The balance between safety and efficacy of T cell therapies re-mains challenging and T cell mediated toxicities have occurred. The stringent selection of tumor-specific targets and careful se... Show moreThe balance between safety and efficacy of T cell therapies re-mains challenging and T cell mediated toxicities have occurred. The stringent selection of tumor-specific targets and careful se-lection of tumor-specific T cells using T cell toxicity screenings are essential. In vitro screening options against vital organs or specialized cell subsets would be preferably included in preclin-ical pipelines, but options remain limited. Here, we set up preclinical models with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, epicardial cells, and kid-ney organoids to investigate toxicity risks of tumor-specific T cells more thoroughly. CD8+T cells reactive against PRAME, HA-1H, CD20, or WT1, currently used or planned to be used in phase I/II clinical studies, were included. Using these hiPSC-derived preclinical models, we demonstrated that WT1-specific T cells caused on-target toxicity that correlated with target gene expression. Multiple measures of T cell reac-tivity demonstrated this toxicity on the level of T cells and hiPSC-derived target cells. In addition, phenotypic analysis illustrated interaction and crosstalk between infiltrated T cells and kidney organoids. In summary, we demonstrated the benefit of hiPSC-derived models in determining toxicity risks of tumor-specific T cells. Furthermore, our data empha-sizes the additional value of other measures of T cell reactivity on top of the commonly used cytokine levels. Show less
Huisman, W.; Gille, I.; Maarel, L.E. van der; Hageman, L.; Morton, L.T.; Jong, R.C.M. de; ... ; Jedema, I. 2022
Background: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated... Show moreBackground: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated malignancies of latency type II/III that express EBV antigens (LMP1/2). Patients who are HLA-A*01:01 positive could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far. Methods: HLA-A*01:01-restricted EBV-LMP2-specific T cells were isolated using peptide major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-gamma) and cytotoxicity when stimulated with EBV-LMP2-expressing cell lines. Functionality of primary T cells transduced with HLA-A*01:01-restricted EBV-LMP2-specific TCRs was optimized by knocking out the endogenous TCRs of primary T cells ( increment TCR) using CRISPR-Cas9 technology. Results: EBV-LMP2-specific T cells were successfully isolated and their TCRs were characterized. TCR gene transfer in primary T cells resulted in specific pMHC tetramer binding and reactivity against EBV-LMP2-expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5-2 fold when the endogenous TCRs of CD8(+) T cells was knocked out. CD8(+/ increment TCR) T cells modified to express EBV-LMP2-specific TCRs showed IFN-gamma secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines. Conclusions: We isolated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can potentially be used in future TCR gene therapy to treat EBV-associated latency type II/III malignancies.Here we identify the first HLA-A*01:01-restricted Epstein-Barr virus Latent Membrane Protein 2 (EBV-LMP2)-specific T-cell population and show that these T-cell populations and T cells modified to express the LMP2-specific T-cell receptor showed IFN-gamma secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines. Show less
Background T cell receptor (TCR)-engineered cells can be powerful tools in the treatment of malignancies. However, tumor resistance by Human Leukocyte antigen (HLA) class I downregulation can... Show moreBackground T cell receptor (TCR)-engineered cells can be powerful tools in the treatment of malignancies. However, tumor resistance by Human Leukocyte antigen (HLA) class I downregulation can negatively impact the success of any TCR-mediated cell therapy. Allogeneic natural killer (NK) cells have demonstrated efficacy and safety against malignancies without inducing graft-versus-host-disease, highlighting the feasibility for an 'off the shelf' cellular therapeutic. Furthermore, primary NK cells can target tumors using a broad array of intrinsic activation mechanisms. In this study, we combined the antitumor effector functions of NK cells with TCR engineering (NK-TCR), creating a novel therapeutic strategy to avoid TCR-associated immune resistance. Methods BOB1, is a transcription factor highly expressed in all healthy and malignant B cell lineages, including multiple myeloma (MM). Expression of an HLA-B*07:02 restricted BOB1-specifc TCR in peripheral blood-derived NK cells was achieved following a two-step retroviral transduction protocol. NK-TCR was then compared with TCR-negative NK cells and CD8-T cells expressing the same TCR for effector function against HLA-B*07:02+ B-cell derived lymphoblastoid cell lines (B-LCL), B-cell acute lymphoblastic leukemia and MM cell lines in vitro and in vivo. Results Firstly, TCR could be reproducibly expressed in NK cells isolated from the peripheral blood of multiple healthy donors generating pure NK-TCR cell products. Secondly, NK-TCR demonstrated antigen-specific effector functions against malignancies which were previously resistant to NK-mediated lysis and enhanced NK efficacy in vivo using a preclinical xenograft model of MM. Moreover, antigen-specific cytotoxicity and cytokine production of NK-TCR was comparable to CD8 T cells expressing the same TCR. Finally, in a model of HLA-class I loss, tumor cells with B2M KO were lysed by NK-TCR in an NK-mediated manner but were resistant to T-cell based killing. Conclusion NK-TCR cell therapy enhances NK cell efficacy against tumors through additional TCR-mediated lysis. Furthermore, the dual efficacy of NK-TCR permits the specific targeting of tumors and the associated TCR-associated immune resistance, making NK-TCR a unique cellular therapeutic. Show less
CAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of multi... Show moreCAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of multi-antigen-targeting approaches. We propose that a T cell receptor (TCR)-based strategy would increase the number of potential antigenic targets, as peptides from both intracellular and extracellular proteins can be recognized. Here, we aimed to isolate a broad range of promising TCRs targeting multiple antigens for treatment of B cell malignancies. As a first step, 28 target genes for B cell malignancies were selected based on gene expression profiles. Twenty target peptides presented in human leukocyte antigen (HLA)-A*01:01, -A*24:02, -B*08:01, or -B*35:01 were identified from the immunopeptidome of B cell malignancies and used to form peptide-HLA (pHLA)-tetramers for T cell isolation. Target-peptide-specific CD8 T cells were isolated from HLA-mismatched healthy donors and subjected to a stringent stepwise selection procedure to ensure potency and eliminate cross-reactivity. In total, five T cell clones specific for FCRL5 in HLA-A*01:01, VPREB3 in HLA-A*24:02, and BOB1 in HLA-B*35:01 recognized B cell malignancies. For all three specificities, TCR gene transfer into CD8 T cells resulted in cytokine production and efficient killing of multiple B cell malignancies. In conclusion, using this systematic approach we successfully identified three promising TCRs for T cell therapy against B cell malignancies. Show less
