RESUMO
Immunological targeting of pathological cells has been successful in oncology and is expanding to other pathobiological contexts. Here, we present a flexible platform that allows labeling cells of interest with the surface-expressed model antigen ovalbumin (OVA), which can be eliminated via either antigen-specific T cells or newly developed OVA antibodies. We demonstrate that hepatocytes can be effectively targeted by either modality. In contrast, pro-fibrotic fibroblasts associated with pulmonary fibrosis are only eliminated by T cells in initial experiments, which reduced collagen deposition in a fibrosis model. This new experimental platform will facilitate development of immune-based approaches to clear potential pathological cell types in vivo.
Assuntos
Anticorpos , Fibrose Pulmonar , Humanos , Fibroblastos , Hepatócitos , CinéticaRESUMO
BACKGROUND AND OBJECTIVES: Anti-CD20 monoclonal antibody (mAb) B-cell depletion is a remarkably successful multiple sclerosis (MS) treatment. Chimeric antigen receptor (CAR)-T cells, which target antigens in a non-major histocompatibility complex (MHC)-restricted manner, can penetrate tissues more thoroughly than mAbs. However, a previous study indicated that anti-CD19 CAR-T cells can paradoxically exacerbate experimental autoimmune encephalomyelitis (EAE) disease. We tested anti-CD19 CAR-T cells in a B-cell-dependent EAE model that is responsive to anti-CD20 B-cell depletion similar to the clinical benefit of anti-CD20 mAb treatment in MS. METHODS: Anti-CD19 CAR-T cells or control cells that overexpressed green fluorescent protein were transferred into C57BL/6 mice pretreated with cyclophosphamide (Cy). Mice were immunized with recombinant human (rh) myelin oligodendrocyte protein (MOG), which causes EAE in a B-cell-dependent manner. Mice were evaluated for B-cell depletion, clinical and histologic signs of EAE, and immune modulation. RESULTS: Clinical scores and lymphocyte infiltration were reduced in mice treated with either anti-CD19 CAR-T cells with Cy or control cells with Cy, but not with Cy alone. B-cell depletion was observed in peripheral lymphoid tissue and in the CNS of mice treated with anti-CD19 CAR-T cells with Cy pretreatment. Th1 or Th17 populations did not differ in anti-CD19 CAR-T cell, control cell-treated animals, or Cy alone. DISCUSSION: In contrast to previous data showing that anti-CD19 CAR-T cell treatment exacerbated EAE, we observed that anti-CD19 CAR-T cells ameliorated EAE. In addition, anti-CD19 CAR-T cells thoroughly depleted B cells in peripheral tissues and in the CNS. However, the clinical benefit occurred independently of antigen specificity or B-cell depletion.
Assuntos
Encefalomielite Autoimune Experimental , Imunoterapia Adotiva , Animais , Humanos , Camundongos , Anticorpos Monoclonais , Antígenos CD19 , Autoimunidade , Sistema Nervoso Central , Encefalomielite Autoimune Experimental/tratamento farmacológico , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Glicoproteína Mielina-Oligodendrócito , Linfócitos T , Linfócitos BRESUMO
Chimeric antigen receptor (CAR) costimulatory domains derived from native immune receptors steer the phenotypic output of therapeutic T cells. We constructed a library of CARs containing ~2300 synthetic costimulatory domains, built from combinations of 13 signaling motifs. These CARs promoted diverse human T cell fates, which were sensitive to motif combinations and configurations. Neural networks trained to decode the combinatorial grammar of CAR signaling motifs allowed extraction of key design rules. For example, non-native combinations of motifs that bind tumor necrosis factor receptor-associated factors (TRAFs) and phospholipase C gamma 1 (PLCγ1) enhanced cytotoxicity and stemness associated with effective tumor killing. Thus, libraries built from minimal building blocks of signaling, combined with machine learning, can efficiently guide engineering of receptors with desired phenotypes.
Assuntos
Aprendizado de Máquina , Biblioteca de Peptídeos , Receptores de Antígenos Quiméricos , Linfócitos T Citotóxicos , Humanos , Fenótipo , Receptores de Antígenos Quiméricos/química , Receptores de Antígenos Quiméricos/imunologia , Transdução de Sinais , Domínios Proteicos , Linfócitos T Citotóxicos/imunologiaRESUMO
Chimeric antigen receptor (CAR) T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the cytokine IL-2. These circuits potently enhance CAR T cell infiltration and clearance of immune-excluded tumors, without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and T cell receptor (TCR)- or CAR-independent manner, bypassing suppression mechanisms including consumption of IL-2 or inhibition of TCR signaling. These engineered cells establish a foothold in the target tumors, with synthetic Notch-induced IL-2 production enabling initiation of CAR-mediated T cell expansion and cell killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for an antitumor response, but in a manner that evades key points of tumor suppression.
