Jean Gariépy

Phototoxic aptamers selectively enter and kill epithelial cancer cells

The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e6 and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells.

Vectorial delivery of macromolecules into cells using peptide-based vehicles

The ability to direct the import of therapeutic agents into cells and target them to specific organelles would greatly enhance their functional efficacy. The available spectrum of peptide-based import signals and intracellular routing signals might provide practical solutions towards achieving a guided or vectorial delivery of molecules. Multiple cell-targeting signals and routing domains can be efficiently displayed on branched peptides. These constructs are typically nonimmunogenic in the absence of adjuvant and can be easily assembled using solid phase synthesis. The vectorial delivery of larger complexes, however, will necessitate the development of alternate templates that favor the optimal presentation of all functional signals.

The Catalytic Subunit of Shiga-like Toxin 1 Interacts with Ribosomal Stalk Proteins and is Inhibited by Their Conserved C-Terminal Domain

Shiga-like toxin 1 (SLT-1) is a type II ribosome-inactivating protein; its A(1) domain blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve interactions with eukaryotic ribosomal proteins. Here, we present evidence that the A(1) chain of SLT-1 binds to the ribosomal proteins P0, P1, and P2. These proteins were identified from a HeLa cell lysate by tandem mass spectrometry, and subsequently confirmed to bind to SLT-1 A(1) chain by yeast-two-hybrid and pull-down experiments using candidate full-length proteins. Moreover, the removal of the last 17 amino acids of either protein P1 or P2 abolishes the interaction with the A(1) chain, whereas P0, lacking this common C terminus, still binds to the A(1) domain. In vitro pull-down experiments using fusion protein-tagged C-terminal peptides corresponding to the common 7, 11, and 17 terminal residues of P1 and P2 confirmed that the A(1) chain of SLT-1 as well as the A chain of ricin bind to this shared C-terminal peptide motif. More importantly, a synthetic peptide corresponding to the 17 amino acid C terminus of P1 and P2 was shown to inhibit the ribosome-inactivating function of the A(1) chain of SLT-1 in an in vitro transcription and translation-coupled assay. These results suggest a role for the ribosomal stalk in aiding the A(1) chain of SLT-1 and other type II ribosome-inactivating proteins in localizing its catalytic domain near the site of depurination in the 28 S rRNA.

Delivering cargoes into cancer cells using DNA aptamers targeting internalized surface portals.

Many evolving treatments for cancer patients are based on the targeted delivery of therapeutic cargoes to and into cancer cells. The advent of monoclonal antibodies and the use of peptide hormones, growth factors and cytokines have historically provided a spectrum of ligands needed to selectively target tumor-associated antigens on cancer cells. However, issues linked to the size, cost and immunogenicity of protein-based ligands have led to the search for alternate ligand families. The advent of short synthetic oligonucleotide ligands known as aptamers now provides a simple strategy to select for membrane-impermeant aptamers tailored to precisely target internalized surface markers present on cancer cells. Here we described how 25-base long, synthetic single-stranded DNA aptamers were derived to bind to known internalized tumor markers such as CD33, CEA, MUC1 and Tn antigens and are imported through these surface portals into cancer cells. The key consequence of using internalized aptamers is their ability to accumulate inside the cells, thus routing their therapeutic cargoes to intracellular sites relevant to their action. Internalized aptamers are discussed in the context of how such ligands have been used to create a range of guided therapeutic agents ranging from drug-based conjugates up to targeted nanoparticles.

Designing peptide-based scaffolds as drug delivery vehicles.

