Sustained cytokine delivery for anticancer vaccination: liposomes as alternative for gene-transfected tumor cells.

Vaccination with tumor cells genetically engineered to produce interleukin (IL)-2 is an attractive strategy to enhance antitumor immune responses. The improved antitumor immunity upon vaccination with IL-2 gene-modified tumor cells may be due to the prolonged presence of the cytokine at the vaccination site. Because liposomes have been used for sustained delivery of a variety of agents, we compared the protective effect of vaccines consisting of IL-2 gene-modified B16 melanoma cells to that of vaccines composed of IL-2 liposomes and irradiated melanoma cells. The results indicate that both approaches equally protect against a lethal challenge with B16 melanoma cells. More than 20% of the protected animals developed vitiligo at the vaccination and/or tumor challenge site.

Interaction of recombinant interleukin-2 with liposomal bilayers.

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.

Liposomes as cytokine-supplement in tumor cell-based vaccines.

Subcutaneous vaccination of C57bl/6 mice with irradiated B16 melanoma cells supplemented with liposomal interleukin-2 (IL2) or murine interferon-gamma (mIFNgamma), resulted in systemic protection in 50% of the animals, against a subsequent tumor cell challenge in a dose dependent manner. The protective efficacy was comparable to the efficacy of cytokine gene-modified cells as tumor vaccine, whereas irradiated B16 cells supplemented with soluble cytokine did not result in protective responses. In vivo evidence was obtained that the beneficial effects mediated by liposome incorporation of the cytokine are the result of a depot function of the liposomal cytokine supplement at the vaccination site. In can be concluded that liposomal delivery of cytokines offers an attractive alternative to cytokine-gene transfection of tumor cells for therapeutic vaccination protocols.

Development of a routine analysis method for liposome encapsulated recombinant interleukin-2.

This paper describes the development of an isocratic reversed-phase high-performance liquid chromatographic method for the routine analysis of recombinant interleukin-2 (rIL-2) in liposome samples. The chromatographic system employed a C4 column maintained at 30 degrees C eluted with 52.5% (w/w) acetonitrile in water, containing 100 mM NaClO4 and 10 mM HClO4. To remove phospholipid interference the chromatographic method was combined with a lipid-extraction procedure. No significant loss of rIL-2 was noted upon inclusion of this extraction step. The protein eluted from the column with a capacity factor (k') of 5.8. The method was validated for robustness, linearity, precision and reproducibility. It was shown that the method was linear over a sample concentration range of 1-100 microg/ml. Upon assessment of the intra-day and inter-day precision, the relative standard deviations (RSD) were within the range of the methodical error (approximately 5%), except at the lower concentration of 10 microg/ml, where the intra-day RSD was relatively high (17.8%). The recovery of rIL-2 upon liposome preparation and subsequent analysis of the samples was in the range 94+/-9%. The results indicate that the method is suitable for routine quantitation of rIL-2 in liposomal samples.

Specific tumor memory induced by polyethylene-glycol-modified interleukin-2 requires both helper and cytotoxic T cells.

Local polyethylene-glycol (PEG)-modified interleukin-2 (IL-2) immunotherapy of the guinea pig Line 10 (L10) tumor was previously demonstrated to evoke long-lasting systemic immunity after cure of the tumor and metastases. T cells, most likely the helper T cell subpopulation, were demonstrated to be crucial to the antitumor effects. Here we show that systemic immunity is induced within 7 days after the start of PEG-IL-2 therapy, indicating a rapid systemic priming of L10-specific T cells. No in vitro cytotoxic activity was detected in cell suspensions obtained from the primary tumor site, the regional lymph node or the spleen when isolated during (days 21 and 28) intratumoral treatment with 200,000 IU PEG-IL-2. These data confirm our earlier results obtained with 60,000 IU PEG-IL-2. Moreover, no cytolytic activity was observed in the chromium-release assay after in vitro restimulation with irradiated tumor cells. Specific L10 immunity can be transferred using spleen cell suspensions. Depletion of such a suspension of helper T cells resulted in rejection of the primary tumor in two out of four animals, but all the guinea pigs developed lymph node metastases. Removal of the cytotoxic/suppressor phenotype caused rejection of the dermal tumor in four of eight guinea pigs, but the capacity to prevent lymph node metastases was retained in all animals. Thus, depletion of either subtype reduces, but does not abrogate, the capacity to transfer L10 immunity with spleen cells. In conclusion, our data suggest that tumor cell killing through direct T cell cytotoxicity is not the main mode of action in PEG-IL-2-induced L10 tumor regression. PEG-IL-2 therapy induces early systemic immunity, resulting in rejection of a distant tumor, and the transfer of this immunity depends mainly on the presence of helper T cells, although cytotoxic T cells may also play a role.

PEG-IL-2 therapy of advanced cancer in the guinea pig. Impact of the primary tumor and beneficial effect of cyclophosphamide.

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.