Tumor Antigen-specific T-cell Receptor Research Articles

Novel adoptive T-cell immunotherapy using a WT1-specific TCR vector encoding silencers for endogenous TCRs shows marked antileukemia reactivity and safety

AbstractAdoptive T-cell therapy for malignancies using redirected T cells genetically engineered by tumor antigen-specific T-cell receptor (TCR) gene transfer is associated with mispairing between introduced and endogenous TCR chains with unknown specificity. Therefore, deterioration of antitumor reactivity and serious autoimmune reactivity are major concerns. To address this problem, we have recently established a novel retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). In this study, to test the clinical application of siTCR gene therapy for human leukemia, we examined in detail the efficacy and safety of WT1-siTCR–transduced T cells. Compared with conventional WT1-TCR (WT1-coTCR) gene-transduced T cells, these cells showed significant enhancement of antileukemia reactivity resulting from stronger expression of the introduced WT1-specific TCR with inhibition of endogenous TCRs. Notably, WT1-siTCR gene-transduced T cells were remarkably expandable after repetitive stimulation with WT1 peptide in vitro, without any deterioration of antigen specificity. WT1-siTCR gene–transduced T cells from leukemia patients successfully lysed autologous leukemia cells, but not normal hematopoietic progenitor cells. In a mouse xenograft model, adoptively transferred WT1-siTCR gene-transduced T cells exerted distinct antileukemia efficacy but did not inhibit human hematopoiesis. Our results suggest that gene-immunotherapy for leukemia using this WT1-siTCR system holds considerable promise.

The development and functions of CD4+ T cells expressing a transgenic TCR specific for an MHC-I-restricted tumor antigenic epitope

It has been reported that the ratio of CD4(+) to CD8(+) T cells has no bias in a few class I major histocompatibility complex (MHC-I)-restricted T-cell receptor (TCR)-transgenic mice specific for alloantigens or autoantigens, in which most CD4(+) T cells express an MHC-I-restricted TCR. In this study, we further showed that more than 50% of CD4(+) T cells in MHC-I-restricted P1A tumor antigen-specific TCR (P1ATCR)-transgenic mice could specifically bind to MHC-I/P1A peptide complex. P1A peptide could stimulate the transgenic CD4(+) T cells to proliferate and secrete both type 1 helper T cell and type 2 helper T cell cytokines. The activated CD4(+) T cells also showed cytotoxicity against P1A-expressing tumor cells. The analysis of TCR α-chains showed that these CD4(+) T cells were selected by co-expressing endogenous TCRs. Our results show that CD4(+) T cells from P1ATCR transgenic mice co-expressed an MHC-I-restricted transgenic TCR and another rearranged endogenous TCRs, both of which were functional.

Requisite considerations for successful adoptive immunotherapy with engineered T-lymphocytes using tumor antigen-specific T-cell receptor gene transfer

Introduction: Although engineered T-cell-based antitumor immunotherapy using tumor-antigen-specific T-cell receptor (TCR) gene transfer is undoubtedly a promising strategy, a number of studies have revealed that it has several drawbacks.Areas covered: This review covers selected articles detailing recent progress in this field, not only for solid tumors, but also for leukemias. In terms of achieving uniform therapeutic quality of TCR gene-modified T cells as an ‘off-the-shelf’ product, the authors abstract and discuss the requisite conditions for successful outcome, including: i) the optimal target choice reflecting the specificity of the introduced TCR, ii) the quality and quantity of expressed TCRs in gene-modified T cells, and additional genetic modification reflecting enhanced antitumor functionality, and iii) ‘on-’ and ‘off-target’ adverse events caused by the quality of the introduced TCRs and other adverse events related to genetic modification itself. Readers will be able to readily abstract recent advances in TCR gene-transferred T-cell therapy, centering notably on efforts to obtain uniformity in the therapeutic functionality of engineered T cellsExpert opinion: Harmonizing the functionality and target specificity of TCR will allow the establishment of clinically useful adoptive immunotherapy in the near future.