Methods for delivering drugs into cells remain an important part of the process of designing drugs. One promising approach is the concept of loligomers, synthetic peptides composed of a branched polylysine core harboring identical arms. Loligomers are typically synthesized with eight arms, each carrying peptide signals guiding their import and localization into cells. The most important advantage of loligomers is the multivalent presentation of targeting signals resulting from a tentacular arrangement. Multivalency increases the efficiency of import and intracellular routing signals as compared to similar linear peptides. Secondly, it reduces and delays the impact of peptide degradation in terms of cellular processing and compartmentalization. The vectorial delivery of nucleus-directed loligomers into cells has recently been confirmed by microscopy and flow cytometry studies. Practical uses of loligomers as intracellular vehicles include the import of plasmid DNA into cells, the conjugation of chemical groups, such as photosensitizers for use in photodynamic therapy, and the incorporation of cytotoxic T-lymphocyte (CTL) epitopes with a view to creating synthetic vaccines. Branched peptides such as loligomers represent simple and versatile molecular vehicles with potential applications in a wide variety of drug design approaches.

A Role for the Protease-sensitive Loop Region of Shiga-like Toxin 1 in the Retrotranslocation of Its A1 Domain from the Endoplasmic Reticulum Lumen

Shiga-like toxin I (Slt-I) is a ribosome-inactivating protein that undergoes retrograde transport to the endoplasmic reticulum to exert its cytotoxic effect on eukaryotic cells. Its catalytically active A1 domain subsequently migrates from the endoplasmic reticulum (ER) lumen to the cytoplasm. To study this final retrotranslocation event, a suicide assay was developed based on the cytoplasmic expression and ER-targeting of the cytotoxic Slt-I A1 fragment in Saccharomyces cerevisiae. Expression of the Slt-I A1 domain (residues 1–251) with and without an ER-targeting sequence was lethal to the host and demonstrated that this domain can efficiently migrate from the ER compartment to the cytosol. Deletion analyses revealed that residues 1–239 represent the minimal A1 segment displaying full enzymatic activity. This fragment, however, accumulates in the ER lumen when directed to this compartment. The addition of residues 240–251 restores the translocation property of the A1 chain in yeast. However, single mutations within this region do not significantly alter this function in the context of the 251-residue long A1 domain or affect the toxicity of the resulting Slt-I variants toward Vero cells in the context of the holotoxin. Since this mechanism of retrotranslocation is common to other protein toxins lacking a peptide motif similar in sequence to residues 240–251, the present results suggest that the ER export mechanism may involve the recognition of a more universal structural element, such as a misfolded or altered peptide domain localized at the C terminus of the A1 chain (residues 240–251) rather than a unique ER export signal sequence.

Vaccine against MUC1 Antigen Expressed in Inflammatory Bowel Disease and Cancer Lessens Colonic Inflammation and Prevents Progression to Colitis-Associated Colon Cancer

Association of chronic inflammation with an increased risk of cancer is well established, but the contributions of innate versus adaptive immunity are not fully delineated. There has furthermore been little consideration of the role played by chronic inflammation–associated antigens, including cancer antigens, and the possibility of using them as vaccines to lower the cancer risk. We studied the human tumor antigen MUC1 which is abnormally expressed in colon cancers and also in inflammatory bowel disease (IBD) that gives rise to colitis-associated colon cancer (CACC). Using our new mouse model of MUC1+ IBD that progresses to CACC, interleukin-10 knockout mice crossed with MUC1 transgenic mice, we show that vaccination against MUC1 delays IBD and prevents progression to CACC. One mechanism is the induction of MUC1-specific adaptive immunity (anti-MUC1 IgG and anti-MUC1 CTL), which seems to eliminate abnormal MUC1+ cells in IBD colons. The other mechanism is the change in the local and the systemic microenvironments. Compared with IBD in vaccinated mice, IBD in control mice is dominated by larger numbers of neutrophils in the colon and myeloid-derived suppressor cells in the spleen, which can compromise adaptive immunity and facilitate tumor growth. This suggests that the tumor-promoting microenvironment of chronic inflammation can be converted to a tumor-inhibiting environment by increasing adaptive immunity against a disease-associated antigen. Cancer Prev Res; 3(4); 438–46. ©2010 AACR.

Identification of an octapeptide involved in homophilic interaction of the cell adhesion molecule gp80 of dictyostelium discoideum.

