ABSTRACT
Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.
Subject(s)
Glioblastoma , Receptors, Chimeric Antigen , Cell Death , Glioblastoma/genetics , Glioblastoma/therapy , Humans , Immunotherapy, Adoptive , T-Lymphocytes/pathologyABSTRACT
Cell-based therapies using chimeric antigen receptor T cells (CAR T) have had dramatic efficacy in the clinic and can even mediate curative responses in patients with hematologic malignancies. As living drugs, engineered cells can still be detected in some patients even years after the original infusion. The excitement around the cell therapy field continues to expand as recent reports have shown that CAR T cells can induce remission in patients with autoimmune disease. While these promising advances in the field garner hope for wide-spread utility of CAR T therapies across diseases, several roadblocks exist that currently limit the access and efficacy of this therapy in the clinic. Herein, we will discuss four major obstacles that the CAR T field faces, including toxicity, identifying tumor-specific antigens, improving function in solid tumors, and reducing manufacturing complexity and cost. CAR T cells have potential for a multitude of diseases, but these glass ceilings will need to be broken in order to improve clinical responses and make this potentially life-saving therapy accessible to a larger patient population.
Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Humans , Receptors, Chimeric Antigen/genetics , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes , Immunotherapy, Adoptive , Antigens, Neoplasm , Tumor MicroenvironmentABSTRACT
COVID-19 breakthrough cases among vaccinated individuals demonstrate the value of measuring long-term immunity to SARS-CoV-2 and its variants. We demonstrate that anti-spike T-cell responses and IgG antibody levels are maintained but decrease over time and are lower in BNT162b2- versus mRNA-1273-vaccinated individuals. T-cell responses to the variants are relatively unaffected.
Subject(s)
COVID-19 , 2019-nCoV Vaccine mRNA-1273 , Antibodies, Viral , BNT162 Vaccine , COVID-19/prevention & control , COVID-19 Vaccines , Humans , SARS-CoV-2/genetics , T-LymphocytesABSTRACT
T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have produced impressive outcomes for the treatment of B cell malignancies, but different products vary in kinetics, persistence, and toxicity profiles based on the co-stimulatory domains included in the CAR. In this study, we performed transcriptional profiling of bulk CAR T cell populations and single cells to characterize the transcriptional states of human T cells transduced with CD3ζ, 4-1BB-CD3ζ (BBζ), or CD28-CD3ζ (28ζ) co-stimulatory domains at rest and after activation by triggering their CAR or their endogenous T cell receptor (TCR). We identified a transcriptional signature common across CARs with the CD3ζ signaling domain, as well as a distinct program associated with the 4-1BB co-stimulatory domain at rest and after activation. CAR T cells bearing BBζ had increased expression of human leukocyte antigen (HLA) class II genes, ENPP2, and interleukin (IL)-21 axis genes, and decreased PD1 compared to 28ζ CAR T cells. Similar to previous studies, we also found BBζ CAR CD8 T cells to be enriched in a central memory cell phenotype and fatty acid metabolism genes. Our data uncovered transcriptional signatures related to costimulatory domains and demonstrated that signaling domains included in CARs uniquely shape the transcriptional programs of T cells.
Subject(s)
4-1BB Ligand/chemistry , 4-1BB Ligand/metabolism , Cell Engineering/methods , Protein Domains/genetics , RNA, Small Cytoplasmic/genetics , Receptors, Chimeric Antigen/genetics , Signal Transduction/genetics , T-Lymphocytes/metabolism , Transcriptome , HEK293 Cells , Humans , K562 Cells , RNA-Seq/methods , Single-Cell Analysis , Transduction, GeneticABSTRACT
CAR-T cell therapy has emerged as a breakthrough therapy for the treatment of relapsed and refractory hematologic malignancies. However, insufficient CAR-T cell expansion and persistence is a leading cause of treatment failure. Exogenous or transgenic cytokines have great potential to enhance CAR-T cell potency but pose the risk of exacerbating toxicities. Here we present a chemical-genetic system for spatiotemporal control of cytokine function gated by the off-patent anti-cancer molecular glue degrader drug lenalidomide and its analogs. When co-delivered with a CAR, a membrane-bound, lenalidomide-degradable IL-7 fusion protein enforced a clinically favorable T cell phenotype, enhanced antigen-dependent proliferative capacity, and enhanced in vivo tumor control. Furthermore, cyclical pharmacologic combined control of CAR and cytokine abundance enabled the deployment of highly active, IL-7-augmented CAR-T cells in a dual model of antitumor potency and T cell hyperproliferation.