Virus Specific T-Lymphocytes Genetically Modified to Target the CD19 Antigen Eradicates Systemic Lymphoma In Mice

AbstractAbstract 2092Human T-cells can be genetically modified to target tumor antigens through tumor antigen-specific artificial T-cell receptors termed chimeric antigen receptors (CARs). To provide a therapeutic option for patients with relapsed leukemia following allogeneic stem cell transplant (allo-SCT) we have developed a novel immunotherapy utilizing donor derived virus specific cytotoxic T-lymphocytes genetically modified to target the CD19 antigen expressed on most B-cell acute lymphoblastic leukemias (B-ALL). We have previously demonstrated that donor T-cells modified to express a CAR specific to the B-cell antigen CD19, termed 19–28z, traffic to systemic sites of tumor and successfully eradicate human CD19+ tumors in a SCID-Beige mouse model. This therapy is currently under clinical investigation using autologous T-cells for adults with chronic lymphocytic leukemia (CLL) and B-ALL (BB-IND 13266). However, in the setting of allo-SCT, a lymphocyte infusion of genetically modified donor T-cells has the potential to cause graft versus host disease (GVHD). In our center's experience with infusions of donor derived EBV-CTLs and EBV-CTLs derived from third party donors for treatment of EBV associated lymphoma we have noted no alloreactivity or development of GVHD in the recipient. Furthermore, we and others have shown EBV-CTLs have long term persistence following adoptive transfer which may enhance the anti-tumor efficacy of genetically modified T-lymphocytes. To this end we postulate the therapeutic use of infusions of donor derived EBV-CTLs genetically modified to target the CD19 antigen in patients with relapsed leukemia post allo-SCT.To investigate the ability of EBV-CTLs to be genetically modified to express our anti-CD19 CAR (19-28z) via gammaretroviral vector gene transfer we tested 3 established EBV-CTL donor cell lines. We compared EBV-CTL activation using autologous EBV B-cell lymphoblastoid cell lines (EBV-BLCL), beads coated with agonistic CD3 + CD28 monoclonal antibodies (Invitrogen Carlsbad, CA), or a combination of BLCL + beads. Transduction efficiency ranged from 25–75% and was consistently higher in the EBV-CTL groups activated using EBV-BLCL alone. In standard 51Cr release cytotoxicity assay 19–28z+ EBV-CTLs exhibited specific cytotoxicity against the CD19+ human tumor cell lines BA-25 (B-ALL), Raji (Burkitt 's lymphoma) and the mouse thymoma cell line EL4 modified to express the human CD19 antigen (EL4-hCD19+). In contrast the untransduced EBV-CTLs failed to lyse these targets. However, both 19–28z+ EBV-CTLs and the untransduced EBV-CTLs retained the ability to specifically lyse autologous BLCL but not autologous PHA-blasts showing retained EBV specificity. Finally we tested the ability of 19–28z+ EBV-CTLs to eradicate established systemic Raji tumor in our SCID-Beige model of disease. Mice were injected with 5×105 Raji-eGFP-fire fly luciferase (Raji-eGFP-FFLuc) tumor cells via tail vein injection six days prior to T-lymphocyte injection. Established tumor was confirmed using bioluminescence imaging (BLI) prior to T-lymphocyte infusion. Mice were treated via tail vein injection with 7.5 × 106 19–28z+ EBV-CTL (n = 5) or control EBV-CTLs (n= 4). Control mice all died of systemic disease (<35 days) following T-lymphocyte infusion while treated mice all showed long term survival (>100 days). These results validate the therapeutic potential of tumor targeted genetically modified EBV-specific T-lymphocytes which may provide a therapeutic option for patients with relapsed CD19+ B-ALL following allo-SCT. Disclosures:No relevant conflicts of interest to declare.

Prevention of Interleukin-2 Withdrawal-Induced Apoptosis in Lymphocytes Retrovirally Cotransduced With Genes Encoding an Antitumor T-cell Receptor and an Antiapoptotic Protein

Adoptive cell transfer using autologous tumor infiltrating lymphocytes or lymphocytes transduced with antitumor T-cell receptor (TCR) is an effective therapy for patients with metastatic melanoma. A limiting factor in the effectiveness of this treatment is the apoptosis of the transferred cells when Interleukin-2 (IL-2) administration is withdrawn. In an attempt to improve persistence of the transferred lymphocytes, we cotransduced human peripheral blood lymphocytes with retroviruses encoding Bcl-2 or Bcl-xL, antiapoptotic genes of the BCL2 family, and the MART-1 melanoma tumor antigen-specific TCR, DMF5. Lymphocytes were cotransduced with 38% to 64% cotransduction efficiency, and exhibited a marked delay in apoptosis after IL-2 withdrawal. Cotransduction with Bcl-2 or Bcl-xL did not affect cytokine secretion or lytic ability of the DMF5-transduced lymphocytes. After 5 days of IL-2 withdrawal, cotransduced lymphocytes produced similar levels of IFN-γ per cell as DMF5-alone transduced lymphocytes in response to tumor cells. Cotransduction did not alter the phenotype of lymphocytes with respect to a panel of T-cell differentiation markers. In a mouse model of melanoma, adoptively transferred T cells transduced with Bcl-2 persisted better in vivo at the site of tumor, 13 and 21 days after adoptive transfer (P=0.0064 and 0.041, respectively), with evidence of enrichment of the Bcl-2-transduced population over time (P<0.0001). Thus, by coexpressing Bcl-2 or Bcl-xL with a tumor-specific TCR, we have engineered a lymphocyte that resists apoptosis owing to IL-2 withdrawal without altering its tumor-specific function or phenotype, and thus may show improved antitumor effectiveness in vivo after cell transfer.