Assuntos
Terapia de Imunossupressão , Imunoterapia Adotiva , Interleucina-2 , Neoplasias , Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Imunoterapia Adotiva/métodos , Interleucina-2/genética , Interleucina-2/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Linfócitos T/transplante , Microambiente Tumoral , Animais , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Engenharia Celular , Receptores Notch/metabolismo , Terapia de Imunossupressão/métodosRESUMO
A new era of biological engineering is emerging in which living cells are used as building blocks to address therapeutic challenges. These efforts are distinct from traditional molecular engineering-their focus is not on optimizing individual genes and proteins as therapeutics, but rather on using molecular components as modules to reprogram how cells make decisions and communicate to achieve higher-order physiological functions in vivo. This cell-centric approach is enabled by a growing tool kit of components that can synthetically control core cell-level functional outputs, such as where in the body a cell should go, what other cells it should interact with, and what messages it should transmit or receive. The power of cell engineering has been clinically validated by the development of immune cells designed to kill cancer. This same tool kit for rewiring cell connectivity is beginning to be used to engineer cell therapies for a host of other diseases and to program the self-organization of tissues and organs. By forcing the conceptual distillation of complex biological functions into a finite set of instructions that operate at the cell level, these efforts also shed light on the fundamental hierarchical logic that links molecular components to higher-order physiological function.
Assuntos
Engenharia Celular , Terapia Baseada em Transplante de Células e Tecidos , Imunoterapia Adotiva , Neoplasias , Linfócitos T , Humanos , Neoplasias/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Linfócitos T/transplanteRESUMO
In the past several decades, the development of cancer therapeutics has largely focused on precision targeting of single cancer-associated molecules. Despite great advances, such targeted therapies still show incomplete precision and the eventual development of resistance due to target heterogeneity or mutation. However, the recent development of cell-based therapies such as chimeric antigen receptor (CAR) T cells presents a revolutionary opportunity to reframe strategies for targeting cancers. Immune cells equipped with synthetic circuits are essentially living computers that can be programmed to recognize tumours based on multiple signals, including both tumour cell-intrinsic and microenvironmental. Moreover, cells can be programmed to launch broad but highly localized therapeutic responses that can limit the potential for escape while still maintaining high precision. Although these emerging smart cell engineering capabilities have yet to be fully implemented in the clinic, we argue here that they will become much more powerful when combined with machine learning analysis of genomic data, which can guide the design of therapeutic recognition programs that are the most discriminatory and actionable. The merging of cancer analytics and synthetic biology could lead to nuanced paradigms of tumour recognition, more akin to facial recognition, that have the ability to more effectively address the complex challenges of treating cancer.
Assuntos
Neoplasias , Humanos , Neoplasias/genética , Imunoterapia Adotiva , Engenharia Celular , Biologia SintéticaRESUMO
Treatment of solid cancers with chimeric antigen receptor (CAR) T cells is plagued by the lack of ideal target antigens that are both absolutely tumor specific and homogeneously expressed. We show that multi-antigen prime-and-kill recognition circuits provide flexibility and precision to overcome these challenges in the context of glioblastoma. A synNotch receptor that recognizes a specific priming antigen, such as the heterogeneous but tumor-specific glioblastoma neoantigen epidermal growth factor receptor splice variant III (EGFRvIII) or the central nervous system (CNS) tissue-specific antigen myelin oligodendrocyte glycoprotein (MOG), can be used to locally induce expression of a CAR. This enables thorough but controlled tumor cell killing by targeting antigens that are homogeneous but not absolutely tumor specific. Moreover, synNotch-regulated CAR expression averts tonic signaling and exhaustion, maintaining a higher fraction of the T cells in a naïve/stem cell memory state. In immunodeficient mice bearing intracerebral patient-derived xenografts (PDXs) with heterogeneous expression of EGFRvIII, a single intravenous infusion of EGFRvIII synNotch-CAR T cells demonstrated higher antitumor efficacy and T cell durability than conventional constitutively expressed CAR T cells, without off-tumor killing. T cells transduced with a synNotch-CAR circuit primed by the CNS-specific antigen MOG also exhibited precise and potent control of intracerebral PDX without evidence of priming outside of the brain. In summary, by using circuits that integrate recognition of multiple imperfect but complementary antigens, we improve the specificity, completeness, and persistence of T cells directed against glioblastoma, providing a general recognition strategy applicable to other solid tumors.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Animais , Encéfalo/metabolismo , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Glioblastoma/terapia , Imunoterapia Adotiva , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Overexpressed tumor-associated antigens [for example, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)] are attractive targets for therapeutic T cells, but toxic "off-tumor" cross-reaction with normal tissues that express low levels of target antigen can occur with chimeric antigen receptor (CAR)-T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive-feedback circuit that allows human cytotoxic T cells to discriminate targets on the basis of a sigmoidal antigen-density threshold. In this circuit, a low-affinity synthetic Notch receptor for HER2 controls the expression of a high-affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells-it increases both CAR expression and activation-leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100 times as much HER2, both in vitro and in vivo.