Subject(s)
Interleukin-7 , Receptors, Antigen, T-Cell , Humans , Lenalidomide/pharmacology , Receptors, Antigen, T-Cell/genetics , Interleukin-7/metabolism , Cell Line, Tumor , T-Lymphocytes/metabolism , Immunotherapy, Adoptive , Cytokines/metabolismABSTRACT
Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.
Subject(s)
Apoptosis , Bridged Bicyclo Compounds, Heterocyclic , Proto-Oncogene Proteins c-bcl-2 , Receptors, Chimeric Antigen , Sulfonamides , Humans , Animals , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Chimeric Antigen/metabolism , Sulfonamides/pharmacology , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Xenograft Model Antitumor Assays , Mice , T-Lymphocytes/metabolism , T-Lymphocytes/immunology , Cell Line, Tumor , Immunotherapy, Adoptive/methods , bcl-X Protein/metabolism , Peptide Fragments , Proto-Oncogene ProteinsABSTRACT
PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.
Subject(s)
CD3 Complex , Endopeptidases , GPI-Linked Proteins , Immunotherapy, Adoptive , Mesothelin , Pancreatic Neoplasms , Receptors, Chimeric Antigen , Tumor Microenvironment , Xenograft Model Antitumor Assays , Humans , Animals , Mice , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/therapy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Tumor Microenvironment/immunology , Immunotherapy, Adoptive/methods , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , CD3 Complex/immunology , CD3 Complex/metabolism , GPI-Linked Proteins/immunology , GPI-Linked Proteins/metabolism , Cell Line, Tumor , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/therapy , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/immunology , Membrane Proteins/immunology , Membrane Proteins/metabolism , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Adenocarcinoma/immunology , Adenocarcinoma/therapy , Adenocarcinoma/pathologyABSTRACT
Chimeric antigen receptor (CAR) T-cell therapy can lead to dramatic clinical responses in B-cell malignancies. However, early clinical trials with CAR T-cell therapy in non-B-cell malignancies have been disappointing to date, suggesting that tumor-intrinsic features contribute to resistance. To investigate tumor-intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T-cell evasion in the clinic. Antigen-independent mediators of CAR T-cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T-cell therapy likely involves alterations in tumor-intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T-cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy. SIGNIFICANCE: The identification and validation of key determinants of CAR T-cell response in pancreatic cancer provide insights into the landscape of tumor cell intrinsic resistance mechanisms and into approaches to improve therapeutic efficacy.
Subject(s)
Immunotherapy, Adoptive , Pancreatic Neoplasms , Receptors, Chimeric Antigen , Humans , Cell Line, Tumor , Cell- and Tissue-Based Therapy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/therapy , Receptors, Antigen, T-Cell , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/therapeutic use , Pancreatic NeoplasmsABSTRACT
Chimeric Antigen Receptor (CAR) T cells directed to B cell maturation antigen (BCMA) mediate profound responses in patients with multiple myeloma, but most patients do not achieve long-term complete remissions. In addition, recent evidence suggests that high-affinity binding to BCMA can result in on-target, off-tumor activity in the basal ganglia and can lead to fatal Parkinsonian-like disease. Here we develop CAR T cells against multiple myeloma using a binder to targeting transmembrane activator and CAML interactor (TACI) in mono and dual-specific formats with anti-BCMA. These CARs have robust, antigen-specific activity in vitro and in vivo. We also show that TACI RNA expression is limited in the basal ganglia, which may circumvent some of the toxicities recently reported with BCMA CARs. Thus, single-targeting TACI CARs may have a safer toxicity profile, whereas dual-specific BCMA-TACI CAR T cells have potential to avoid the antigen escape that can occur with single-antigen targeting.