Coexpression of the T-cell receptor constant α domain triggers tumor reactivity of single-chain TCR-transduced human T cells

AbstractTransfer of tumor antigen–specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRα/β chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRα constant α (Cα) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Cα domain preceded by a TCRα signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Cα were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector ζ-chain–associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Cα transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Lack of specific γ-retroviral vector long terminal repeat promoter silencing in patients receiving genetically engineered lymphocytes and activation upon lymphocyte restimulation

Retroviral transduction of tumor antigen-specific T-cell receptor (TCR) genes into lymphocytes redirects T cells to lyse tumors. Furthermore, adoptive transfer of these lymphocytes has mediated objective responses in patients with metastatic cancer. From 2004 to 2006, more than 40 patients were treated with autologous gene-modified lymphocytes expressing a melanoma antigen-specific TCR at the National Cancer Institute. Eighteen such patients were analyzed for persistence and gene expression in vivo. In addition, the impact of epigenetic silencing and of lymphocyte restimulation was studied. Although gene-modified lymphocytes persisted in vivo, the shutdown of TCR transgene expression was observed. Bisulfite sequencing analysis and ex vivo DNA methyltransferase inhibition demonstrated that the decrease in gene expression did not result from DNA methylation. Surprisingly, down-regulation of vector-driven transgene transcriptional activity was not vector specific but mimicked that of endogenous genes. The decrease in TCR transgene expression, however, was reversed upon lymphocyte stimulation. These data demonstrate a lack of gamma-retroviral promoter-specific gene silencing in adoptively transferred human lymphocytes and support that transgene expression is largely affected by global cellular mechanisms. The use of immunomodulatory adjuvants, eg, vaccination or cytokine therapy, for in vivo T-cell activation may help overcome this metabolic quiescence and thus augment cellular immunotherapy-based cancer therapy.

Development of a Wilms' tumor antigen-specific T-cell receptor for clinical trials: engineered patient's T cells can eliminate autologous leukemia blasts in NOD/SCID mice

The Wilms' tumor antigen (WT1) is an attractive target for immunotherapy of leukemia. In the past, we isolated and characterized the specificity and function of a WT1-specific T-cell receptor. The goal of this translational study was to develop a safe and efficient WT1-T-cell receptor retroviral vector for an adoptive immunotherapy trial with engineered T cells. We generated a panel of retroviral constructs containing unmodified or codon-optimized WT1-T-cell receptor alpha and beta genes, linked via internal ribosome entry sites or 2A sequences, with or without an additional inter-chain disulfide bond in the T-cell receptor constant domains. These constructs were functionally analyzed in vitro, and the best one was tested in an autologous primary leukemia model in vivo. We identified a WT1-T-cell receptor construct that showed optimal tetramer staining, antigen-specific cytokine production and killing activity when introduced into primary human T cells. Fresh CD34(+) cells purified from a patient with leukemia were engrafted into NOD/SCID mice, followed by adoptive immunotherapy with patient's autologous T cells transduced with the WT1-T-cell receptor. This therapeutic treatment evidently decreased leukemia engraftment in mice and resulted in a substantial improvement of leukemia-free survival. This is the first report that patient's T cells, engineered to express the WT1-T-cell receptor, can eliminate autologous leukemia progenitor cells in an in vivo model. This study provides a firm basis for the planned WT1-T-cell receptor gene therapy trial in leukemia patients.

Efficient nonviral Sleeping Beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity

Genetically engineered lymphocytes hold promise for the treatment of genetic disease, viral infections and cancer. However, current methods for genetic transduction of peripheral blood lymphocytes rely on viral vectors, which are hindered by production and safety-related problems. In this study, we demonstrated an efficient novel nonviral platform for gene transfer to lymphocytes. The Sleeping Beauty transposon-mediated approach allowed for long-term stable expression of transgenes at approximately 50% efficiency. Utilizing transposon constructs expressing tumor antigen-specific T-cell receptor genes targeting p53 and MART-1, we demonstrated sustained expression and functional reactivity of transposon-engineered lymphocytes on encountering target antigen presented on tumor cells. We found that transposon- and retroviral-modified lymphocytes had comparable transgene expression and phenotypic function. These results demonstrate the promise of nonviral ex vivo genetic modification of autologous lymphocytes for the treatment of cancer and immunologic disease.

