Generation of Epstein-Barr virus antigen-specific T cell receptors recognizing immunodominant epitopes of LMP1, LMP2A, and EBNA3C for immunotherapy

Epstein-Barr virus (EBV) infections in healthy individuals are usually cleared by immune cells, wherein CD8+ T cells play the most important role. However, in some immunocompromised individuals, EBV infections can lead to the development of cancer in B, T, NK cells and epithelial cells. Most EBV-associated cancers express a limited number of virus-specific antigens such as latent membrane proteins (LMP1, LMP2) and nuclear proteins (EBNA1, -2, EBNA3A, -B, -C, EBNA-LP). These antigens represent true tumor-specific antigens and can be considered useful targets for TCR gene therapy to treat EBV-associated diseases. We used a TCR isolation platform based on a single major histocompatibility class I complexe (MHC I) K562 cell library for the detection, isolation, and re-expression of TCRs targeting immunodominant peptide-MHC (pMHC) complexes. Mature dendritic cells (mDCs) were pulsed with in vitro-transcribed (ivt) RNA encoding for the selected antigen to stimulate autologous T cells. The procedure allowed the mDCs to select an immunogenic epitope of the antigen for processing and presentation on the cell surface in combination with the most suitable MHC I molecule. We isolated eight EBV-specific TCRs. They recognize various pMHC complexes of EBV antigens LMP1, LMP2A, and EBNA3C, some of them described previously and some newly identified in this study. The TCR genes were molecularly cloned into retroviral vectors and the resultant TCR-engineered T cells secreted interferon-γ after antigen contact and were able to lyse tumor cells. The EBV-specific TCRs can be used as a basis for the generation of a TCR library, which provides a valuble source of TCRs for the production of EBV-specific T cells to treat EBV-associated diseases in patients with different MHC I types

A polyvalent cellular vaccine induces T-cell responses against specific self-antigens overexpressed in chronic lymphocytic B-cell leukemia.

B cell-derived chronic lymphocytic leukemia (CLL) is an incurable disease that requires innovative therapeutic regimens. Experimental approaches of immunotherapy aiming at induction of systemic T-cell responses have been developed. Trioma cells provide a potent vaccine derived from malignant B cells that allows multiple antigens (Ags) from the parental tumor to be ingested by Ag-presenting cells. Like other strategies using modified whole tumor cells, this approach induces polyvalent responses. Using trioma cell-pulsed dendritic cells (DCs) for T-cell activation in vitro, we asked whether specific Ags overexpressed by CLL can be identified as target structures of such responses and what is the nature of these Ags. Expression levels of several genes in CLL samples were quantitated by reverse transcriptase-polymerase chain reaction. T lymphocytes were polyvalently stimulated by trioma-pulsed DCs and specificities were tested by determining cytokine secretion in the presence of target cells transfected with RNA coding for those Ags that were found to be overexpressed. We demonstrate that DCs pulsed with the modified tumor cells efficiently activate T lymphocytes against CLL and that overexpressed Ags related to leukemogenesis, such as BCL-2, MDM2, and ETV5, serve as targets for those T cells. Immune escape by Ag loss or mutation is less likely to occur if immunity is directed against altered self-proteins that are involved in malignant transformation. Therefore, vaccines based on modified tumor cells such as triomas hold promise for immunotherapy of CLL and other malignancies. Polyvalent vaccines originally designed as individualized therapeutics may be more broadly applicable, at least in patients showing similar Ag patterns

T-cell receptor gene-modified T cells with shared renal cell carcinoma specificity for adoptive T-cell therapy.

PURPOSE: Adoptive therapy with genetically engineered T cells carrying redirected antigen specificity is a new option for the treatment of cancer. This approach is not yet available for metastatic renal cell carcinoma (RCC), due to the scarcity of therapeutically useful reagents. We analyzed tumor-infiltrating lymphocytes (TIL) from RCC to identify T-cell specificities with shared tumor-specific recognition to develop T-cell receptor (TCR)-engineered T lymphocytes for adoptive therapy of RCC. EXPERIMENTAL DESIGN: We established a T-cell clone from TIL that recognized a human leukocyte antigen (HLA)-A2-restricted tumor antigen. The TCR alpha- and beta-chain genes were isolated, modified by codon optimization and murinization, and retrovirally transduced into peripheral blood lymphocytes (PBL). A TCR-expressing indicator line (B3Z-TCR53) was established to screen for antigen prevalence in RCC, other malignancies, and normal cell counterparts. RESULTS: TCR53-engineered PBL recapitulated the specificity of the TIL and showed tumor-specific HLA-A2-restricted effector activities (IFN-gamma, tumor necrosis factor-alpha, interleukin-2, macrophage inflammatory protein-1beta, cytotoxicity). PBL-TCR53 of healthy donors and RCC patients exhibited similar transduction efficiency, expansion, and polyfunctional profile. Using B3Z-TCR53 cells, 130 tumor and normal cells were screened and shared TCR53 peptide: MHC expression was found in >60% of RCC and 25% of tumor lines of other histology, whereas normal tissue cells were not recognized. CONCLUSIONS: To date, TCR53 is the only TCR with shared HLA-A2-restricted recognition of RCC. It fulfills the criteria for utilization in TCR gene therapy and advances T cell-based immunotherapy to patients with RCC and other malignancies expressing the TCR ligand

Vaccination against B-cell Chronic Lymphocytic Leukemia with Trioma Cells : Preclinical Evaluation.

Purpose: Trioma cells are lymphoma cells that have been fused to a hybridoma and have thereby been modified to express an immunoglobulin directed against surface receptors of antigen-presenting cells. Trioma cells that potentially include all lymphoma-derived antigens will be targeted to professional antigen-presenting cells in vivo. This allows uptake, processing, and presentation of tumor-derived antigens to T lymphocytes. In a mouse model, vaccination with trioma cells conferred long-lasting, T cell-dependent tumor immunity and was even able to eradicate established lymphomas. Here, we investigated whether this potent approach is effective in the human system. Experimental design: Malignant cells from 11 patients with B cell chronic-lymphocytic leukemia (B-CLL) were fused to an anti-Fc receptor hybridoma. The resulting trioma cells were extensively characterized with respect to their clonal origin. The induction of autologous tumor-specific T lymphocytes in the presence of trioma and antigen-presenting cells was examined in vitro by determining cytokine secretion in coculture assays. Results: In seven cases, trioma cells could successfully be generated from B-CLL cells. Stimulation of autologous lymphocytes with trioma cells induced a leukemia-specific T-cell response. Immunostimulatory trioma cells were also obtained from two patients with solid B-cell lymphoma. Conclusions: Trioma-mediated immunization may be a promising adjuvant treatment of human malignancies of the B-cell lineage, particularly of B-CLL, which has still a very poor prognosis. Our in vitro results pave the way for clinical application. &nbsp