radiolabeled liposomes for pretargeted radioimmunotherapy.

The aim of this study was to design liposomes as radioactivity carriers for pretargeted radioimmunotherapy with favorable pharmacokinetic parameters. To monitor the liposomes integrity in vivo, their surface was radiolabeled with indium-111 bound to DTPA-derivatized phosphatidylethanolamine (DSPE-DTPA) and the aqueous phase was labeled by using an original active loading technique of radioiodinated Bolton-Hunter reagent (BH) that reacts with pre-encapsulated arginine to form a positively charged conjugate ((125)I-BH-arginine). Different formulations of doubly radiolabeled liposomes were tested in vitro and in vivo to evaluate radiolabeling stability, integrity of the vesicles and their pharmacokinetics. Radiolabeling yields were high (surface >75%, encapsulation >60%) and stable (>85% after 24 h in serum 37 degrees C). In vivo, the pharmacokinetic behavior of doubly radiolabeled liposomes was strongly dependant on the formulation. Blood clearance of PEGylated liposomes (DSPC/Chol/DSPE-DTPA/DSPE-PEG5%) was 0.15 mL/h compared to a conventional formulation (DSPC/Chol/DSPE-DTPA: clearance 1.44 mL/h). Non-encapsulated BH-arginine conjugate was quickly eliminated in urine (clearance 6.04 mL/h). Blood kinetics of the two radionuclides were similar and radiochromatographic profiles of mice serum confirmed the integrity of circulating liposomes. The significant reduction of activity uptake in organs after liposome catabolism (liver and spleen), achieved by the rapid renal elimination of (125)I-BH-arginine, should bring significant improvements for targeted radionuclide therapy with sterically-stabilized liposomes.

Influence of pegylation and hapten location at the surface of radiolabelled liposomes on tumour immunotargeting using bispecific antibody.

INTRODUCTION: This paper proposes liposomes as a potential new tool for radioimmunotherapy in solid tumours with a two step targeting system. Tumour pretargeting is obtained by using a monoclonal bispecific antibody (BsmAb, anti CEA x anti-DTPA-In) and pegylated liposomes containing lipid-hapten (DSPE-DTPA-In or DSPE-PEG-DTPA-In). To optimise at the same time in vivo behaviour and specific targeting, the study focuses on the liposome formulation in order to determine more precisely the role of pegylation on both the blood half-life and the specific recognition with the BsmAb. METHODS: Different liposome formulations containing two PEG length (1000 and 2000) in varying amount (1.5-6 mol%) were prepared with DTPA directly coupled to DSPE or at the end of the PEG chain (DSPE-DTPA or DSPE-PEG-DTPA). Liposomes were immobilized on an L1 chip to measure by SPR (Surface Plasmon Resonance) the effect of pegylation on the BsmAb recognition of the DTPA-In hapten. Pharmacokinetic studies were performed in mice. Tumour targeting was studied in nude mice xenografted with human colorectal adenocarcinoma cells that express CEA, and doubly radiolabelled liposomes (with (111)In and (125)I) injected 24h after the BsmAb. RESULTS: The best in vitro apparent dissociation constant was obtained with liposomes bearing DTPA at the end of the PEG chain (KD=6.3 nM), which showed significant specific tumour uptake after BsmAb injection (8.6 ± 2.4% ID/g at 24h versus 4.5 ± 0.5%ID/g for passive targeting, α=0.01). All tumour/organ ratios were superior to 1 at 24h for this formulation, except for the spleen. CONCLUSION: The feasibility of specific tumour targeting in mice with a BsmAb and radiolabelled liposomes was demonstrated and the interest of SPR to predict their targeting performance in vivo was highlighted. This original and new approach provides promising prospects for the radioimmunotherapy of solid tumours.

Radiolabeling and targeting of lipidic nanocapsules for applications in radioimmunotherapy.

AIM: Radioimmunotherapy is limited in some cases by the low radioactive doses delivered to tumor cells by antibodies or pretargeted haptens. In order to increase this dose, lipidic nanocapsules (LNC) with a hydrophobic core are proposed as radionuclide vectors that could be targeted to cancer cells by a bispecific anti-tumor x anti-hapten antibody after incorporation of different haptens in the nanocapsule membrane. METHODS: To bind different radionuclides to the nanocapsules, several bifunctional chelating agents (BCA) were used to form stable complexes with the radionuclides. Some of them are hydrophilic for LNC shell while others are lipophilic to radiolabel the core. Poly(ethylene glycols) (PEG) were used to increase the residence time in blood. Since PEG can modify haptens recognition by the bispecific antibody and radiolabeling efficiency, haptens, BCA or Bolton-Hunter reagent (BH) were attached to the PEG extremity to optimize accessibility. Specific constructs (DSPE-PEG-haptens, DSPE-PEG-BCA, and DSPE-PEG-BH) were synthesized to develop these new radiolabeled vector formulations. Large amounts of PEG have been introduced by a postinsertion method without important change in nanocapsule size and properties. The nanocapsule core was radiolabeled with a lipophilic [(99m)Tc]SSS complex. RESULTS: Serum stability studies showed that this (99m)Tc-labeling method was efficient for at least 20 h. Concerning the nanocapsule surface, several methods have been performed for (111)In-labeling by using DSPE-PEG-DTPA and for (125)I-labeling with DSPE-PEG-BH. CONCLUSIONS: The nanocapsules labeling feasibility with a variety of radionuclides and their stability were demonstrated in this paper.