Subject(s)
Multiple Myeloma , Receptors, Chimeric Antigen , Humans , Multiple Myeloma/metabolism , Immunotherapy, Adoptive , B-Cell Maturation Antigen/genetics , T-LymphocytesABSTRACT
Background: Chimeric antigen receptor (CAR) T cells have achieved remarkable responses in patients with hematological malignancies; however, the potential of this therapeutic platform for solid tumors like glioblastoma (GBM) has been limited, due in large part to the targeting of single antigens in a heterogeneous disease. Strategies that allow CAR T cells to engage multiple antigens concomitantly may broaden therapeutic responses and mitigate the effects of immune escape. Methods: Here we have developed a novel, dual-specific, tandem CAR T (TanCART) cell with the ability to simultaneously target both EGFRvIII and IL-13Rα2, two well-characterized tumor antigens that are frequently found on the surface of GBM cells but completely absent from normal brain tissues. We employed both standard immunological assays and multiple orthotopic preclinical models including patient-derived xenograft to demonstrate efficacy of this approach against heterogeneous tumors. Results: Tandem CAR T cells displayed enhanced cytotoxicity in vitro against heterogeneous GBM populations, including patient-derived brain tumor cultures (P < .05). Compared to CAR T cells targeting single antigens, dual antigen engagement through the tandem construct was necessary to achieve long-term, complete, and durable responses in orthotopic murine models of heterogeneous GBM, including patient-derived xenografts (P < .05). Conclusions: We demonstrate that TanCART is effective against heterogeneous tumors in the brain. These data lend further credence to the development of multi-specific CAR T cells in the treatment of GBM and other cancers.
ABSTRACT
CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.
Subject(s)
Burkitt Lymphoma , Leukemia , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Chimeric Antigen , Humans , Animals , Mice , RNA, Guide, CRISPR-Cas Systems , Immunotherapy, Adoptive , T-Lymphocytes , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Tumor MicroenvironmentABSTRACT
The cellular processes that govern tumor resistance to immunotherapy remain poorly understood. To gain insight into these processes, here we perform a genome-scale CRISPR activation screen for genes that enable human melanoma cells to evade cytotoxic T cell killing. Overexpression of four top candidate genes (CD274 (PD-L1), MCL1, JUNB, and B3GNT2) conferred resistance in diverse cancer cell types and mouse xenografts. By investigating the resistance mechanisms, we find that MCL1 and JUNB modulate the mitochondrial apoptosis pathway. JUNB encodes a transcription factor that downregulates FasL and TRAIL receptors, upregulates the MCL1 relative BCL2A1, and activates the NF-κB pathway. B3GNT2 encodes a poly-N-acetyllactosamine synthase that targets >10 ligands and receptors to disrupt interactions between tumor and T cells and reduce T cell activation. Inhibition of candidate genes sensitized tumor models to T cell cytotoxicity. Our results demonstrate that systematic gain-of-function screening can elucidate resistance pathways and identify potential targets for cancer immunotherapy.
