RESUMEN
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.
Asunto(s)
Alergia e Inmunología , Vacunas contra el Cáncer/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Biología Sintética , Linfocitos T/inmunología , Animales , Ingeniería Genética , Humanos , Activación de Linfocitos , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T/trasplanteRESUMEN
Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Receptores Artificiales , Humanos , Receptores de Antígenos de Linfocitos T/genética , Receptores Artificiales/genética , Biología Sintética , Linfocitos TRESUMEN
Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.
Asunto(s)
Técnicas de Sustitución del Gen/métodos , Ingeniería Genética/métodos , Inmunoterapia/métodos , Animales , Células Sanguíneas , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Guía de Kinetoplastida/genética , Análisis de la Célula Individual/métodos , Linfocitos T , Transcriptoma/genéticaRESUMEN
T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.
Asunto(s)
Inmunoterapia/métodos , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T/metabolismo , Animales , Antígenos CD19/metabolismo , Antígenos de Superficie/inmunología , Efecto Espectador , Comunicación Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI/metabolismo , Humanos , Células Jurkat , Activación de Linfocitos , Mesotelina , Ratones , Receptores Notch/metabolismoRESUMEN
The Notch protein is one of the most mechanistically direct transmembrane receptors-the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.
Asunto(s)
Ingeniería Celular , Receptores Notch/química , Transducción de Señal , Biología Sintética/métodos , Animales , Línea Celular , Perros , Humanos , Ratones , Neuronas/metabolismo , Receptores Notch/metabolismo , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Ingeniería Celular , Neoplasias/terapia , Receptores Artificiales/inmunología , Receptores Notch/inmunología , Anticuerpos/inmunología , Línea Celular Tumoral , Citocinas/inmunología , Citotoxicidad Inmunológica , Humanos , Inmunoterapia/métodos , Activación de Linfocitos , Receptores Artificiales/genética , Receptores Notch/genética , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Células TH1/inmunología , Transcripción Genética , Microambiente TumoralRESUMEN
Adoptive T cell therapies have produced exceptional responses in a subset of patients with cancer. However, therapeutic efficacy can be hindered by poor T cell persistence and function1. In human T cell cancers, evolution of the disease positively selects for mutations that improve fitness of T cells in challenging situations analogous to those faced by therapeutic T cells. Therefore, we reasoned that these mutations could be co-opted to improve T cell therapies. Here we systematically screened the effects of 71 mutations from T cell neoplasms on T cell signalling, cytokine production and in vivo persistence in tumours. We identify a gene fusion, CARD11-PIK3R3, found in a CD4+ cutaneous T cell lymphoma2, that augments CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy of therapeutic T cells in several immunotherapy-refractory models in an antigen-dependent manner. Underscoring its potential to be deployed safely, CARD11-PIK3R3-expressing cells were followed up to 418 days after T cell transfer in vivo without evidence of malignant transformation. Collectively, our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies.
Asunto(s)
Evolución Molecular , Inmunoterapia Adoptiva , Linfoma Cutáneo de Células T , Mutación , Linfocitos T , Humanos , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Citocinas/metabolismo , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Inmunoterapia Adoptiva/métodos , Linfoma Cutáneo de Células T/genética , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/patología , Linfoma Cutáneo de Células T/terapia , Fosfatidilinositol 3-Quinasas , Transducción de Señal/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplanteRESUMEN
Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno CTLA-4/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Interferón gamma/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/metabolismo , Línea Celular Tumoral , Supresión Clonal/efectos de los fármacos , Supresión Clonal/inmunología , Resistencia a Antineoplásicos/inmunología , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunologíaRESUMEN
Synthetic biology (synbio) tools, such as chimeric antigen receptors (CARs), have been designed to target, activate, and improve immune cell responses to tumors. These therapies have demonstrated an ability to cure patients with blood cancers. However, there are significant challenges to designing, testing, and efficiently translating these complex cell therapies for patients who do not respond or have immune refractory solid tumors. The rapid progress of synbio tools for cell therapy, particularly for cancer immunotherapy, is encouraging but our development process should be tailored to increase translational success. Particularly, next-generation cell therapies should be rooted in basic immunology, tested in more predictive preclinical models, engineered for potency with the right balance of safety, educated by clinical findings, and multi-faceted to combat a range of suppressive mechanisms. Here, we lay out five principles for engineering future cell therapies to increase the probability of clinical impact, and in the context of these principles, we provide an overview of the current state of synbio cell therapy design for cancer. Although these principles are anchored in engineering immune cells for cancer therapy, we posit that they can help guide translational synbio research for broad impact in other disease indications with high unmet need.