Assuntos
Engenharia Celular , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva , Células K562 , Camundongos , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Receptores Artificiais/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Esferoides Celulares , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Biomaterials can improve the safety and presentation of therapeutic agents for effective immunotherapy, and a high level of control over surface functionalization is essential for immune cell modulation. Here, we developed biocompatible immune cell-engaging particles (ICEp) that use synthetic short DNA as scaffolds for efficient and tunable protein loading. To improve the safety of chimeric antigen receptor (CAR) T cell therapies, micrometre-sized ICEp were injected intratumorally to present a priming signal for systemically administered AND-gate CAR-T cells. Locally retained ICEp presenting a high density of priming antigens activated CAR T cells, driving local tumour clearance while sparing uninjected tumours in immunodeficient mice. The ratiometric control of costimulatory ligands (anti-CD3 and anti-CD28 antibodies) and the surface presentation of a cytokine (IL-2) on ICEp were shown to substantially impact human primary T cell activation phenotypes. This modular and versatile biomaterial functionalization platform can provide new opportunities for immunotherapies.
Assuntos
Materiais Biocompatíveis/química , DNA/química , Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Materiais Biocompatíveis/uso terapêutico , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva , Ativação Linfocitária , Camundongos , Nanopartículas/química , Neoplasias/terapia , Proteínas/química , Proteínas/imunologia , Proteínas/uso terapêutico , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplanteRESUMO
Living cells often identify their correct partner or target cells by integrating information from multiple receptors, achieving levels of recognition that are difficult to obtain with individual molecular interactions. In this study, we engineered a diverse library of multireceptor cell-cell recognition circuits by using synthetic Notch receptors to transcriptionally interconnect multiple molecular recognition events. These synthetic circuits allow engineered T cells to integrate extra- and intracellular antigen recognition, are robust to heterogeneity, and achieve precise recognition by integrating up to three different antigens with positive or negative logic. A three-antigen AND gate composed of three sequentially linked receptors shows selectivity in vivo, clearing three-antigen tumors while ignoring related two-antigen tumors. Daisy-chaining multiple molecular recognition events together in synthetic circuits provides a powerful way to engineer cellular-level recognition.
Assuntos
Comunicação Celular/imunologia , Engenharia Celular , Receptores de Antígenos Quiméricos/imunologia , Receptores Notch/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Humanos , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores Notch/genética , Transcrição GênicaRESUMO
Precise discrimination of tumor from normal tissues remains a major roadblock for therapeutic efficacy of chimeric antigen receptor (CAR) T cells. Here, we perform a comprehensive in silico screen to identify multi-antigen signatures that improve tumor discrimination by CAR T cells engineered to integrate multiple antigen inputs via Boolean logic, e.g., AND and NOT. We screen >2.5 million dual antigens and â¼60 million triple antigens across 33 tumor types and 34 normal tissues. We find that dual antigens significantly outperform the best single clinically investigated CAR targets and confirm key predictions experimentally. Further, we identify antigen triplets that are predicted to show close to ideal tumor-versus-normal tissue discrimination for several tumor types. This work demonstrates the potential of 2- to 3-antigen Boolean logic gates for improving tumor discrimination by CAR T cell therapies. Our predictions are available on an interactive web server resource (antigen.princeton.edu).
Assuntos
Antígenos de Neoplasias/metabolismo , Imunoterapia Adotiva/métodos , HumanosRESUMO
Many processes controlling cell growth and death are well characterized for individual cell lineages, but how ensembles of different cell types in a tissue regulate collective size and composition remains unclear. In this issue of Cell, Zhou et al. employ experiments and theory to uncover design principles of tissue homeostasis arising from cross-talk between fibroblasts and macrophages.
