Vaccine 24 (2006) 3191–3202 Delivery of tumor associated antigens to antigen presenting cells using penetratin induces potent immune responses Vasso Apostolopoulos ∗,1 , Dodie S. Pouniotis 1 , Peter J. van Maanen 2 , Rene W. Andriessen 2 , Jodie Lodding, Pei-xiang Xing, Ian F.C. McKenzie, Bruce E. Loveland, Geoffrey A. Pietersz The Austin Research Institute, Kronheimer Building, Studley Road, Heidelberg, Vic. 3084, Australia Received 13 July 2005; received in revised form 11 January 2006; accepted 14 January 2006 Available online 25 January 2006 Abstract Cytoplasmic delivery of proteins or CTL epitopes is crucial for the presentation of antigen for the generation of CTL. We previously described the use of the 16-amino acid peptide penetratin from the Drosophila Antennapedia domain (Int) to transport CTL epitopes into cells. Here we show that, Int, incorporating MUC1 CTL epitopes in tandem is able to facilitate their rapid uptake by macrophages and dendritic cells (DC) in an energy-dependent endocytic pathway. We also demonstrate for the first time that Int conjugated proteins are also able to be efficiently taken up by DC. Furthermore, C57BL/6 and HLA-A2 transgenic mice immunized with the Int-peptides or Int-proteins induce strong IFN- secreting T cells and weak IgG1 antibodies. Immunized C57BL/6 mice were protected against the growth of a MUC1 + tumor cell line. © 2006 Elsevier Ltd. All rights reserved. Keywords: Penetratin; Antennapedia; MUC1; Membrane translocating peptide; Trojan 1. Introduction The delivery of immunogens to antigen presenting cells and their subsequent uptake into the cells, processing and presentation by the MHC class I and/or II molecules is cru- cial for the development of vaccines. Peptide or protein based vaccines without a mechanism of delivery into antigen pre- senting cells have limited uptake. To overcome this problem, methods such as targeting cell surface receptors or the use of membrane translocating peptides to deliver proteins into the Abbreviations: Int, RQIKIWFQNRRMKWKK; MUC1, Mucin 1; MUC1FP, GST fusion protein containing 5 VNTR repeats; NFP, GST fusion protein of the N-terminal of MUC1 (amino acids 33–103); pVNTR, recom- binant MUC1 VNTR protein containing 5 repeats; VNTR, variable number of tandem repeat ∗ Corresponding author. Tel.: +61 3 92870666; fax: +61 3 92870600. E-mail address: v.apostolopoulos@ari.unimelb.edu.au (V. Apostolopoulos). 1 These authors contributed equally to this work. 2 Present address: Vrije Universiteit, Faculteit der Geneeskunde, Van der Boechorststraat 7, 1081 Amsterdam, The Netherlands. cytoplasm of cells, including, antigen presenting cells have been investigated [1]. Penetratin provides a unique approach for the transport of peptides and proteins into the cytoplasm of cells. The TAT protein from human immunodeficiency virus, the VP22 pro- tein from herpes simplex virus and Pep-1, an amphipathic peptide have been shown to deliver peptides and proteins into cells [2–11]. The 60-mer DNA binding domain (home- odomain) of the Drosophila transcription factor Antenna- pedia, consists of 3 -helices, the region responsible for internalization having been mapped to a 16-amino acid pep- tide, RQIKIWFQNRRMKWKK (penetratin or Int) within the third helix [12]. A recombinant fusion protein consist- ing of the 60-amino acid homeodomain fused to an influenza nucleoprotein cytotoxic T cell (CTL) peptide generated CTLs to the peptide, however SDS was required to denature the antigen [13,14]. We had previously demonstrated that the 16-mer Int-peptide linked in tandem with the H-2K b CTL epi- tope of ovalbumin, SIINFEKL (RQIKIWFQNRRMKWKK- SIINFEKL; IntSIINFEKL), was rapidly internalized by cells [15]. Injection with IntSIINFEKL resulted in targeting the 0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.01.032