T-Cell Receptor Gene Therapy of Established Tumors in a Murine Melanoma Model

Adoptive cell transfer therapy using tumor-infiltrating lymphocytes for patients with metastatic melanoma has demonstrated significant objective response rates. One major limitation of these current therapies is the frequent inability to isolate tumor-reactive lymphocytes for treatment. Genetic engineering of peripheral blood lymphocytes with retroviral vectors encoding tumor antigen-specific T-cell receptors (TCRs) bypasses this restriction. To evaluate the efficacy of TCR gene therapy, a murine treatment model was developed. A retroviral vector was constructed encoding the pmel-1 TCR genes targeting the B16 melanoma antigen, gp100. Transduction of C57BL/6 lymphocytes resulted in efficient pmel-1 TCR expression. Lymphocytes transduced with this retrovirus specifically recognized gp100-pulsed target cells as measured by interferon-gamma secretion assays. Upon transfer into B16 tumor-bearing mice, the genetically engineered lymphocytes significantly slowed tumor development. The effectiveness of tumor treatment was directly correlated with the number of TCR-engineered T cells administered. These results demonstrated that TCR gene therapy targeting a native tumor antigen significantly delayed the growth of established tumors. When C57BL/6 lymphocytes were added to antigen-reactive pmel-1 T cells, a reduction in the ability of pmel-1 T cell to treat B16 melanomas was seen, suggesting that untransduced cells may be deleterious to TCR gene therapy. This model may be a powerful tool for evaluating future TCR gene transfer-based strategies.

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

AbstractMalignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8+ T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus–based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28−, CD45RA−, CD45RO+, and CD62L−, a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen–specific T-cell receptors, the clone secreted IFN-γ upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15Rα expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation.

Extrathymic Generation of Tumor-Specific T Cells from Genetically Engineered Human Hematopoietic Stem Cells via Notch Signaling

Adoptive cell transfer (ACT) of tumor-reactive lymphocytes has been shown to be an effective treatment for cancer patients. Studies in murine models of ACT indicated that antitumor efficacy of adoptively transferred T cells is dependent on the differentiation status of the cells, with lymphocyte differentiation inversely correlated with in vivo antitumor effectiveness. T-cell in vitro development technologies provide a new opportunity to generate naive T cells for the purpose of ACT. In this study, we genetically modified human umbilical cord blood-derived hematopoietic stem cells (HSCs) to express tumor antigen-specific T-cell receptor (TCR) genes and generated T lymphocytes by coculture with a murine cell line expressing Notch-1 ligand, Delta-like-1 (OP9-DL1). Input HSCs were differentiated into T cells as evidenced by the expression of T-cell markers, such as CD7, CD1a, CD4, CD8, and CD3, and by detection of TCR excision circles. We found that such in vitro differentiated T cells expressed the TCR and showed HLA-A2-restricted, specific recognition and killing of tumor antigen peptide-pulsed antigen-presenting cells but manifested additional natural killer cell-like killing of tumor cell lines. The genetic manipulation of HSCs has broad implications for ACT of cancer.

Simultaneous Generation of CD8+ and CD4+ Melanoma-Reactive T Cells by Retroviral-Mediated Transfer of a Single T-Cell Receptor

Adoptive immunotherapy of cancer requires the generation of large numbers of tumor antigen-reactive T cells for transfer into cancer patients. Genes encoding tumor antigen-specific T-cell receptors can be introduced into primary human T cells by retroviral mediated gene transfer as a potential method of providing any patient with a source of autologous tumor-reactive T cells. A T-cell receptor-specific for a class I MHC (HLA-A2)-restricted epitope of the melanoma antigen tyrosinase was isolated from a CD4(+) tumor-infiltrating lymphocyte (TIL 1383I) and introduced into normal human peripheral blood lymphocytes by retroviral transduction. T-cell receptor-transduced T cells secreted various cytokines when cocultured with tyrosinase peptide-loaded antigen-presenting cells as well as melanoma cells in an HLA-A2-restricted manner, and could also lyse target cells. Furthermore, T-cell clones isolated from these cultures showed both CD8(+) and CD4(+) transduced T cells could recognize HLA-A2(+) melanoma cells, giving us the possibility of engineering class I MHC-restricted effector and T helper cells against melanoma. The ability to confer class I MHC-restricted tumor cell recognition to CD4(+) T cells makes the TIL 1383I TCR an attractive candidate for T-cell receptor gene transfer-based immunotherapy.