Subject(s)
Melanoma , Proto-Oncogene Proteins c-bcl-2 , Animals , Apoptosis/genetics , Cell Line, Tumor , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Humans , Melanoma/genetics , Melanoma/pathology , Mice , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , N-Acetylglucosaminyltransferases/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
Chimeric antigen receptor (CAR) T cells induce impressive responses in patients with hematologic malignancies but can also trigger cytokine release syndrome (CRS), a systemic toxicity caused by activated CAR T cells and innate immune cells. Although IFNγ production serves as a potency assay for CAR T cells, its biologic role in conferring responses in hematologic malignancies is not established. Here we show that pharmacologic blockade or genetic knockout of IFNγ reduced immune checkpoint protein expression with no detrimental effect on antitumor efficacy against hematologic malignancies in vitro or in vivo. Furthermore, IFNγ blockade reduced macrophage activation to a greater extent than currently used cytokine antagonists in immune cells from healthy donors and serum from patients with CAR T-cell-treated lymphoma who developed CRS. Collectively, these data show that IFNγ is not required for CAR T-cell efficacy against hematologic malignancies, and blocking IFNγ could simultaneously mitigate cytokine-related toxicities while preserving persistence and antitumor efficacy. SIGNIFICANCE: Blocking IFNγ in CAR T cells does not impair their cytotoxicity against hematologic tumor cells and paradoxically enhances their proliferation and reduces macrophage-mediated cytokines and chemokines associated with CRS. These findings suggest that IFNγ blockade may improve CAR T-cell function while reducing treatment-related toxicity in hematologic malignancies. See related content by McNerney et al., p. 90 (17). This article is highlighted in the In This Issue feature, p. 85.
Subject(s)
Hematologic Neoplasms , Immunotherapy, Adoptive , Cytokine Release Syndrome , Cytokines/metabolism , Hematologic Neoplasms/therapy , Humans , Immunotherapy, Adoptive/adverse effects , Interferon-gamma/metabolism , Macrophage Activation , T-Lymphocytes/metabolismABSTRACT
Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. Approximately half of patients with refractory large B cell lymphomas achieve durable responses from CD19-targeting CAR-T treatment; however, failure mechanisms are identified in only a fraction of cases. To gain new insights into the basis of clinical response, we performed single-cell transcriptome sequencing of 105 pretreatment and post-treatment peripheral blood mononuclear cell samples, and infusion products collected from 32 individuals with large B cell lymphoma treated with either of two CD19 CAR-T products: axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). Expansion of proliferative memory-like CD8 clones was a hallmark of tisa-cel response, whereas axi-cel responders displayed more heterogeneous populations. Elevations in CAR-T regulatory cells among nonresponders to axi-cel were detected, and these populations were capable of suppressing conventional CAR-T cell expansion and driving late relapses in an in vivo model. Our analyses reveal the temporal dynamics of effective responses to CAR-T therapy, the distinct molecular phenotypes of CAR-T cells with differing designs, and the capacity for even small increases in CAR-T regulatory cells to drive relapse.
Subject(s)
Biological Products , Lymphoma, Large B-Cell, Diffuse , Receptors, Chimeric Antigen , Antigens, CD19 , Humans , Immunotherapy, Adoptive/adverse effects , Leukocytes, Mononuclear , Lymphoma, Large B-Cell, Diffuse/pathology , Neoplasm Recurrence, Local/drug therapy , Receptors, Chimeric Antigen/geneticsABSTRACT
This Review discusses the major advances and changes made over the past 3 years to our understanding of chimeric antigen receptor (CAR) T cell efficacy and safety. Recently, the field has gained insight into how various molecular modules of the CAR influence signalling and function. We report on mechanisms of toxicity and resistance as well as novel engineering and pharmaceutical interventions to overcome these challenges. Looking forward, we discuss new targets and indications for CAR T cell therapy expected to reach the clinic in the next 1-2 years. We also consider some new studies that have implications for the future of CAR T cell therapies, including changes to manufacturing, allogeneic products and drug-regulatable CAR T cells.
Subject(s)
Immunotherapy, Adoptive , Receptors, Chimeric Antigen/immunology , Animals , Clinical Trials as Topic , Humans , Immunotherapy, Adoptive/adverse effects , Lymphocyte Activation , Signal Transduction/physiologyABSTRACT
Recently, two mRNA vaccines to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become available, but there is also an emergence of SARS-CoV-2 variants with increased transmissibility and virulence1-6. A major concern is whether the available vaccines will be equally effective against these variants. The vaccines are designed to induce an immune response against the SARS-CoV-2 spike protein7,8, which is required for viral entry to host cells9. Immunity to SARS-CoV-2 is often evaluated by antibody production, while less is known about the T-cell response. Here we developed, characterized, and implemented two standardized, functional assays to measure T-cell immunity to SARS-CoV-2 in uninfected, convalescent, and vaccinated individuals. We found that vaccinated individuals had robust T-cell responses to the wild type spike and nucleocapsid proteins, even more so than convalescent patients. We also found detectable but diminished T-cell responses to spike variants (B.1.1.7, B.1.351, and B.1.1.248) among vaccinated but otherwise healthy donors. Since decreases in antibody neutralization have also been observed with some variants10-12, investigation into the T-cell response to these variants as an alternative means of viral control is imperative. Standardized measurements of T-cell responses to SARS-CoV-2 are feasible and can be easily adjusted to determine changes in response to variants.
