Search

Context Target identification for CAR-T cell therapies, especially shared targets among different malignancies, remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through TCR-mimic antibodies. Objective and Design We hypothesized that mass spectrometry (MS)-based analysis of the presented HLA ligands of multiple cancer cell lines can be utilized to identify a shared, tumor-associated HLA ligand, which could then be targeted by TCR-mimic CAR-T cells specific for this particular HLA ligand. Results MS of HLA ligands from eight hematological and non-hematological cancer cell lines identified a shared, non-immunogenic, HLA-A*02 restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene, which was later identified in >90% (20/22) of all A*02-positive cell lines tested. A TCR-mimic scFv was prepared against this epitope, termed “NDC80-C.” CAR-T cells transduced with the NDC80-C, directed against the ALNEQIARL:HLA-A*02 complex on cancer cells, demonstrated high sensitivity and specificity for recognizing and killing multiple cancer types with a high preference for hematological malignancies (e.g., AML, ALL, DLBCL, and ALCL). In contrast, healthy leukocytes, activated B and T-cells, or hematopoietic stem cells from A*02-positive and -negative donors were not lysed. NDC80-C CAR-T cells suppressed colony formation of primary AML cells but not of healthy stem cells. Additionally, NDC80-C CAR-T cells were efficacious in mouse models against human mesothelioma and leukemia. Conclusions Our study demonstrates how MS can inform the design of tumor-agnostic TCR-mimic therapeutic platforms that target structures that are currently not druggable by small molecules, conventional CAR-T cells, T-cells, or antibodies. This strategy lays the groundwork for a potential antibody platform therapy or CAR-T cell with efficacy against highly proliferative A*02-positive cancer cells, independent of the respective cancer type.

Carbon nanotubes display characteristics that are potentially useful in their development as scaffolds for vaccine compositions. These features include stability in vivo, lack of intrinsic immunogenicity, low toxicity, and the ability to be appended with multiple copies of antigens. In addition, the particulate nature of carbon nanotubes and their unusual properties of rapid entry into antigen-presenting cells, such as dendritic cells, make them especially useful as carriers of antigens. Early attempts demonstrating carbon nanotube-based vaccines can be used in both infectious disease settings and cancer are promising.

Adoptive transfer of in vivo generated antigen-specific donor-derived T-cells is increasingly recognized as an effective approach for the treatment or prevention of EBV lymphomas and cytomegalovirus infections complicating allogeneic hematopoietic cell transplants. This review examines evidence from preclinical experiments and initial clinical trials to critically assess both the potential and current limitations of adoptive transfer of donor T-cells sensitized to selected minor alloantigens of the host or to peptide epitopes of proteins, differentially expressed by clonogenic leukemia cells, such as the Wilms tumor protein, WT-1, as a strategy to treat or prevent recurrence of leukemia in the post-transplant period.

The ability to reliably identify the peptides that can bind to MHC molecules is of practical importance for rapid vaccine development. Several computer-based prediction methods have been applied to study the interaction of MHC class I/peptide binding. Here we have compared the binding of peptides predicted by three algorithms (BIMAS, SYFPEITHI and Rankpep) to the binding of the peptides to HLA-A*0201 molecules in vitro, assessed using a MHC stabilization assay on live T2 cells. Fifty HLA-A*0201 peptides were selected from several target oncoproteins: Wilms' tumor protein (WT1), native and imatinib-mutated bcr-abl p210, JAK2 protein and Ewing's sarcoma fusion protein type 1. The sensitivity and specificity of BIMAS, SYFPEITHI and Rankpep respectively, were: 86%, and 82%; 75% and 73%; 64% and 82%. Combining two or more computer methods did not appear to significantly improve the predictive value.

Introduction: The Wilms' tumor 1 (WT1) protein is a tumor associated antigen that is potentially targetable by immunotherapeutic approaches. We have demonstrated the overexpression of WT1 in myeloma cells by IHC and in HLA-A*0201+ pts by staining with a high-affinity fully human IgG1 mAb (ESK1) specific to the RMFPNAPYL/HLA-A*0201 complex on malignant plasma cells. We report initial results from pts with plasma cell leukemia (PCL) or relapsed/refractory multiple myeloma (rMM) who have been treated with CD34-selected allo transplants followed by the administration of donor-derived WT1-specific T-cell infusions to induce an immunotherapeutic effect. Methods: In situ expression of WT1 was assessed by IHC analyses using a sequential double staining technique of MoAbs specific for CD138 and WT1.For staining with the RMFPNAPYL/HLA-A*0201 complex, BM samples were blocked with human FcR Blocking Reagent and then directly stained with MoAbs specific for CD38, CD56, CD45 and ESK1 or its isotype control human IgG1 and were analyzed by flow cytometry. WT1-specific T cells were generated from the original stem cell donors by sensitization of CD3+ enriched T-cell fractions with autologous APCs loaded with the pool of overlapping pentadecapeptides of WT1 (Invitrogen, Boston, MA). Cells were propagated in vitro with weekly restimulation and supplementation with IL-2 beginning at day 10-16. After 35-49 days, T-cells were harvested, counted and tested for antigen specific cytotoxicity, HLA-restriction, lack of alloreactivity and sterility. Pts received CD34-selected PBSC allografts after myeloablative cytoreduction with busulfan, melphalan and fludarabine. Pts were treated with 3 infusions of donor-derived WT1-specific T-cell infusions (5x10e6 cells/kg) starting 6 weeks post allo HSCT and at 4 weekly thereafter. Results: Marrow from all pts with immunohistochemical documented plasma cell involvement stained positive for WT1 IHC while WT1 staining remained negative in pts in CR. Only pts expressing HLA-A*0201 that stained positively for WT1 by IHC also demonstrated expression of WT1 by the RMFPNAPYL/HLA-A*0201 complex, whereas pts lacking HLA-A*0201 but with active disease stained positive for WT1 IHC but not ESK1 staining. Of 7 pts, 3 PCL and 4 rMM, treated with WT1-specific T cells, 4 pts had persistent disease post CD34-selected allotransplant. Of these 4 pts 2 pts developed a striking rise of WT1-specific T-cell frequencies and developed a complete remission post WT1 CTL infusions lasting for >2years. Conclusion: WT1 is overexpressed on malignant plasma cells and serves as a target for potential immunotherapeutic approaches in pts with multiple myeloma. Pts with persistent PCL following CD34-selected allografts treated with adoptive transfer of donor-derived WT1-specific cytotoxic T cells can achieve long lasting remission underscoring the therapeutic potential of T-cells specific for immunogenic WT1 peptides expressed on malignant plasma cells. Disclosures O'Reilly: Atara Biotherapeutics: Research Funding.

