ABSTRACT
TCR gene therapy is a promising approach for the treatment of various human malignancies. However, the tumoricidal activity of TCR-modified T cells may be limited by local immunosuppressive mechanisms within the tumor environment. In particular, many malignancies induce T cell suppression in their microenvironment by TGF-ß secretion. In this study, we evaluate whether blockade of TGF-ß signaling in TCR-modified T cells enhances TCR gene therapy efficacy in an autochthonous mouse tumor model. Treatment of mice with advanced prostate cancer with T cells genetically engineered to express a tumor-reactive TCR and a dominant-negative TGF-ß receptor II induces complete and sustained tumor regression, enhances survival, and leads to restored differentiation of prostate epithelium. These data demonstrate the potential to tailor the activity of TCR-modified T cells by additional genetic modification and provide a strong rationale for the clinical testing of TGF-ß signaling blockade to enhance TCR gene therapy against advanced cancers.
Subject(s)
Genetic Therapy/methods , Immunotherapy, Adoptive/methods , Neoplasms, Experimental/therapy , Prostatic Neoplasms/immunology , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Flow Cytometry , Immunohistochemistry , Male , Mice , Mice, Transgenic , Neoplasms, Experimental/genetics , Neoplasms, Experimental/immunology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/therapy , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes/transplantation , Transduction, GeneticABSTRACT
Genetically modified T cells that express a transduced T cell receptor (TCR) α/ß heterodimer in addition to their endogenous TCR are used in clinical studies to treat cancer. These cells express two TCR-α and two TCR-ß chains that do not only compete for CD3 proteins but also form potentially self-reactive mixed TCR dimers, composed of endogenous and transferred chains. To overcome these deficits, we developed an RNAi-TCR replacement vector that simultaneously silences the endogenous TCR and expresses an RNAi-resistant TCR. Transduction of the virus-specific P14 TCR without RNAi resulted in unequal P14 TCR-α and -ß chain surface levels, indicating heterodimerization with endogenous TCR chains. Such unequal expression was also observed following TCR gene optimization. Equal surface levels of the introduced TCR chains were however achieved by silencing the endogenous TCR. Importantly, all mice that received cells transduced with the native or optimized P14 TCR developed lethal TCR gene transfer-induced graft-versus-host-disease (TI-GVHD) due to formation of mixed TCR dimers. In contrast, TI-GVHD was almost completely prevented when using the RNAi-TCR replacement vector. Our data demonstrate that RNAi-assisted TCR replacement reduces the formation of mixed TCR dimers, and thereby significantly reduces the risk of TI-GVHD in TCR gene therapy.
Subject(s)
Graft vs Host Disease/prevention & control , Lung Neoplasms/secondary , Lung Neoplasms/therapy , Melanoma, Experimental/therapy , Receptors, Antigen, T-Cell/antagonists & inhibitors , T-Lymphocytes/immunology , Animals , Autoimmunity , Cells, Cultured , Disease Models, Animal , Gene Knockdown Techniques , Genetic Vectors/administration & dosage , Lung Neoplasms/immunology , Melanoma, Experimental/immunology , Mice , RNA Interference , Receptors, Antigen, T-Cell/genetics , Transduction, GeneticABSTRACT
Regulatory T cells (Tregs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment of autoimmunity. The potential clinical translation of targeted therapy with antigen-specific Tregs is hampered by the difficulties of isolating rare specificities from the natural polyclonal T cell repertoire. Moreover, the initiating antigen is often unknown in autoimmune disease. Here we tested the ability of antigen-specific Tregs generated by retroviral gene transfer to ameliorate arthritis through linked suppression and therefore without cognate recognition of the disease-initiating antigen. We explored two distinct strategies: T cell receptor (TCR) gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs; and co-transfer of FoxP3 and TCR genes served to convert conventional CD4(+) T cells into antigen-specific regulators. Following adoptive transfer into recipient mice, the gene-modified T cells engrafted efficiently and retained TCR and FoxP3 expression. Using an established arthritis model, we demonstrate antigen-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, we describe a robust strategy to rapidly generate antigen-specific regulatory T cells capable of highly targeted inhibition of tissue damage in the absence of systemic immune suppression. This opens the possibility to target Tregs to tissue-specific antigens for the treatment of autoimmune tissue damage without the knowledge of the disease-causing autoantigens recognized by pathogenic T cells.