Asunto(s)
Neoplasias Hematológicas , Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva , Neoplasias/terapia , InmunoterapiaRESUMEN
T cells are activated through interaction with antigen-presenting cells (APCs). During activation, receptors and signaling intermediates accumulate in diverse spatiotemporal distributions. These distributions control the probability of signaling interactions and thus govern information flow through the signaling system. Spatiotemporally resolved system-scale investigation of signaling can extract the regulatory information thus encoded, allowing unique insight into the control of T-cell function. Substantial technical challenges exist, and these are briefly discussed herein. While much of the work assessing T-cell spatiotemporal organization uses planar APC substitutes, we focus here on B-cell APCs with often stark differences. Spatiotemporal signaling distributions are driven by cell biologically distinct structures, a large protein assembly at the interface center, a large invagination, the actin-supported interface periphery as extended by smaller individual lamella, and a newly discovered whole-interface actin-driven lamellum. The more than 60 elements of T-cell activation studied to date are dynamically distributed between these structures, generating a complex organization of the signaling system. Signal initiation and core signaling prefer the interface center, while signal amplification is localized in the transient lamellum. Actin dynamics control signaling distributions through regulation of the underlying structures and drive a highly undulating T-cell/APC interface that imposes substantial constraints on T-cell organization. We suggest that the regulation of actin dynamics, by controlling signaling distributions and membrane topology, is an important rheostat of T-cell signaling.
Asunto(s)
Actinas/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Comunicación Celular , Humanos , Activación de LinfocitosRESUMEN
CD4(+) Th2 development is regulated by the zinc finger transcription factor GATA3. Once induced by acute priming signals, such as IL-4, GATA3 poises the Th2 cytokine locus for rapid activation and establishes a positive-feedback loop that maintains elevated GATA3 expression. Type I IFN (IFN-α/ß) inhibits Th2 cells by blocking the expression of GATA3 during Th2 development and in fully committed Th2 cells. In this study, we uncovered a unique mechanism by which IFN-α/ß signaling represses the GATA3 gene in human Th2 cells. IFN-α/ß suppressed expression of GATA3 mRNA that was transcribed from an alternative distal upstream exon (1A). This suppression was not mediated through DNA methylation, but rather by histone modifications localized to a conserved noncoding sequence (CNS-1) upstream of exon 1A. IFN-α/ß treatment led to a closed conformation of CNS-1, as assessed by DNase I hypersensitivity, along with enhanced accumulation of H3K27me3 mark at this CNS region, which correlated with increased density of total nucleosomes at this putative enhancer. Consequently, accessibility of CNS-1 to GATA3 DNA binding activity was reduced in response to IFN-α/ß signaling, even in the presence of IL-4. Thus, IFN-α/ß disrupts the GATA3-autoactivation loop and promotes epigenetic silencing of a Th2-specific regulatory region within the GATA3 gene.
Asunto(s)
Metilación de ADN/inmunología , Elementos de Facilitación Genéticos/inmunología , Factor de Transcripción GATA3/inmunología , Interferón-alfa/inmunología , Transducción de Señal/inmunología , Células Th2/inmunología , Transcripción Genética/inmunología , Adulto , Metilación de ADN/genética , Exones/inmunología , Femenino , Factor de Transcripción GATA3/genética , Humanos , Interferón-alfa/genética , Interferón beta/genética , Interferón beta/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Masculino , Transducción de Señal/genética , Células Th2/citología , Transcripción Genética/genéticaRESUMEN
ABSTRACT: Multiple myeloma is characterized by frequent clinical relapses after conventional therapy. Recently, chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, although >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune-intrinsic mechanisms may contribute to this resistance. Although there were no preexisting T-cell features associated with clinical outcomes, we found that patients with a durable response to CAR T-cell treatment had greater persistence of their CAR T cells than patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T-effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR T cells. These cells expand in vivo early after infusion but express exhaustion markers (hepatitis A virus cellular receptor 2 [HAVCR2] and T-cell immunoglobulin and mucin domain-containing-3 [TIGIT]) and remain polyclonal. Finally, we demonstrate that nonclassical monocytes are enriched in the myeloma niche and may induce CAR T-cell dysfunction through mechanisms that include transforming growth factor ß. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR T-cell therapy.
Asunto(s)
Antígeno de Maduración de Linfocitos B , Linfocitos T CD4-Positivos , Inmunoterapia Adoptiva , Mieloma Múltiple , Receptores Quiméricos de Antígenos , Mieloma Múltiple/terapia , Mieloma Múltiple/inmunología , Humanos , Antígeno de Maduración de Linfocitos B/metabolismo , Antígeno de Maduración de Linfocitos B/inmunología , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Recurrencia , Masculino , Femenino , Agotamiento de Células TRESUMEN
T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.
Asunto(s)
Complejo CD3/metabolismo , Sinapsis Inmunológicas , Fosfatidilinositoles/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Aminoácidos Básicos , Animales , Sitios de Unión/inmunología , Complejo CD3/química , Complejo CD3/inmunología , Línea Celular , Humanos , Activación de Linfocitos/inmunología , Ratones , Fosfatidilinositoles/inmunología , Unión Proteica/inmunología , Transducción de Señal/inmunología , TransfecciónRESUMEN
The immune system includes an array of specialized cells that keep us healthy by responding to pathogenic cues. Investigations into the mechanisms behind immune cell behavior have led to the development of powerful immunotherapies, including chimeric-antigen receptor (CAR) T cells. Although CAR T cells have demonstrated efficacy in treating blood cancers, issues regarding their safety and potency have hindered the use of immunotherapies in a wider spectrum of diseases. Efforts to integrate developments in synthetic biology into immunotherapy have led to several advancements with the potential to expand the range of treatable diseases, fine-tune the desired immune response, and improve therapeutic cell potency. Here, we examine current synthetic biology advances that aim to improve on existing technologies and discuss the promise of the next generation of engineered immune cell therapies.