ABSTRACT
Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.
Subject(s)
Lenalidomide , Receptors, Chimeric Antigen , T-Lymphocytes , Humans , Jurkat Cells , Receptors, Antigen, T-Cell , Ubiquitin-Protein LigasesABSTRACT
BACKGROUND: Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure. METHODS: We created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies. RESULTS: We found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma. CONCLUSIONS: Our aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.
Subject(s)
Antigen-Presenting Cells/metabolism , Immunotherapy, Adoptive/methods , Lentivirus/genetics , Transduction, Genetic/methods , HumansABSTRACT
Regulatory T cells (Tregs) are key modulators of inflammation and are important for the maintenance of peripheral tolerance. Adoptive immunotherapy with polyclonal Tregs holds promise in organ transplantation, graft-versus-host disease, and autoimmune diseases, but may be enhanced by antigen-specific, long-lived Treg cells. We modified primary human Tregs with chimeric antigen-receptors (CARs) bearing different costimulatory domains and performed in vitro analyses of their phenotype and function. While neither the presence of a CAR nor the type of costimulation domain influenced Foxp3 expression in Tregs, the costimulation domain of the CARs affected CAR Treg surface phenotype and functions such as cytokine production. Furthermore, signaling from the CD28 costimulation domain maintained CAR Treg suppressor function, whereas 4-1B costimulation did not. In vivo, CAR Tregs accumulated at sites expressing target antigen, and suppressed antigen specific effector T cell responses; however, only CAR Tregs with CD28 signaling domains were potent inhibitors of effector T cell mediated graft rejection in vivo. Our findings support the use of CD28 based CAR-Tregs for tissue specific immune suppression in the clinic.
Subject(s)
Receptors, Chimeric Antigen/chemistry , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Animals , Autoimmune Diseases , Autoimmunity , CD28 Antigens , Cell Line , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Graft vs Host Disease , Granzymes , Heterografts , Humans , Immunosuppression Therapy , Male , Mice , Mice, Inbred NOD , Organ Transplantation , Phenotype , Skin/pathologyABSTRACT
Chimeric antigen receptor (CAR) T cells (CARTs) have shown tremendous potential for the treatment of certain B-cell malignancies, including patients with relapsed/refractory multiple myeloma (MM). Targeting the B-cell maturation antigen (BCMA) has produced the most promising results for CART therapy of MM to date, but not all remissions are sustained. Emergence of BCMA escape variants has been reported under the selective pressure of monospecific anti-BCMA CART treatment. Thus, there is a clinical need for continuous improvement of CART therapies for MM. Here, we show that a novel trimeric APRIL (a proliferation-inducing ligand)-based CAR efficiently targets both BCMA+ and BCMA- MM. Modeled after the natural ligand-receptor pair, APRIL-based CARs allow for bispecific targeting of the MM-associated antigens BCMA and transmembrane activator and CAML interactor (TACI). However, natural ligands as CAR antigen-binding domains may require further engineering to promote optimal binding and multimerization to adequately trigger T-cell activation. We found that using a trimeric rather than a monomeric APRIL format as the antigen-binding domain enhanced binding to BCMA and TACI and CART activity against MM in vitro and in vivo. Dual-specific, trimeric APRIL-based CAR are a promising therapeutic approach for MM with potential for preventing and treating BCMA escape.