Whole protein and defined CD8⁺ and CD4⁺ peptides linked to penetratin targets both MHC class I and II antigen presentation pathways

Abstract

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4 + and/or CD8 + T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8 + or CD4 + T-cell responses in vitro. Furthermore, interferon (IFN)-c-secreting CD4 + and CD8 + T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8 + T-cell-dependent mechanism and did not require CD4 + help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4 + help enhanced antigen-specific IFN-g-secreting CD8 + T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model. Keywords: Antennapedia; penetratin; cell-penetrating peptides; Antp; vaccine Peptide-based vaccines for cancer immunotherapy using defined CD8 + T-cell epitopes from tumor antigens have been utilized in many clinical trials for a number of different cancers, particularly melanoma. [1][2][3][4][5][6][7][8] Although the use of peptides in immunotherapy has provided promising pre-clinical results, there are limitations for their use in clinical trials as cytotoxic T lymphocyte (CTL) responses were lacking or weak with low clinical response rates. 9-11 The delivery of immunogenic tumor antigens can utilize dendritic cell (DC) pulsing ex vivo for the induction of CTL responses. This process is labor intensive and expensive and required multiple immunizations for the patient. 12 Although peptide-based vaccines can induce remarkable CTL responses, poor clinical responses have been observed because of a number of tumor evasion strategies utilized by the host, which results in an ineffective antitumor response. 13,14 Thus, it may be more advantageous to use intact proteins or peptides that incorporate both CD4 + and CD8 + epitopes in peptide-based vaccines to induce CD4 + mediated immune responses. 15,16 Peptide or protein-based vaccines without a mechanism of delivery into antigen-presenting cells have limited uptake, which results in inefficient presentation and stimulation of CTL responses. In addition, the absence of an adjuvant in peptide or protein-based vaccines can also limit costimulation and induce an inappropriate i...

Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells

“…This mechanism for Antp based peptide immunogens was different to TAT based peptide immunogens in that they were tapasin independent but required further processing in transGolgi and ER compartments [30]. In addition, immunogenicity studies in mice with penetratin covalently linked to ovalbumin and tandem linked to OVA CD4 and/or CD8 epitopes demonstrated OVA-specific class I and class II responses resulting in prophylactic and therapeutic protection of mice from a tumor challenge [21].…”

“…The cell penetrating property of these peptides have been used to deliver antigenic peptide and proteins into antigen presenting cells including DCs for the development of vaccine delivery systems [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. For this purpose peptides chemically conjugated to protein antigens or synthetic peptides of CPP fused in tandem with cytotoxic (Tc) or helper (Th) T cell epitopes have been used.…”

Tetraspanin CD37 contributes to the initiation of cellular immunity by promoting dendritic cell migration

“…Two weeks after immunization, 5 × 10 5 splenocytes were stimulated in triplicate with either 2.5 μg/mL con A, 20 μg SIINFEKL peptide, 20 μg Helper peptide, or 2 × 10 5 irradiated RMA-Muc1 cells [39]. Naïve splenocytes were stimulated in triplicate with 0.5-1.0 μg/mL Con A.…”

“…2013. 43: 1208-1219 described previously [39]. Mucin-1 expression in RMA-Muc1 cells was maintained during culture by hygromycin treatment and assessed via flow cytometry.…”

“…Mice were challenged subcutaneously with 1-5 × 10 6 cells from either RMA (C57BL/6 -T-cell lymphoma) or RMA-Muc1 as described previously [39]. Mucin-1 expression in RMA-Muc1 cells was maintained during culture by hygromycin treatment and assessed via flow cytometry.…”