During development of Dictyostelium discoideum, a surface glycoprotein of Mr 80,000 (gp80) is known to mediate EDTA-resistant cell-cell adhesion via homophilic interaction. Antibodies directed against a 13 amino acid sequence (13-mer) near the NH2 terminus of the protein were found to inhibit cell reassociation. This 13-mer also inhibited gp80-cell interaction and gp80-gp80 interaction. The cell binding site was mapped to the octapeptide sequence YKLNVNDS by using shorter peptide sequences to inhibit gp80 interaction. High salt concentrations inhibited homophilic interactions of both the 13-mer and gp80, suggesting that ionic interactions are involved in the forward binding reaction. Since disruption of homophilic interactions between the bound molecules required the presence of Triton X-100, hydrophobic interactions may occur after the initial ionic binding.

Tumor-associated MUC1 glycopeptide epitopes are not subject to self-tolerance and improve responses to MUC1 peptide epitopes in MUC1 transgenic mice.

Abstract Human adenocarcinomas overexpress a hypoglycosylated, tumor-associated form of the mucin-like glycoprotein MUC1 containing abnormal mono- and disaccharide antigens, such as Tn, sialyl-Tn, and TF, as well as stretches of unglycosylated protein backbone in the variable number of tandem repeats (VNTR) region. Both peptide and glycopeptide epitopes generated from the VNTR are candidates for cancer vaccines and we performed experiments to evaluate their relative potential to elicit tumor-MUC1-specific immunity. We show here that immunization with the 100 amino acid-long VNTR peptide (MUC1p) elicits weaker responses in MUC1 transgenic mice compared to wild type mice suggesting self-tolerance. In contrast, when glycosylated with tumor-associated Tn antigen (GalNAc-O-S/T), TnMUC1 induces glycopeptide-specific T cell and antibody responses in both strains of mice and helps enhance responses to MUC1p in MUC1 transgenic mice. Using newly derived MUC1-specific mouse T cell hybridomas we show that the only antigen-presenting cells able to cross-present TnMUC1 glycopeptide are dendritic cells (DCs). This is likely due to their exclusive expression of receptors capable of binding TnMUC1. We conclude that MUC1 glycopeptides induce stronger immunity in MUC1-Tg mice because they are recognized as `foreign rather than `self and because they are cross-presented preferentially by DCs.

Tumor Antigen Epitopes Interpreted by the Immune System as Self or Abnormal-Self Differentially Affect Cancer Vaccine Responses

Epitope selection is an important consideration in the design of cancer vaccines, but factors affecting selection are not fully understood. We compared the immune responses to peptides and glycopeptides from the common human tumor antigen MUC1, a mucin that is coated with O-linked carbohydrates in its variable number of tandem repeats (VNTR) region. MUC1 expressed on tumor cells is characteristically underglycosylated, creating peptide and glycopeptide neoepitopes that are recognized by the immune system. The response to VNTR peptides is weaker in MUC1-transgenic mice (MUC1-Tg mice) than in wild-type (WT) mice, whereas the response to VNTR glycopeptides is equally strong in the two strains. Thus, glycopeptides seem to be recognized as foreign, whereas peptides, although immunogenic, are perceived as self. To explore this further, we generated MUC1 peptide- and glycopeptide-specific T-cell receptor transgenic mice and studied the function of their CD4 T cells when adoptively transferred into MUC1-Tg or WT mice. Peptide-specific T-cell precursors were not centrally deleted in MUC1-Tg mice and did not acquire a T regulatory phenotype. However, their response to the cognate peptide was reduced in MUC1-Tg mice compared with WT mice. In contrast, glycopeptide-specific CD4 T cells responded equally well in the two hosts and, when simultaneously activated, also enhanced the peptide-specific T-cell responses. Our data show that the immune system differentially recognizes various epitopes of tumor-associated antigens either as self or as foreign, and this controls the strength of antitumor immunity. This represents an important consideration for designing safe and effective cancer vaccines.