Delivery of tumor associated antigens to antigen presenting cells using penetratin induces potent immune responses
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 crucial for the development of vaccines. Peptide or protein based vaccines without a mechanism of delivery into antigen presenting 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 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 protein from herpes simplex virus and Pep-1, an amphipathic peptide have been shown to deliver peptides and proteins into cells [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. The 60-mer DNA binding domain (homeodomain) of the Drosophila transcription factor Antennapedia, consists of 3 α-helices, the region responsible for internalization having been mapped to a 16-amino acid peptide, RQIKIWFQNRRMKWKK (penetratin or Int) within the third helix [12]. A recombinant fusion protein consisting 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-2Kb CTL epitope of ovalbumin, SIINFEKL (RQIKIWFQNRRMKWKK-SIINFEKL; IntSIINFEKL), was rapidly internalized by cells [15]. Injection with IntSIINFEKL resulted in targeting the MHC class I pathway, the induction of CTL and immunity against E.G7-OVA tumor cells [15]. It is important to note that in these studies there was no requirement for the addition of denaturing agents or adjuvants.
Studies on the uptake mechanism of peptides such as penetratin and Tat has indicated that these peptides are transported into cells by a receptor-dependent mechanism [16], [17], [18]. In contrast to earlier studies indicating receptor and energy-independent uptake, new studies have shown that penetratin and Tat are internalized via negative-charged receptors [17], [19]. However, the mechanism of uptake is more complex dependent on the peptides used (e.g. penetratin, Tat), the peptide cargo (e.g. peptide, protein) and different cell types used for assessing uptake [17], [20], [21], [22].
Our laboratory has been interested in receptor-mediated uptake of antigens to induce CTL specific to tumor associated antigens such as MUC1 which is over-expressed on epithelial tumors of breast, colon, ovary [23], [24], [25]. We have identified CTL epitopes of 5 H-2 alleles (H-2Kb, H-2Db, H-2Kd, H-2Dd, H-2Kk) and to HLA-A2 within the variable number of tandem repeat (VNTR) region after conjugation of MUC1 fusion protein (containing 5 VNTR repeats) to oxidized mannan [26], [27], [28], [29], [30], [31]. In addition, we have identified CTL epitopes to H-2d, H-2b and to HLA-A2 which are outside the VNTR region [32]. We have demonstrated that immunization of mice with MUC1 GST fusion protein or MUC1 peptides (from VNTR and non-VNTR region) generated high antibody responses with no cellular immunity [23], [24], [25], [33], [34], [35], [36], [37]. Peptides or fusion proteins alone, without the use of in vivo grown dendritic cells (DC) or adjuvants do not efficiently induce CTL responses and tumor protection. To induce cellular immune responses (CTL, CD8+ T cells), MUC1 fusion protein was conjugated to oxidized mannan and required 3 injections in mice [1], [28], [29], [31], [33], [36], [37], [38], [39], [40], [41]. DC pulsed with MUC1 peptides or MUC1 coupled to mannan was required to induce cellular responses after 3 injections [26], [27], [28], [29], [33], [37]. In this study we linked these MUC1 GST fusion proteins [N-terminus (region 33–103) and VNTR (comprising 5 repeats)] and MUC1 CTL epitopes from both the VNTR and non-VNTR regions to Int, to induce IFN-γ secreting T cells in both C57BL/6 and HLA-A2 transgenic mice. Furthermore, C57BL/6 mice immunized once with these proteins showed protection against a MUC1+ tumor challenge. It is demonstrated for the first time that Int conjugated to proteins is able to generate effective T cell responses. The Int-peptide/protein combination is a very powerful method of inducing CTLs and tumor immunity.
Section snippets
Peptide and proteins
Peptides (Table 1) were synthesized at the Austin Research Institute and the purity of the peptides (>95%) was determined by mass spectrometry. MUC1FP, a GST fusion protein comprising of 5 VNTR repeats [35] and NFP, a GST fusion protein from the N-terminus (extracellular region, 33–103) of MUC1 were produced using the bacterial vectors PGEX-3X and PGEX-2T, respectively. An alternative source of the MUC1FP antigen, not containing GST, was prepared to assay VNTR specific immune responses in
Immunofluoresence
The internalization of proteins (IntMUC1FP and IntNFP) and Int-MUC1 peptide (IntMUC1Kb) in peritoneal macrophages was examined by immunofluoresence. Cells incubated with IntMUC1FP, IntMUC1Kb and IntNFP (Fig. 1A, C, E) showed increased internalization (higher % of cells positive and higher fluorescence intensity, >90% cells) compared to cells that were incubated with MUC1FP, MUC1Kb or NFP (Fig. 1B, D, F) (<10% cells). IntMUC1Kb peptide demonstrating intracellular staining (Fig. 1I); similar
Discussion
To generate effector T cells to exogenous antigens, they must be processed in the cytoplasm of antigen presenting cells (primarily DCs) and presented to CD8 T cells in the context of MHC class I molecules [1]. Exogenous antigens are usually processed in late endosomes/lysosomes and presented by MHC class II molecules to CD4 cells [50]. DC in particular are capable of processing antigens and peptides for presentation by class I molecules via the cross presentation pathway. A number of approaches
Acknowledgements
This work was supported by the National Breast Cancer Foundation Kathleen Cunningham project grant (V.A., G.P., B.L., I.F.C.M.), NH&MRC project grant 223310 (V.A.), Susan Komen Breast Cancer Foundation grant (G.P.), Equity Trustees of Australia (V.A.) and State Trustees of Australia Foundation (V.A.). V.A. is an NH&MRC R. Douglas Wright Fellow 223316, B.L. is an NH&MRC Senior Research Fellow and R.A. and J.P.V. were supported by Vrje University Medical Center, The Netherlands. All authors were
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These authors contributed equally to this work.
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Present address: Vrije Universiteit, Faculteit der Geneeskunde, Van der Boechorststraat 7, 1081 Amsterdam, The Netherlands.