Subject(s)
Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/therapy , Immunotherapy, Adoptive/methods , T-Lymphocytes, Regulatory/immunology , Adoptive Transfer , Animals , Autoantigens/immunology , Flow Cytometry , Gene Transfer Techniques , Genetic Vectors/genetics , Mice , Mice, Inbred C57BL , Retroviridae , T-Cell Antigen Receptor Specificity/geneticsABSTRACT
Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/transplantation , Melanoma, Experimental/immunology , Melanoma, Experimental/therapy , Receptors, Antigen, T-Cell/administration & dosage , Receptors, Antigen, T-Cell/genetics , Transduction, Genetic , Adoptive Transfer , Animals , CD8-Positive T-Lymphocytes/radiation effects , Cancer Vaccines/administration & dosage , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Cell Line, Tumor , Gamma Rays , Genetic Vectors/radiation effects , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Transgenic , Ovalbumin/genetics , Receptors, Antigen, T-Cell/radiation effects , Receptors, Antigen, T-Cell/therapeutic use , Retroviridae/genetics , Retroviridae/immunology , Transplantation Conditioning , Viral Vaccines/administration & dosage , Viral Vaccines/genetics , Viral Vaccines/immunology , Whole-Body IrradiationABSTRACT
Analogous to the clinical use of recombinant high-affinity Abs, transfer of TCR genes may be used to create a T cell compartment specific for self-Ags to which the endogenous T cell repertoire is immune tolerant. In this study, we show in a spontaneous prostate carcinoma model that the combination of vaccination with adoptive transfer of small numbers of T cells that are genetically modified with a tumor-specific TCR results in a marked suppression of tumor development, even though both treatments are by themselves without effect. These results demonstrate the value of TCR gene transfer to target otherwise nonimmunogenic tumor-associated self-Ags provided that adoptive transfer occurs under conditions that allow in vivo expansion of the TCR-modified T cells.
Subject(s)
Immunotherapy, Adoptive , Lymphocyte Activation/immunology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/therapy , Receptors, Antigen, T-Cell/therapeutic use , T-Lymphocytes/immunology , Transduction, Genetic , Adenocarcinoma/genetics , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Adenocarcinoma/therapy , Animals , Antigens, Viral, Tumor/biosynthesis , Antigens, Viral, Tumor/genetics , Clone Cells , Immunotherapy, Adoptive/methods , Influenza A virus/immunology , Lymphocyte Activation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Prostatic Neoplasms/immunology , Prostatic Neoplasms/pathology , Receptors, Antigen, T-Cell/administration & dosage , Simian virus 40/immunology , T-Lymphocytes/virology , Transduction, Genetic/methods , Vaccinia/immunologyABSTRACT
T-cell-based antigen-specific immunotherapy targeting tumour-associated antigens offers the potential for cancer immunotherapy. However, the majority of identified tumour-associated antigens are also expressed at low levels in normal tissues and mechanisms of tolerance induction are likely to affect the quality of T-cell responses to such antigens. In this study a T-cell receptor transgenic model was developed to determine the magnitude of T-cell tolerance to the tumour-associated antigen murine double minute-2 (MDM2), a widely expressed protein that is found at elevated levels in many tumours. The analysis of transgenic mice showed that thymic deletion was responsible for purging large numbers of MDM2-specific T cells from the repertoire. However, some T cells with specificity for MDM2 were able to escape thymic deletion and persisted in the peripheral T-cell pool. Functional analysis revealed that these T cells displayed defects in antigen-driven expansion. This functional impairment of the MDM2-specific T cells was maintained following adoptive transfer of the T cells into hosts that were unable to present the T-cell-receptor-recognized antigen. This study demonstrates that thymic deletion and the functional impairment of T cells present in the periphery both operate to establish T-cell tolerance to the tumour-associated antigen MDM2. Furthermore, the tolerant phenotype was stable and did not require continuous MDM2 peptide presentation in normal tissues.
Subject(s)
Proto-Oncogene Proteins c-mdm2/immunology , Receptors, Antigen, T-Cell/immunology , Animals , Antigen Presentation/immunology , Antigens, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Clonal Deletion/immunology , Immune Tolerance , Mice , Mice, Transgenic , Spleen/immunology , Thymus Gland/immunologyABSTRACT
Mutation-derived neoantigens represent an important class of tumour-specific, tumour rejection antigens, and are attractive targets for TCR gene therapy of cancer. The majority of such mutations are patient-specific and targeting these requires a fully personalized approach. However, some mutations are found recurrently among cancer patients, and represent potential targets for neoantigen-specific TCR gene therapy that is more widely applicable. Therefore, we have investigated if some cancer mutations found recurrently in hematological malignancies encode immunogenic neoantigens presented by common European Caucasoid HLA class I alleles and can form targets for TCR gene therapy. We initially focused on identifying HLA class I neoepitopes derived from calreticulin (CALR) exon 9 mutations, found in ~ 80% of JAK2wt myeloproliferative neoplasms (MPN). Based on MHC class I peptide predictions, a number of peptides derived from mutant CALR (mCALR) were predicted to bind to HLA-A*03:01 and HLA-B*07:02. However, using mass spectrometry and ex vivo pMHC multimer staining of PBMC from MPN patients with CALR exon 9 mutations, we found no evidence that these peptides were naturally processed and presented on the surface of mCALR-expressing target cells. We next developed a protocol utilizing pMHC multimers to isolate CD8+ T cells from healthy human donor PBMC that are specific for mCALR and additional putative neoepitopes found recurrently in hematological malignancies. Using this approach, CD8+ T cells specific for HLA-A*03:01- and HLA-B*07:02-presented mCALR peptides and an HLA-A*11:01-presented mutant FBXW7 (mFBXW7) peptide were successfully isolated. TCRs isolated from mCALR-specific CD8+ T cell populations were not able to recognize target cells engineered to express mCALR. In contrast, mFBXW7-specific CD8+ T cells were able to recognize target cells engineered to express mFBXW7. In conclusion, while we found no evidence for mCALR derived neoepitope presentation in the context of the HLA class I alleles studied, our data suggests that the recurrent pR465H mutation in FBXW7 may encode an HLA-A*11:01 presented neoepitope, and warrants further investigation as a target for T cell based immunotherapy of cancer.
Subject(s)
Antigens, Neoplasm/immunology , Hematologic Neoplasms/immunology , Hematologic Neoplasms/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Epitopes, T-Lymphocyte/immunology , HLA Antigens/genetics , HLA Antigens/immunology , Hematologic Neoplasms/pathology , Humans , Lymphocyte Activation/immunology , Mutation , Peptides/genetics , Peptides/immunology , Receptors, Antigen, T-Cell/genetics , Recurrence , T-Cell Antigen Receptor SpecificityABSTRACT
There is evidence showing that high avidity CTLs can be more effective than low avidity CTLs for adoptive tumor immunotherapy. Because many T cell-recognized tumor antigens are nonmutated self-proteins, tolerance mechanisms are likely to render high avidity T cells unresponsive or cause T cell elimination by clonal deletion. We recently used the allo-restricted strategy to circumvent immunologic tolerance to a ubiquitously expressed tumor-associated protein, MDM2, and raised high avidity CTLs in humans and in mice. In this study, we investigated whether high avidity MDM2-specific CTLs can mediate tumor protection without causing damage to normal tissues in mice. Although the CTLs prolonged survival of tumor-bearing mice without causing damage to normal tissues, tumor protection was incomplete. We show that tumor growth occurred despite the continued presence of MDM2-specific CTLs and the continued susceptibility of tumor cells to CTL killing. However, analysis of the CTLs revealed that they had been rendered unresponsive in vivo because they did not produce interferon gamma in response to antigen-specific stimulation. These experiments suggest that induction of unresponsiveness may be an important mechanism limiting the efficacy of adoptive CTL therapy.
Subject(s)
Immunotherapy, Adoptive , Neoplasms, Experimental/therapy , Nuclear Proteins/immunology , Proto-Oncogene Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Cell Line, Tumor , DNA-Binding Proteins/physiology , Interferon-gamma/biosynthesis , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Proto-Oncogene Proteins c-mdm2ABSTRACT
The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.
Subject(s)
Genes, T-Cell Receptor , High-Throughput Nucleotide Sequencing/methods , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Animals , Antigens, Neoplasm/metabolism , Gene Library , Genetic Therapy , Humans , Mice , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/therapy , T-Lymphocytes/immunologyABSTRACT
T-cell receptor (TCR) gene therapy aims to induce immune reactivity against tumors by introducing genes encoding a tumor-reactive TCR into patient T cells. This approach has been extensively tested in preclinical mouse models, and initial clinical trials have demonstrated the feasibility and potential of TCR gene therapy as a cancer treatment. However, data obtained from preclinical and clinical studies suggest that both the therapeutic efficacy and the safety of TCR gene therapy can be and needs to be further enhanced. This review highlights those strategies that can be followed to develop TCR gene therapy into a clinically relevant treatment option for cancer patients.
Subject(s)
Genetic Therapy/methods , Genetic Therapy/trends , Neoplasms/genetics , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , Animals , Humans , Neoplasms/immunology , Receptors, Antigen, T-Cell/immunology , Treatment OutcomeABSTRACT
The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy-induced autoimmune pathology.
Subject(s)
Genes, T-Cell Receptor , Genetic Therapy/methods , Graft vs Host Disease/pathology , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , Animals , Graft vs Host Disease/immunology , Humans , Mice , Mice, Inbred C57BL , Models, Animal , Neoplasms/genetics , Neoplasms/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/metabolismABSTRACT
The adoptive transfer of TCR gene-modified T cells has been developed with the aim to induce immune reactivity toward defined tumor-associated antigens to which the endogenous T cell repertoire is non-responsive. Here we discuss in which areas preclinical studies in mouse models can or cannot be expected to be of value to guide clinical trial design, and how the available data from preclinical studies should influence forthcoming clinical trials.