Asunto(s)
Neoplasias , Linfocitos T , Humanos , Receptores de Antígenos de Linfocitos T/genética , Inmunoterapia Adoptiva , Inmunoterapia , Neoplasias/terapiaRESUMEN
T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.
Asunto(s)
Microvellosidades , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Linfocitos T , Microvellosidades/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Anticuerpos de Cadena Única/metabolismo , Humanos , AntígenosRESUMEN
For the past several decades, chimeric antigen receptor T-cell therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Methods: Using synthetic biology, we engineered T cells with a chimeric receptor synthetic intramembrane proteolysis receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette on recognition of specific tumor markers. We then applied a SNIPR-based PET reporter system to 2 cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors, respectively. Results: Antigen-specific reporter induction of the SNIPR PET T cells was confirmed in vitro using green fluorescent protein fluorescence, luciferase luminescence, and the HSV-TK PET reporter with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG). T cells associated with their target antigens were successfully imaged using PET in dual-xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with more than 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. Conclusion: The main innovation found in this work was PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.
Asunto(s)
Neoplasias de la Mama , Glioblastoma , Animales , Humanos , Femenino , Linfocitos T , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/genética , Línea Celular Tumoral , Tomografía de Emisión de Positrones/métodos , Genes ReporterosRESUMEN
Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.
RESUMEN
Mycobacterial shuttle vectors contain dual origins of replication for growth in both Escherichia coli and mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-kan-MCS2) enabled green fluorescent protein expression in E. coli, Mycobacterium smegmatis, and M. avium at levels up to 50-fold higher than that detected with the parental vector, which was originally developed with a lacZα promoter. This high-level fluorescent protein expression allowed easy visualization of M. smegmatis and M. avium in infected macrophages. The M. tuberculosis gene esat-6 was cloned in place of the green fluorescence protein gene (gfp) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-kan-MCS2) yielded high levels of ESAT-6 expression in M. smegmatis. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide. M. smegmatis clones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced in E. coli, was insufficient to suppress a strong inflammatory response elicited by M. smegmatis or lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species.
Asunto(s)
Expresión Génica , Vectores Genéticos , Genética Microbiana/métodos , Macrófagos/microbiología , Biología Molecular/métodos , Mycobacterium/genética , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/genética , Fluorescencia , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Evasión Inmune , Tolerancia Inmunológica , Mycobacterium/patogenicidad , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Factores de Virulencia/biosíntesis , Factores de Virulencia/genéticaRESUMEN
IL-12 is an essential cytokine involved in the generation of memory or memory-like NK cells. Mouse cytomegalovirus infection triggers NK receptor-induced, ligand-specific IL-12-dependent NK cell expansion, yet specific IL-12 stimulation ex vivo leading to NK cell proliferation and expansion is not established. Here, we show that IL-12 alone can sustain human primary NK cell survival without providing IL-2 or IL-15 but was insufficient to promote human NK cell proliferation. IL-12 signaling analysis revealed STAT5 phosphorylation and weak mTOR activation, which was enhanced by activating NK receptor upregulation and crosslinking leading to STAT5-dependent, rapamycin-sensitive, or TGFß-sensitive NK cell IL-12-dependent expansion, independently of IL-12 receptor upregulation. Prolonged IL-2 culture did not impair IL-12-dependent ligand-specific NK cell expansion. These findings demonstrate that activating NK receptor stimulation promotes differential IL-12 signaling, leading to human NK cell expansion, and suggest adopting strategies to provide IL-12 signaling in vivo for ligand-specific IL-2-primed NK cell-based therapies.
Asunto(s)
Interleucina-12 , Factor de Transcripción STAT5 , Proliferación Celular , Humanos , Interleucina-2/farmacología , Ligandos , Receptores de Células Asesinas NaturalesRESUMEN
Synthetic gene circuits that precisely control human cell function could expand the capabilities of gene- and cell-based therapies. However, platforms for developing circuits in primary human cells that drive robust functional changes in vivo and have compositions suitable for clinical use are lacking. Here, we developed synthetic zinc finger transcription regulators (synZiFTRs), which are compact and based largely on human-derived proteins. As a proof of principle, we engineered gene switches and circuits that allow precise, user-defined control over therapeutically relevant genes in primary T cells using orthogonal, US Food and Drug Administration-approved small-molecule inducers. Our circuits can instruct T cells to sequentially activate multiple cellular programs such as proliferation and antitumor activity to drive synergistic therapeutic responses. This platform should accelerate the development and clinical translation of synthetic gene circuits in diverse human cell types and contexts.