Liposomes have been employed as a delivery system for recombinant interleukin-2 (rIL-2) in cancer immunotherapy. In this study the effects of the rIL-2-bilayer interaction on protein structure were investigated. It was shown that rIL-2 adsorbs to liposomal membranes when added to preformed liposomes. Polarized fluorescence decay studies showed that the single tryptophan in "native" rIL-2 has a relatively large motional freedom, although iodide quenching of this residue's fluorescence was relatively ineffective. However, adsorption of rIL-2 to liposomes alters this situation dramatically- fluorescence intensity increased 2-fold and the residue became more susceptible to iodide quenching. At the same time, the average fluorescence lifetime of the fluorophore is extended. Interestingly, circular dichroism studies showed that no major conformational changes occurred in rIL-2's secondary structure upon adsorption. These observations support the hypothesis that intramolecular quenching takes place in the native rIL-2 molecule, which is abrogated upon adsorption to the liposomal membrane, resulting in a higher fluorescence intensity. Fluorescence anisotropy decay experiments indicate that the protein forms self-aggregates under the low-ionic strength conditions used, confirming the earlier observations on the tendency of the protein to precipitate in salt-containing media.

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The efficacy of tumor therapy using polyethylene-glycol-modified interleukin-2 (PEG-IL-2), alone or in combination with cyclophosphamide, was studied in advanced metastatic disease in the guinea pig. Line 10 (L10) tumor cells appeared in the axillary lymph node only 7 days after intradermal tumor-cell inoculation, and lymph-node leukocytes were almost completely replaced by tumor cells on day 28. Local treatment of the intradermally growing L10 hepatocarcinoma in the guinea pig with a relatively low dose of PEG-IL-2 resulted in regression of the primary tumor and prevention of lymph-node metastases. Therapy was completely curative (4 out of 5 animals) when started on day 7 or 14 after tumor-cell inoculation. When started on day 21, therapy was effective in only some (2 out of 5 cured) of the treated animals. Anti-tumor effects against the primary tumor and against lymph-node metastases were observed only after intratumoral (i.t.) administration of PEG-IL-2. Injection of the agent into or near lymph-node metastases in the absence of the primary tumor had no curative effect. In PBS/BSA-treated control animals the primary tumor and metastases grew progressively. In the treatment of far advanced metastatic disease, the combination of i.t. administration of PEG-IL-2 and i.p. injection of cyclophosphamide (Cy) resulted in improved anti-tumoral effects (5/5 guinea pigs were cured) when compared with monotherapy using either agent (one and none out of 5 animals cured, respectively). PBS/BSA heated controls showed progressive tumor-growth. We conclude that large primary tumors and lymph-node metastases can be treated effectively with PEG-IL-2. The i.t. route of administration is of major importance in the treatment of metastases, since administration of PEG-IL-2 near or into the lymph node had no therapeutic effect. Combination of PEG-IL-2 therapy with systemic injections of Cy significantly improved the curative effects of the treatment of advanced metastatic cancer.

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