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.

Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell–based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE.

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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.

A tumor-specific, bcr-abl-derived fusion peptide vaccine can be safely administered to patients with chronic myelogenous leukemia (CML) and can elicit a bcr-abl peptide-specific T-cell immune response. In the present phase 2 trial, 14 patients with CML in chronic phase were vaccinated with 6 fusion peptides mixed with Quillaja saponaria (QS-21). No significant toxic effects were observed. In 14 of 14 patients, delayed-type hypersensitivity (DTH) and/or CD4 proliferative responses developed after beginning vaccinations, and 11 of 14 patients showed interferon-gamma (IFN-gamma) release by CD4 enzyme-linked immunospot (ELIS-POT) at one or more time points. These responses were CD4 ؉ CD45RO ؉ . A peptidespecific CD8 ؉ interferon-gamma ELIS-POT was found in 4 patients. Four patients in hematologic remission had a decrease in Philadelphia chromosome (Ph) percentage (3 concurrently receiving interferon-alpha and 1 on imatinib mesylate), and 3 patients in molecular relapse after allogenic transplantation became transiently polymerase chain reaction (PCR) negative after vaccination; 2 of these patients received concurrent donor lymphocyte infusion (DLI). All 5 patients on IFN-alpha ultimately reached a complete cytogenetic remission. In conclusion, a tumor-specific bcr-abl breakpoint peptide-derived vaccine can be safely administered and can reliably elicit measurable peptide-specific CD4 immune responses, including in patients after bone marrow transplantation, on interferon, or on imatinib mesylate. A relationship between the clinical responses and vaccination cannot be determined from this trial. IntroductionChronic myelogenous leukemia (CML) presents a unique opportunity to develop therapeutic strategies using vaccination against a truly tumor-specific antigen that is also the oncogenic protein. The chimeric fusion protein is a tumor-specific antigen because the junctional regions of p210 contain a sequence of amino acids that is not expressed in a normal cell; in addition, as a result of the codon split on the fused message, a new amino acid (lysine in bcr-abl [b3a2]) is found. [1][2][3][4][5][6][7][8] Fusion peptides from the junctional sequences of CML can bind to several class I and class II molecules and also elicit human leukocyte antigen (HLA)-restricted cytotoxicity and proliferation in vitro. [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] Recently, Clark et al 24 have confirmed our previous supposition that peptides derived from the bcr-abl protein can be processed in the cytosol and loaded in HLA molecules that will be transported to the surface of the CML cell for potential T-cell recognition.These features provided the immunologic basis for our initial phase 1 trial in which a bcr-abl (b3a2)-derived peptide vaccine elicited a bcr-abl peptide-specific T-cell immune response, despite the presence of active disease in these patients. 25 The current phase 2 trial was designed to answer the following questions in a larger cohort of patients with CML in chronic phase: (1) whether a fixed dose of p...