This gene encodes a class 1 aminoacyl-tRNA synthetase, cysteinyl-tRNA synthetase. Each of the twenty aminoacyl-tRNA synthetases catalyzes the aminoacylation of a specific tRNA or tRNA isoaccepting family with the cognate amino acid. This gene is one of several located near the imprinted gene domain on chromosome 11p15.5, an important tumor-suppressor gene region. Alterations in this region have been associated with Beckwith-Wiedemann syndrome, Wilms tumor, rhabdomyosarcoma, adrenocortical carcinoma, and lung, ovarian and breast cancers. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, Aug 2010]
CARS is implicated in: - ATP binding
- cysteine-tRNA ligase activity
- cysteinyl-tRNA aminoacylation
- gene expression
- metal ion binding
- protein binding
- protein homodimerization activity
- tRNA aminoacylation for protein translation
- tRNA binding
Data from Gene Ontology via CGAP [Hide]
What pathways are this gene/protein implicaed in? Show (2)
NOPHO, publications, links, details of meetings, a section for young doctors etc.
CARS OMIM, Johns Hopkin University Referenced article focusing on the relationship between phenotype and genotype.
CARS International Cancer Genome Consortium. Summary of gene and mutations by cancer type from ICGC
CARS Cancer Genome Anatomy Project, NCI Gene Summary
CARS COSMIC, Sanger Institute Somatic mutation information and related details
CARS TICdb, Universidad de Navarra Search the database of Translocation breakpoints In Cancer for "CARS"
CARS GEO Profiles, NCBI Search the gene expression profiles from curated DataSets in the Gene Expression Omnibus (GEO) repository.
Latest Publications: CARS (cancer-related)
Nakazawa Y Gene-modified T-cell therapy using chimeric antigen receptors for pediatric hematologic malignancies. Rinsho Ketsueki. 2016; 57(6):701-8 [PubMed] Related Publications
Chimeric antigen receptor (CAR) is the generic name for synthetic T cell receptors redirected to tumor-associated antigens. Most CARs consist of an ectodomain (scFv or ligand), a hinge region, a transmembrane domain, and signaling endodomains derived from one or two co-stimulatory molecules (CD28, 4-1BB, etc) and from a CD3-ζ chain. CD19-targeted CAR T cell therapy has achieved major success in the treatment of B cell malignancies. CD19 CAR-T cells elicited complete remission in 70-90% of adult and pediatric patients with relapsed/refractory acute lymphoblastic leukemia (ALL). CD19 CAR T cell therapy from allogeneic donors including third party donors is a potential option for B-cell malignancies. CAR T cell therapies for myeloma, acute myeloid leukemia, and T-cell leukemia are still under development. Our group is currently preparing a phase I study of CD19 CAR T cell therapy in pediatric and young adult patients with ALL using a non-viral gene transfer method, the piggyBac-transposon system.
Maus MV, June CH Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy. Clin Cancer Res. 2016; 22(8):1875-84 [PubMed] Article available free on PMC after 15/04/2017 Related Publications
Chimeric antigen receptors (CAR) are engineered fusion proteins constructed from antigen recognition, signaling, and costimulatory domains that can be expressed in cytotoxic T cells with the purpose of reprograming the T cells to specifically target tumor cells. CAR T-cell therapy uses gene transfer technology to reprogram a patient's own T cells to stably express CARs, thereby combining the specificity of an antibody with the potent cytotoxic and memory functions of a T cell. In early-phase clinical trials, CAR T cells targeting CD19 have resulted in sustained complete responses within a population of otherwise refractory patients with B-cell malignancies and, more specifically, have shown complete response rates of approximately 90% in patients with relapsed or refractory acute lymphoblastic leukemia. Given this clinical efficacy, preclinical development of CAR T-cell therapy for a number of cancer indications has been actively investigated, and the future of the CAR T-cell field is extensive and dynamic. Several approaches to increase the feasibility and safety of CAR T cells are currently being explored, including investigation into the mechanisms regulating the persistence of CAR T cells. In addition, numerous early-phase clinical trials are now investigating CAR T-cell therapy beyond targeting CD19, especially in solid tumors. Trials investigating combinations of CAR T cells with immune checkpoint blockade therapies are now beginning and results are eagerly awaited. This review evaluates several of the ongoing and future directions of CAR T-cell therapy.
Hinrichs CS Molecular Pathways: Breaking the Epithelial Cancer Barrier for Chimeric Antigen Receptor and T-cell Receptor Gene Therapy. Clin Cancer Res. 2016; 22(7):1559-64 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.
Geldres C, Savoldo B, Dotti G Chimeric Antigen Receptors for Cancer Immunotherapy. Methods Mol Biol. 2016; 1393:75-86 [PubMed] Related Publications
The adoptive transfer of T lymphocytes expressing chimeric antigen receptors (CARs) has emerged as a promising treatment for various lymphoid and solid malignancies. Patients treated with CAR-T cells have achieved dramatic responses and in some cases, complete tumor eradication. Given that CARs combine the specificity of a monoclonal antibody with the internal signaling domains of T cells, there is flexibility for choice of target antigen and strength of T-cell activation. This targeting mechanism also relinquishes the need for antigen processing and presentation via the major histocompatibility complex (MHC), making CARs a very attractive therapeutic option for the majority of patients. This review describes current methodological strategies used to generate CAR molecules, how to insert these molecules in T lymphocytes and how to evaluate the functionality of these CAR-T cells using various in vitro and in vivo experiments.
Di S, Li Z Treatment of solid tumors with chimeric antigen receptor-engineered T cells: current status and future prospects. Sci China Life Sci. 2016; 59(4):360-9 [PubMed] Related Publications
Chimeric antigen receptors (CARs) are artificial recombinant receptors that generally combine the antigen-recognition domain of a monoclonal antibody with T cell activation domains. Recent years have seen great success in clinical trials employing CD19-specific CAR-T cell therapy for B cell leukemia. Nevertheless, solid tumors remain a major challenge for CAR-T cell therapy. This review summarizes the preclinical and clinical studies on the treatment of solid tumors with CAR-T cells. The major hurdles for the success of CAR-T and the novel strategies to address these hurdles have also been described and discussed.
The tumor-killing properties of T cells provide tremendous opportunities to treat cancer. Adoptive T cell therapies have begun to harness this potential by endowing a functionally diverse repertoire of T cells with genetically modified, tumor-specific recognition receptors. Normally, this antigen recognition function is mediated by an αβ T cell receptor (TCR), but the dominant therapeutic forms currently in development are synthetic constructs called chimeric antigen receptors (CARs). While CAR-based adoptive cell therapies are already showing great promise, their basic mechanistic properties have been studied in less detail compared with those of αβ TCRs. In this review, we compare and contrast various features of TCRs versus CARs, with a goal of highlighting issues that need to be addressed to fully exploit the therapeutic potential of both.
CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.
Frey NV, Porter DL CAR T-cells merge into the fast lane of cancer care. Am J Hematol. 2016; 91(1):146-50 [PubMed] Related Publications
Chimeric antigen receptors (CARs) can be introduced into T-cells redirecting them to target specific tumor antigens. CAR-modified T cells targeting CD19 have shown remarkable activity against CD19+ malignancies including B cell acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphomas (NHL). Complete remission rates as high as 90% have been observed for patients with relapsed and refractory ALL and greater than 50% response rates have been seen in heavily pre-treated CLL and NHL. Excitingly, some remissions have been durable without any additional therapy, a finding which correlates with in-vivo T-cell persistence and B-cell aplasia. The major treatment related toxicities include B-cell aplasia, neurologic toxicities, and a potentially severe cytokine release syndrome. This review summarizes outcomes for patients treated with CD19-CAR T-cells while exploring the field's challenges and future directions.
Pinz K, Liu H, Golightly M, et al. Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells. Leukemia. 2016; 30(3):701-7 [PubMed] Related Publications
Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.
Krug C, Birkholz K, Paulus A, et al. Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone. Cancer Immunol Immunother. 2015; 64(12):1623-35 [PubMed] Related Publications
Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration.
Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies.
Autologous stem cell transplantation (ASCT) is indicated in a number of hematologic malignancies, including multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma. Relapse, however, remains 1 of the main causes of post-ASCT failure, and several strategies are being investigated to decrease the risk of relapse of progression. Recent advances in the treatment of hematological malignancies have included adoptive transfer of genetically modified T cells that express chimeric antigen receptors or T cell receptors, as well the use of checkpoint inhibitors. Early clinical results in nontransplantation patients have been very promising. This review will focus on the use of gene-modified T cells and checkpoint inhibitors in stem cell transplantation.
T cells are a new and promising antigen-specific therapeutic option for the treatment of malignant diseases. To achieve antigen specificity against tumor antigens, T cells can be manipulated by gene transfer to express chimeric antigen receptors (CARs). CAR-expressing T cells are called redirected T cells. CARs are composed of an extracellular antibody-derived antigen recognition domain, a transmembrane domain and a cytoplasmatic signal domain. Therefore, redirected T cells combine the exchangeable specificity of an antibody with the cytotoxic machinery of a T cell. Early clinical trials with redirected T cells targeting cluster of differentiation (CD) 19 have shown impressive results in CD19-positive hematological cancers. However, for solid cancers only limited clinical experience exists and new and innovative concepts have to be developed to overcome tumor-mediated immune suppression. Herein, we describe the general design of a CAR, the function of the different domains and the different strategies to produce redirected T cells. Furthermore, we summarize and discuss the preclinical and clinical data indicating the tremendous potential of redirected T cells to become a mainstay of cancer immunotherapy.
Schutsky K, Song DG, Lynn R, et al. Rigorous optimization and validation of potent RNA CAR T cell therapy for the treatment of common epithelial cancers expressing folate receptor. Oncotarget. 2015; 6(30):28911-28 [PubMed] Free Access to Full ArticleRelated Publications
Using lentiviral technology, we recently demonstrated that incorporation of CD27 costimulation into CARs greatly improves antitumor activity and T cell persistence. Still, virus-mediated gene transfer is expensive, laborious and enables long-term persistence, creating therapies which cannot be easily discontinued if toxic. To address these concerns, we utilized a non-integrating RNA platform to engineer human T cells to express FRα-specific, CD27 CARs and tested their capacity to eliminate human FRα(+) cancer. Novel CARs comprised of human components were constructed, C4-27z and C4opt-27z, a codon-optimized variant created for efficient expression. Following RNA electroporation, C4-27z and C4opt-27z CAR expression is initially ubiquitous but progressively declines across T cell populations. In addition, C4-27z and C4opt-27z RNA CAR T cells secrete high levels of Th-1 cytokines and display strong cytolytic function against human FRα(+) cancers in a time- and antigen-dependent manner. Further, C4-27z and C4opt-27z CAR T cells exhibit significant proliferation in vivo, facilitate the complete regression of fully disseminated human ovarian cancer xenografts in mice and reduce the progression of solid ovarian cancer. These results advocate for rapid progression of C4opt-27z RNA CAR to the clinic and establish a new paradigm for preclinical optimization and validation of RNA CAR candidates destined for clinical translation.
Stauss HJ, Morris EC, Abken H Cancer gene therapy with T cell receptors and chimeric antigen receptors. Curr Opin Pharmacol. 2015; 24:113-8 [PubMed] Related Publications
Viral and non-viral gene transfer technologies have been used to efficiently generate therapeutic T cells with desired cancer-specificity. Chimeric antigen receptors (CARs) redirect T cell specificity toward antibody-recognized antigens expressed on the surface of cancer cells, while T cell receptors (TCRs) extend the range of targets to include intracellular tumor antigens. CAR redirected T cells specific for the B cell differentiation antigen CD19 have shown dramatic efficacy in the treatment of B cell malignancies, while TCR-redirected T cells have shown benefits in patients suffering from solid cancer. In this review we will present strategies to optimize CAR and TCR function, and discuss the importance of target antigen selection to enhance tumor specificity, while reducing on-target and off-target toxicity.
Lymphomas arise from clonal expansions of B, T, or NK cells at different stages of differentiation. Because they occur in the immunocyte-rich lymphoid tissues, they are easily accessible to antibodies and cell-based immunotherapy. Expressing chimeric antigen receptors (CARs) on T cells is a means of combining the antigen-binding site of a monoclonal antibody with the activating machinery of a T cell, enabling antigen recognition independent of major histocompatibility complex restriction, while retaining the desirable antitumor properties of a T cell. Here, we discuss the basic design of CARs and their potential advantages and disadvantages over other immune therapies for lymphomas. We review current clinical trials in the field and consider strategies to improve the in vivo function and safety of immune cells expressing CARs. The ultimate driver of CAR development and implementation for lymphoma will be the demonstration of their ability to safely and cost-effectively cure these malignancies.
Many tumors overexpress tumor-associated antigens relative to normal tissue, such as EGFR. This limits targeting by human T cells modified to express chimeric antigen receptors (CAR) due to potential for deleterious recognition of normal cells. We sought to generate CAR(+) T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies that differ in affinity. T cells with low-affinity nimotuzumab-CAR selectively targeted cells overexpressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high-affinity cetuximab-CAR was not affected by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from nonmalignant cells.
Twenty-five years after its inception, the genetic engineering of T cells is now a therapeutic modality pursued at an increasing number of medical centers. This immunotherapeutic strategy is predicated on gene transfer technology to instruct T lymphocytes to recognize and reject tumor cells. Chimeric antigen receptors (CARs) are synthetic receptors that mediate antigen recognition, T cell activation, and - in the case of second-generation CARs - costimulation to augment T cell functionality and persistence. We demonstrated over a decade ago that human T cells engineered with a CD19-specific CAR eradicated B cell malignancies in mice. Several phase I clinical trials eventually yielded dramatic results in patients with leukemia or lymphoma, especially acute lymphoblastic leukemia (ALL). This review recounts the milestones of CD19 CAR therapy and summarizes lessons learned from the CD19 paradigm.
Suerth JD, Morgan MA, Kloess S, et al. Efficient generation of gene-modified human natural killer cells via alpharetroviral vectors. J Mol Med (Berl). 2016; 94(1):83-93 [PubMed] Related Publications
UNLABELLED: Natural killer (NK) cells play an important role in tumor immunotherapy with their unique capability of killing transformed cells without the need for prior sensitization and without major histocompatibility complex (MHC)/peptide restriction. However, tumor cells can escape NK cell cytotoxicity by various tumor immune escape mechanisms. To overcome these escape mechanisms, NK cells can be modified to express chimeric antigen receptors (CARs), enhancing their tumor-specific cytotoxicity. To determine the most efficacious method to modify human NK cells, we compared different retroviral vector systems, retroviral pseudotypes, and transduction protocols. Using optimized transduction conditions, the highest transduction levels (up to 60%) were achieved with alpharetroviral vectors. Alpharetroviral-modified primary human NK cells exhibited no alteration in receptor expression and had similar degranulation activity as untransduced NK cells, thus demonstrating that alpharetroviral modification did not negatively affect NK cell cytotoxicity. Transduction of NK cells with an alpharetroviral vector containing a CD19 CAR expression cassette selectively enhanced NK cell cytotoxicity towards CD19-expressing leukemia cells, achieving nearly complete elimination of leukemia cells after 48 h. Taken together, alpharetroviral vectors are promising tools for NK cell-mediated cancer immunotherapy applications. KEY MESSAGES: Efficient modification of human NK cells using alpharetroviral vectors. Anti-CD19-CAR-NK cells exhibited improved cytotoxicity towards CD19(+) leukemia cells. Alpharetroviral vectors are promising tools for immunotherapy applications using NK cells.
Chimeric antigen receptor (CAR) T-cells have shown remarkable results in patients with B-cell leukemia and lymphoma. However, while CAR T-cells have shown complete responses in a majority of patients with acute lymphoblastic leukemia (ALL), lymphomas are more difficult to treat. Different CAR designs and conditioning protocols seem to affect the persistence of patient responses. However, factors that determine if patients receiving the same CARs will respond or not remain obscure. In Sweden, a phase I/IIa trial using third-generation CAR T-cells is ongoing in which we intend to compare tumor biology and immunology, in each patient, to treatment response. CAR T-cell therapy is a powerful tool to add to the treatment options for this patient group but we need to perform the necessary basic research on the multifactorial mechanisms of action to give patients the best possible option of survival. Such studies are also crucial to expand the success of CAR T-cells beyond CD19+ B-cell malignancy. This review will focus on possible barriers of treating lymphoma to define factors that need to be investigated to develop the next generation of CAR T-cell therapy.
Options for targeted therapy of T-cell malignancies remain scarce. Recent clinical trials demonstrated that chimeric antigen receptors (CARs) can effectively redirect T lymphocytes to eradicate lymphoid malignancies of B-cell origin. However, T-lineage neoplasms remain a more challenging task for CAR T cells due to shared expression of most targetable surface antigens between normal and malignant T cells, potentially leading to fratricide of CAR T cells or profound immunodeficiency. Here, we report that T cells transduced with a CAR targeting CD5, a common surface marker of normal and neoplastic T cells, undergo only limited fratricide and can be expanded long-term ex vivo. These CD5 CAR T cells effectively eliminate malignant T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma lines in vitro and significantly inhibit disease progression in xenograft mouse models of T-ALL. These data support the therapeutic potential of CD5 CAR in patients with T-cell neoplasms.
Tumours use many strategies to evade the host immune response, including downregulation or weak immunogenicity of target antigens and creation of an immune-suppressive tumour environment. T cells play a key role in cell-mediated immunity and, recently, strategies to genetically modify T cells either through altering the specificity of the T cell receptor (TCR) or through introducing antibody-like recognition in chimeric antigen receptors (CARs) have made substantial advances. The potential of these approaches has been demonstrated in particular by the successful use of genetically modified T cells to treat B cell haematological malignancies in clinical trials. This clinical success is reflected in the growing number of strategic partnerships in this area that have attracted a high level of investment and involve large pharmaceutical organisations. Although our understanding of the factors that influence the safety and efficacy of these therapies has increased, challenges for bringing genetically modified T-cell immunotherapy to many patients with different tumour types remain. These challenges range from the selection of antigen targets and dealing with regulatory and safety issues to successfully navigating the routes to commercial development. However, the encouraging clinical data, the progress in the scientific understanding of tumour immunology and the improvements in the manufacture of cell products are all advancing the clinical translation of these important cellular immunotherapies.
Deniger DC, Yu J, Huls MH, et al. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations. PLoS One. 2015; 10(6):e0128151 [PubMed] Free Access to Full ArticleRelated Publications
T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.
Müller N, Michen S, Tietze S, et al. Engineering NK Cells Modified With an EGFRvIII-specific Chimeric Antigen Receptor to Overexpress CXCR4 Improves Immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma. J Immunother. 2015; 38(5):197-210 [PubMed] Free Access to Full ArticleRelated Publications
Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell-resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII glioblastoma cells in vitro and to established subcutaneous U87-MG tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor-engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.
Although T cells expressing CD19-specific chimeric antigen receptors (CARs) are a promising new therapy for B-cell malignancies, objective responses are observed at lower frequencies in patients with lymphoma than in those with acute B-cell leukemia. We postulated that the tumor microenvironment suppresses CAR-expressing T cells (CARTs) through the activity of indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme that converts tryptophan into metabolites that inhibit T -: cell activity. To investigate the effects of tumor IDO on CD19-CART therapy, we used a xenograft lymphoma model expressing IDO as a transgene. CD19-CARTs inhibited IDO-negative tumor growth but had no effect on IDO-positive tumors. An IDO inhibitor (1-methyl-tryptophan) restored IDO-positive tumor control. Moreover, tryptophan metabolites inhibited interleukin (IL)-2-, IL-7-, and IL-15-dependent expansion of CARTs; diminished their proliferation, cytotoxicity, and cytokine secretion in vitro in response to CD19 recognition; and increased their apoptosis. Inhibition of CD19-CARTs was not mitigated by the incorporation of costimulatory domains, such as 4-1BB, into the CD19-CAR. Finally, we found that fludarabine and cyclophosphamide, frequently used before CART administration, downregulated IDO expression in lymphoma cells and improved the antitumor activity of CD19-CART in vivo. Because tumor IDO inhibits CD19-CARTs, antagonizing this enzyme may benefit CD19-CART therapy.
Buckley SA, Walter RB Update on antigen-specific immunotherapy of acute myeloid leukemia. Curr Hematol Malig Rep. 2015; 10(2):65-75 [PubMed] Related Publications
Among the few drugs that have shown a benefit for patients with acute myeloid leukemia (AML) in randomized clinical trials over the last several decades is the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Undoubtedly, this experience has highlighted the value of antigen-specific immunotherapy in AML. A wide variety of therapeutics directed against several different antigens on AML cells are currently explored in preclinical and early clinical studies. On the one hand, these include passive strategies such as unconjugated antibodies targeting one or more antigens, antibodies armed with drugs, toxic proteins, or radionuclides, or adoptive immunotherapies, in particular utilizing T cells engineered to express chimeric antigen receptors (CARs) or modified T cell receptor (TCR) genes; on the other hand, these include active strategies such as vaccinations. With the documented benefit for GO and the emerging data with several classes of therapeutics in other leukemias, in particular small bispecific antibodies and CAR T cells, the future is bright. Nevertheless, a number of important questions related to the choice of target antigen(s), patient population, exact treatment modality, and supportive care needs remain open. Addressing such questions in upcoming studies will ultimately be required to optimize the clinical use of antigen-specific immunotherapies in AML and ensure that such treatments become an effective, versatile tool for this disease for which the outcomes have remained unsatisfactory in many patients.
Koneru M, O'Cearbhaill R, Pendharkar S, et al. A phase I clinical trial of adoptive T cell therapy using IL-12 secreting MUC-16(ecto) directed chimeric antigen receptors for recurrent ovarian cancer. J Transl Med. 2015; 13:102 [PubMed] Free Access to Full ArticleRelated Publications
PURPOSE: Recurrent platinum-resistant ovarian cancer has no curative options, necessitating the development of novel treatments, including immunotherapy. RATIONALE: Patient-derived T cells can be genetically modified to express chimeric antigen receptors (CARs) specific to tumor-associated antigens in an HLA-independent manner, with promising preclinical results. MUC16(ecto) is highly expressed on most epithelial ovarian carcinomas but at low levels on normal tissues, offering an excellent immunotherapeutic target for this cancer. CAR T cells further modified to secrete IL-12 show enhanced cytotoxicity, persistence, and modulation of the tumor microenvironment. DESIGN: We propose a dose escalation phase I clinical trial for patients with recurrent MUC-16(ecto+) ovarian cancer to test the safety of intravenous and intraperitoneal administration and the preliminary efficacy of autologous IL-12 secreting, MUC-16(ecto) CAR T cells containing a safety elimination gene. INNOVATION: This trial targets MUC-16(ecto), a novel and promising tumor-associated antigen. This will be the first time CAR T cells are injected intraperitoneally directly into the site of the tumor within the abdomen in humans. Furthermore, the ability of genetically modified cells to secrete IL-12 will potentially enhance CAR T cell persistence and modulate the tumor microenvironment. For safety purposes, an elimination gene has been incorporated into the CAR T cells to mitigate any on-target, off-tumor or other unforeseen toxicity.
Chimeric antigen receptor (CAR) T-cell therapies have demonstrated durable and potentially curative therapeutic efficacy against B-cell leukemia in clinical trials. A CAR strategy can target any tumor surface antigens as long as an antigen-binding receptor can be generated. New CARs that target solid tumors and have the potential to target multiple tumor types are needed. In this study, B7H6, a ligand for the NK cell activating receptor NKp30, was targeted to create a CAR that targets multiple tumor types. B7H6 is expressed on various primary human tumors, including leukemia, lymphoma and gastrointestinal stromal tumors, but it is not constitutively expressed on normal tissues. B7H6-specific CAR T cells have robust cellular cytotoxicity and interferon-γ secretion when co-cultured with B7H6+ tumor cells, and they exhibit little self-reactivity to immature dendritic cells or pro-inflammatory monocytes. In vivo, B7H6-specific CAR T cells greatly enhanced the survival of RMA/B7H6 lymphoma-bearing mice. The long-term survivor mice were protected against a B7H6-deficient tumor re-challenge. This CAR therapy also decreased tumor burden in a murine ovarian cancer model. In conclusion, B7H6-specific CARs have the potential to treat B7H6+ hematologic and solid tumors.
Levine BL Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells. Cancer Gene Ther. 2015; 22(2):79-84 [PubMed] Related Publications
Performance enhancement of the immune system can now be generated through ex vivo gene modification of T cells in order to redirect native specificity to target tumor antigens. This approach combines the specificity of antibody therapy, the expanded response of cellular therapy and the memory activity of vaccine therapy. Recent clinical trials of chimeric antigen receptor (CAR) T cells directed toward CD19 as a stand-alone therapy have shown sustained complete responses in patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. As these drug products are individually derived from a patient's own cells, a different manufacturing approach is required for this kind of personalized therapy compared with conventional drugs. Key steps in the CAR T-cell manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of modified T cells and cryopreservation in infusible media. In this review, the steps involved in isolating, genetically modifying and scaling-out the CAR T cells for use in a clinical setting are described in the context of in-process and release testing and regulatory standards.
INTRODUCTION: Over 20 years ago, chimeric antigen receptors (CARs) were created to endow T cells with new antigen-specificity and create a therapy that could eradicate cancer and provide life-long protection against recurrence. Steady progress has led to significant improvements with CAR design and CAR T-cell production, allowing evaluation of CAR T cells in patients. The initial trials have targeted CD19, which is expressed on normal and malignant B cells. AREAS COVERED: We review data from trials for patients with chronic lymphocytic leukemia (CLL) and B-cell acute lymphoblastic leukemia (B-ALL). In addition, we discuss the on-target toxicities, B-cell aplasia and cytokine release syndrome (CRS), which is uniquely associated with T-cell immunotherapies. EXPERT OPINION: We compare the results when targeting the same antigen in CLL or B-ALL and speculate on reasons for outcome differences and future directions to enhance outcomes. Furthermore, the dramatic results targeting B-ALL require further analysis in Phase II trials, and we discuss important components of these future trials. We also suggest a management scheme for CRS. The next several years will be critical and may lead to the first clinical indication of a gene-engineered cell therapy for cancer.