Assuntos
Homeostase , Macrófagos , Linhagem da Célula , Fenômenos Fisiológicos Celulares , FibroblastosRESUMO
Although oscillatory circuits are prevalent in transcriptional regulation, it is unclear how a circuit's structure and the specific parameters that describe its components determine the shape of its oscillations. Here, we engineer a minimal, inducible human nuclear factor κB (NF-κB)-based system that is composed of NF-κB (RelA) and degradable inhibitor of NF-κB (IκBα), into the yeast, Saccharomyces cerevisiae. We define an oscillation's waveform quantitatively as a function of signal amplitude, rest time, rise time, and decay time; by systematically tuning RelA concentration, the strength of negative feedback, and the degradation rate of IκBα, we demonstrate that peak shape and frequency of oscillations can be controlled in vivo and predicted mathematically. In addition, we show that nested negative feedback loops can be employed to specifically tune the frequency of oscillations while leaving their peak shape unchanged. In total, this work establishes design principles that enable function-guided design of oscillatory signaling controllers in diverse synthetic biology applications.
Assuntos
NF-kappa B/genética , Transdução de Sinais/genética , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica/genética , Humanos , Modelos Biológicos , Inibidor de NF-kappaB alfa/genética , Saccharomyces cerevisiae/genética , Fator de Transcrição RelA/genéticaRESUMO
The ability of immune cells to survey tissues and sense pathologic insults and deviations makes them a unique platform for interfacing with the body and disease. With the rapid advancement of synthetic biology, we can now engineer and equip immune cells with new sensors and controllable therapeutic response programs to sense and treat diseases that our natural immune system cannot normally handle. Here we review the current state of engineered immune cell therapeutics and their unique capabilities compared to small molecules and biologics. We then discuss how engineered immune cells are being designed to combat cancer, focusing on how new synthetic biology tools are providing potential ways to overcome the major roadblocks for treatment. Finally, we give a long-term vision for the use of synthetic biology to engineer immune cells as a general sensor-response platform to precisely detect disease, to remodel disease microenvironments, and to treat a potentially wide range of challenging diseases.
Assuntos
Alergia e Imunologia , Vacinas Anticâncer/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Biologia Sintética , Linfócitos T/imunologia , Animais , Engenharia Genética , Humanos , Ativação Linfocitária , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Linfócitos T/transplanteRESUMO
Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 [PD-1]) have both opened new avenues for cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies.
Assuntos
Sistemas CRISPR-Cas , Marcação de Genes , Neoplasias/genética , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígeno B7-H1/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imunofenotipagem , Camundongos , Camundongos Knockout , Neoplasias/patologia , Neoplasias/terapia , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Chimeric antigen receptor (CAR) T cells have proven that engineered immune cells can serve as a powerful new class of cancer therapeutics. Clinical experience has helped to define the major challenges that must be met to make engineered T cells a reliable, safe, and effective platform that can be deployed against a broad range of tumors. The emergence of synthetic biology approaches for cellular engineering is providing us with a broadly expanded set of tools for programming immune cells. We discuss how these tools could be used to design the next generation of smart T cell precision therapeutics.
Assuntos
Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Animais , Antígenos CD19/análise , Engenharia Celular/métodos , Humanos , Linfócitos do Interstício Tumoral/imunologia , Medicina de Precisão , Receptores de Antígenos de Linfócitos T/imunologia , Biologia Sintética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente TumoralRESUMO
Engineered T cells are currently in clinical trials to treat patients with cancer, solid organ transplants, and autoimmune diseases. However, the field is still in its infancy. The design, and manufacturing, of T cell therapies is not standardized and is performed mostly in academic settings by competing groups. Reliable methods to define dose and pharmacokinetics of T cell therapies need to be developed. As of mid-2016, there are no US Food and Drug Administration (FDA)-approved T cell therapeutics on the market, and FDA regulations are only slowly adapting to the new technologies. Further development of engineered T cell therapies requires advances in immunology, synthetic biology, manufacturing processes, and government regulation. In this review, we outline some of these challenges and discuss the contributions that pathologists can make to this emerging field.
Assuntos
Doenças Autoimunes/terapia , Terapia Baseada em Transplante de Células e Tecidos , Ensaios Clínicos como Assunto , Neoplasias/terapia , Biologia Sintética , Linfócitos T/imunologia , Animais , Doenças Autoimunes/imunologia , Humanos , Neoplasias/imunologiaRESUMO
Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However, the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic, whereas other responses, such as the ability to overcome tumor immunosuppression, are absent. Thus, the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles, biased T cell differentiation, and local delivery of non-native therapeutic payloads, such as antibodies, in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases.