As shown previously, encapsulation of a peptide derived from tyrosinase-related protein2 (TRP2) into liposomes (artificial virus envelope (AVE) 3) resulted in combination with CpG-oligodeoxynucleotides in the induction of higher numbers of antigen-specific T cells compared to vaccination with free TRP2.Here, we present further data with regard to optimal antigen dose, the relevance of vaccine injection site and on the T cell stimulatory synergism of liposomal adjuvant combinations.Compared to an aqueous solution liposomal TRP2 was more potent in the induction of TRP2-specific T cells at an optimal dose but showed a narrow dose optimum with profoundly impaired T cell responses at higher vaccine doses.Higher T cell numbers were induced when mice were vaccinated into their hint foodpads compared to intradermal vaccination, the site used routinely in murine tumor vaccination models.A synergistic adjuvant effect was observed when CpG-oligodeoxynucleotides were admixed with liposomal monophosphoryl lipid A (MPLA) and the lipopeptide Pam 3 Cys, respectively.In summary our data demonstrate that liposomes as carriers for peptide-antigen and adjuvant induce a strong antigen-specific T cell response and are superior over vaccine formulations composed of free peptide and adjuvant.

The induction of a potent and specific T cell response is a major challenge in the development of efficacious cancer vaccine strategies. We applied a novel liposomal formulation (AVE3) for efficient delivery of antigenic peptides into APCs of the skin. These liposomes resulted in a long-lasting deposition of encapsulated compounds at the injection site and the draining lymph nodes. Using a peptide from the melanocyte differentiation antigen tyrosinase-related protein (TRP2) 2 we could show that vaccination with liposome-encapsulated peptide in combination with oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs) as adjuvant leads to the induction of tumor cell-specific cytotoxic T cells. The most potent immune response was observed when both, TRP2 peptide and CpG ODNs, were encapsulated into AVE3. Importantly, in contrast to vaccination with free TRP2 liposomal TRP2 peptide generated T cells which respond to 1000-fold lower antigen concentration. Using the poorly immunogenic B16 melanoma model we could demonstrate that vaccination with liposomal TRP2 peptide plus CpG ODNs but not vaccination with free peptide or adjuvant alone resulted in tumor protection in subcutaneous and metastatic tumor models. In summary, vaccination with liposome-encapsulated peptide antigen and CpG ODN allows for the in vivo loading and activation of DC, thereby generating reactive CTL populations even against poorly immunogenic self-peptide presenting tumors resulting in a potent anti-tumor immune response.

About a 100 years ago, Coley observed tumor regression after application of a mixture of bacterial toxins. This experiment made the foundation of immune therapy for cancer, which only recently became a promising treatment and an efficient alternative to chemotherapy. In contrast to chemotherapy (1), this new treatment does not kill tumor cells directly; rather it enhances the sensitivity of the patient’s own immune system against tumor cells with all the potential positive aspects like high selectivity of treatment and much less side effects (2).

Artificial virus-like envelopes (AVEs) are liposomal carriers that may be useful for target-site-specific delivery of contrast agents. We speculated that T1 relaxation times of a suspension of Gadolinium-filled AVEs might be shortened after internalization and lysosomal breakdown. To test this hypothesis we evaluated the T1 relaxation times of Gadobutrol-containing AVEs before and after degradation in vitro and after receptor-mediated cellular uptake. AVEs were filled with 1 M Gadobutrol (Gadovist; Schering AG, Berlin, Germany) yielding Gd-chelate-AVEs. T1-relaxation times were calculated using an inversion recovery technique for different concentrations of the liposomal suspension. AVEs were degraded in vitro to mimic the release of the encapsulated Gadolinium in cells and to determine a putatitive increase of the T1-effect. Finally, Gd-chelate-AVEs where equipped with integrin-binding RGD ligands and the T1 relaxation times of these Gd-chelate-RDG-AVEs were determined after cellular uptake into endothelial or melanoma cells. Gadobutrol could be encapsulated into AVEs at a high concentration of 1 M (Gd-chelate-AVEs). The Gd-chelate-AVEs could be visualized by MRI. Concentrations down to 1:4 × 103 showed a significant T1-shortening effect. The degradation of the liposomes with Triton X-100 resulted in a further reduction down to concentrations of 1:10 × 103. In addition, cellular uptakes of Gd-chelate-RGD-AVEs also lead to a significant T1-shortening. Our study shows that Gadolinium can be efficiently encapsulated into AVEs and that Gd-chelate-AVEs can be detected by MRI T1-weighted measurements. The MRI detectability is enhanced by degradation. Gd-chelate-RGD-AVEs can be used to enhance the Gd uptake in cells expressing the αvβ3 receptor.

Ligand-targeted anticancer therapeutics represent an opportunity for the selective and efficient delivery of drugs to tumours. The chemical coupling of ligands to drugs or drug carrier systems is, however, often hampered by the presence of multiple reactive groups within the ligand, for example, epsilon-NH(2) groups in lysine side chains. In this paper, we describe the isolation by phage display of human epidermal growth factor (EGF) variants without lysine and a reduced number of arginine residues. The selection on A431 carcinoma cells also revealed that R41 is indispensable for EGF binding activity as all EGF variants contained an arginine residue at this position. One EGF variant (EGFm1) with K28Q, R45S, K48S and R53S mutations was expressed in bacteria and showed an identical binding activity as wild-type EGF. EGFm1 could be labelled with fluorescein isothiocyanate demonstrating the accessibility of the N-terminal amino group for coupling reagents. Furthermore, coupling of EGFm1 to PEGylated liposomes resulted in target cell-specific binding and internalization of the liposomes. These human EGF variants should be advantageous for the generation of anticancer therapeutics targeting the EGF receptor, which is overexpressed by a wide variety of different tumours.