Subject(s)
Genetic Therapy/methods , Immunotherapy, Adoptive/methods , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/metabolism , Animals , Antigens, Neoplasm/immunology , Disease Models, Animal , Humans , Mice , Neoplasms/genetics , Neoplasms/metabolism , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , T-Lymphocytes/transplantationABSTRACT
BACKGROUND: Most tumor-associated antigens (TAA) currently used for immunotherapy of cancer are also expressed in normal tissues, which may induce tolerance and impair T cell-mediated immunity. However, there is limited information about how physiological expression in normal tissues alters the function of TAA-specific T cells. METHODOLOGY/PRINCIPAL FINDINGS: We used a T cell receptor transgenic model to study how MDM2 expression in normal tissues affects the function of T cells specific for this TAA that is found at high levels in many different types of tumors. We found that some MDM2-specific T cells escaped thymic deletion and persisted in the peripheral T cell pool. When stimulated with antigen, these T cells readily initiated cell division but failed to proliferate and expand, which was associated with a high rate of apoptosis. Both IL-2 and IL-15 efficiently rescued T cell survival and antigen-specific T cell proliferation, while IL-7 and IL-21 were ineffective. Antigen-stimulated T cells showed impaired expression of the effector molecules CD43, granzyme-B and IFN-gamma, a defect that was completely restored when T cells were stimulated in the presence of IL-2. In contrast, IL-15 and IL-21 only restored the expression of CD43 and granzyme-B, but not IFN-gamma production. Finally, peptide titration experiments with IL-2 rescued T cells indicated that they were of lower avidity than non-tolerant control T cells expressing the same TCR. CONCLUSIONS/SIGNIFICANCE: These data indicate that cytokines can rescue the antigen-specific proliferation and effector function of MDM2-specific T cells, although this does not lead to the recovery of high avidity T cell function. This study sheds light on possible limitations of immunotherapy approaches that target widely expressed TAA, such as MDM2.
Subject(s)
Antibody Affinity , Cytokines/physiology , Immune Tolerance , Proto-Oncogene Proteins c-mdm2/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Base Sequence , Cell Differentiation , Cell Proliferation , DNA Primers , Flow Cytometry , Mice , Mice, Transgenic , Polymerase Chain Reaction , T-Lymphocytes, Cytotoxic/cytologyABSTRACT
Adoptive transfer of antigen-specific CD4(+) and CD8(+) T cells is one of the most efficient forms of cancer immunotherapy. However, the isolation of antigen-specific CD4(+) T cells is limited because only few tumor-associated helper epitopes are identified. Here, we used T cell antigen receptor gene transfer to target CD4(+) T cells against an MHC class I-presented epitope of a model tumor antigen. IFN-gamma-producing CD4(+) T cells were unable to expand in vivo and to provide help for tumor rejection. In contrast, CD4(+) T cells producing high levels of IL-2 expanded in vivo, provided help for cytotoxic T lymphocyte-mediated tumor rejection, and developed T cell memory. The data demonstrate in vivo synergy between T cell antigen receptor-transduced CD4(+) and CD8(+) T cells specific for the same epitope resulting in long-term tumor protection.
Subject(s)
Immunologic Memory/immunology , Immunotherapy, Adoptive/methods , Neoplasms/prevention & control , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Helper-Inducer/metabolism , Animals , Antigens, Neoplasm/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Epitopes/metabolism , Histocompatibility Antigens Class I/immunology , Interleukin-2/metabolism , Mice , Mice, Inbred C57BL , Neoplasms/therapy , Retroviridae , Spleen/cytology , T-Lymphocytes, Helper-Inducer/immunology , Transduction, GeneticABSTRACT
T cell-based antigen-specific immunotherapy targeting self-proteins aberrantly expressed in many tumours offers the potential for widely applicable cancer immunotherapy, but carries the risk of autoimmunity. Immunological tolerance represents an inherent limitation of cancer vaccines targeting such broadly expressed tumour-associated proteins. Therefore, strategies to circumvent T cell tolerance have been developed and, when combined with T cell receptor (TCR) gene transfer technology, can generate highly avid tumour-reactive patient cytotoxic T lymphocytes (CTLs) specific for peptide epitopes of tumour-associated proteins. This review analyses the level of tolerance to broadly expressed tumour-associated proteins in the autologous T cell repertoire, assesses strategies that have been developed to circumvent T cell tolerance to such antigens, and evaluates the prospects for effective immunotherapy targeting broadly expressed tumour-associated proteins.