Adoptive therapy with chimeric antigen receptor (CAR) T cells specific for CD19 is clinically successful in a limited set of leukemias and most CARs studied are targeted against external antigens. Wilms Tumor Antigen 1 (WT1) protein (WT1) is an intracellular antigen overexpressed in many cancers, including leukemias and solid malignancies and is thus an appealing, broadly applicable target. We have engineered the first armored T cell receptor-mimic (TCRm) CAR against WT1. Derived from the ESK1 antibody, the second generation CAR, WT1-28z, is reactive with the RMFPNAPYL peptide of WT1 that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1-28z was further modified to secrete human IL-12 cytokine, thus creating the armored CAR WT1-28z/IL-12. T cells expressing WT1-28z or WT1-28z/IL-12 are cytotoxic against a range of both hematological and solid tumors. Importantly, both WT1-directed T cells are specific for the WT1-HLA-A*02:01 complex and are not reactive against cells that do not express both HLA-A*02:01 and WT1. In established SCID/Beige mouse models of either acute leukemia or ovarian cancer, one dose of WT1-28z T cells prolongs survival of mice over untreated or irrelevant antigen-specific CAR T cell treated mice. Furthermore, one dose of the armored WT1-28z/IL12 CAR T cells further significantly prolongs survival of mice in both models over WT1-28z CAR T cell treated mice, with a subset of mice whose disease was eradicated. The armored TCRm CAR T cells against WT1 are effective in eradicating disease in both hematologic and solid tumors and may hold great clinical potential to expand on the success of CAR T cell therapy.

The production of interleukin-2 (IL-2) by phytohemaglutinin (PHA)-stimulated cells of human leukemia T cell line MOLT-16 can be significantly increased by interleukin-1 (IL-1) or interferon-alpha (IFN-alpha). The enhancing effect of IL-1 and IFN-alpha on IL-2 production was studied at the IL-2 mRNA level. We show that IL-1 enhances considerably and transiently, with the maximum level between 1 and 2 hr after stimulation, the expression of IL-2 mRNA in the PHA-stimulated cells. The level of IL-2 mRNA declined rapidly within 4 to 6 hr after stimulation in both PHA- and PHA plus IL-1-stimulated cell cultures. On the contrary, IFN-alpha does not elevate the level of IL-2 mRNA above the level in PHA-stimulated cultures, but maintains an enhanced level of IL-2 mRNA in the activated cells for more than 6 hr after stimulation. These observations correlate well with the kinetics of IL-2 protein production into the culture media. The results thus suggest that IL-1 and IFN-alpha may exert an enhancing effect on IL-2 production by distinct mechanisms. In addition, none of the five other lymphokines tested (i.e., IL-2, IL-3, IL-4, IL-5, and IL-6) had any significant effect on IL-2 mRNA expression in the activated MOLT-16 cells.

The production of interleukin 2 (IL-2) by phytohemagglutinin (PHA)-stimulated human leukemia T cell lines MOLT-13 and MOLT-14, the T cell receptor (TcR) gamma delta-bearing clones, was significantly increased by adding human natural interferon-gamma (IFN-gamma) to the cell cultures. In addition, the IL-2 production by PHA-stimulated MOLT-13 and MOLT-14 cells was also augmented by human natural tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner but was inhibited by IFN-alpha even at low concentrations (10 IU/ml). This pattern of reactivity is different from the responsiveness of the MOLT-16 cells, a TcR alpha beta-bearing cell line, which respond to IFN-alpha and TNF-alpha, but not to IFN-gamma, by augmentation of IL-2 production. These results suggest that (1) the addition of IFN-gamma has a direct effect on PHA-stimulated MOLT-13 and MOLT-14, resulting in an augmentation of IL-2 production; (2) phenotypically different T cell lines vary in their susceptibility to IFN-alpha and IFN-gamma regulation; and (3) the augmentation of IL-2 production by TNF-alpha appears to have no correlation with phenotypically different T cell clones.