MicroRNAs (miRNAs), an important class of small regulatory molecules for gene expression, are transcribed by RNA polymerase II. But little is known about the mechanisms that control miRNA expression. Comparing miRNA expression profiles between colon cancer cell line HCT 116 and its derivative, DNA methyltransferase 1 and 3b (DNMT1 and DNMT3b) double knockout cell line, we found that the expression of about 10% miRNAs was regulated by DNA methylation. In addition, neither 5-aza-2'-deoxycytidine treatment nor deletion of DNMT1 alone recapitulated miRNA expression profile seen in the double knockout cell line, suggesting that miRNA expression was tightly controlled by DNA methylation and partial methylation reduction was not sufficient for miRNA reexpression. We also found that HOXA3 and HOXD10 were putative targets of mir-10a, one of the differentially expressed miRNAs that is located in HOX gene cluster.

The Wilms tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is overexpressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell human leukocyte antigen (HLA)–A0201 epitope, is a validated target for T cell–based immunotherapy. Using phage display technology, we discovered a fully human “T cell receptor–like” monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex. ESK1 bound to several leukemia and solid tumor cell lines and primary leukemia cells, in a WT1-and HLA-A0201–restricted manner, with high avidity [dissociation constant (Kd) = 0.1 nM]. ESK1 mediated antibody-dependent human effector cell cytotoxicity in vitro. Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Philadelphia chromosome–positive leukemia in nonobese diabetic/severe combined immunodeficient γc−/− (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A0201 transgenic mice. ESK1 is a potential therapeutic agent for a wide range of cancers overexpressing the WT1 oncoprotein. This finding also provides preclinical validation for the strategy of developing therapeutic mAbs targeting intracellular oncogenic proteins.

We studied the feasibility of using single-wall carbon nanotubes (SWNTs) as antigen carriers to improve immune responses to peptides that are weak immunogens, a characteristic typical of human tumor antigens. Binding and presentation of peptide antigens by the MHC molecules of antigen presenting cells (APCs) is essential to mounting an effective immune response. The Wilm’s tumor protein (WT1) is upregulated in many human leukemias and cancers and several vaccines directed at this protein are in human clinical trials. WT1 peptide 427 induces human CD4 T cell responses in the context of multiple human HLA-DR.B1 molecules, but the peptide has a poor binding affinity to BALB/c mouse MHC class II molecules. We used novel, spectrally quantifiable chemical approaches to covalently append large numbers of peptide ligands (0.4 mmol/g) onto solubilized SWNT scaffolds. Peptide-SWNT constructs were rapidly internalized into professional APCs (dendritic cells and macrophages) within minutes in vitro, in a dose dependent manner. Immunization of BALB/c mice with the SWNT–peptide constructs mixed with immunological adjuvant induced specific IgG responses against the peptide, while the peptide alone or peptide mixed with the adjuvant did not induce such a response. The conjugation of the peptide to SWNT did not enhance the peptide-specific CD4 T cell response in human and mouse cells, in vitro. The solubilized SWNTs alone were nontoxic in vitro, and we did not detect antibody responses to SWNT in vivo. These results demonstrated that SWNTs are able to serve as antigen carriers for delivery into APCs to induce humoral immune responses against weak tumor antigens.

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Single-walled carbon nanotubes (SWNTs) can deliver imaging agents or drugs to tumours and offer significant advantages over approaches based on antibodies or other nanomaterials. In particular, the nanotubes can carry a substantial amount of cargo (100 times more than a monoclonal antibody), but can still be rapidly eliminated from circulation by renal filtration, like a small molecule, due to their high aspect ratio. Here we show that SWNTs can target tumours in a two-step approach in which nanotubes modified with morpholino oligonucleotide sequences bind to cancer cells that have been pre-targeted with antibodies modified with oligonucleotide strands complementary to those on the nanotubes. The nanotubes can carry fluorophores or radioisotopes, and were shown to selectively bind to cancer cells in vitro and in tumour-bearing xenografted mice. The binding process is also found to lead to antigen capping and internalization of the antibody/nanotube complexes. The nanotube conjugates were labelled with both alpha-particle and gamma-ray emitting isotopes, at high specific activities. Conjugates labelled with alpha-particle generating 225Ac were found to clear rapidly, thus mitigating radioisotope toxicity, and were shown to be therapeutically effective in vivo.