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1.
Front Pharmacol ; 15: 1380000, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38887559

RESUMEN

Introduction: Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities. Methods: We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15. This "PD1/IL15" selectively delivers IL-15 signaling to lymphocytes expressing PD-1. We then investigated the pharmacokinetic (PK) and pharmacodynamic (PD) effects of PD1/IL15 TaCk on immune cell subsets in cynomolgus monkeys after single and repeat intravenous dose administrations. We used these results to determine the first-in-human (FIH) dose and dosing frequency for early clinical trials. Results: The PD1/IL15 TaCk exhibited a nonlinear multiphasic PK profile, while the untargeted isotype control TaCk, containing an anti-respiratory syncytial virus antibody (RSV/IL15), showed linear and dose proportional PK. The PD1/IL15 TaCk also displayed a considerably prolonged PK (half-life range ∼1.0-4.1 days) compared to wild-type IL-15 (half-life ∼1.1 h), which led to an enhanced cell expansion PD response. The PD was dose-dependent, durable, and selective for PD-1+ lymphocytes. Notably, the dose- and time-dependent PK was attributed to dynamic TMDD resulting from test article-induced lymphocyte expansion upon repeat administration. The recommended first-in-human (FIH) dose of PD1/IL15 TaCk is 0.003 mg/kg, determined based on a minimum anticipated biological effect level (MABEL) approach utilizing a combination of in vitro and preclinical in vivo data. Conclusion: This work provides insight into the complex PK/PD relationship of PD1/IL15 TaCk in monkeys and informs the recommended starting dose and dosing frequency selection to support clinical evaluation of this novel targeted cytokine.

3.
Mol Cancer Ther ; 2024 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-38739434

RESUMEN

Insufficient quantity of functional T cells is a likely factor limiting clinical activity of T cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T cell dependent (TDB) antibodies. Activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in upregulation of IL-2/15Rß (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to the IL-15. XmAb24306 enhanced T cell bispecific antibody induced CD8+ and CD4+ T cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T cell numbers were normalized, suggesting that T cell expansion is the main mechanism for the observed benefit. Pre-treatment of immune competent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in significant increase of T cells in blood, spleen and in tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis where the number of tumor infiltrating T cells is rate limiting for the activity of solid tumor targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T cell bispecific antibodies supports clinical evaluation of this novel immunotherapy combination.

5.
Nature ; 627(8004): 646-655, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38418879

RESUMEN

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.


Asunto(s)
Anticuerpos Monoclonales , Antineoplásicos , Antígeno B7-H1 , Células Mieloides , Neoplasias , Receptores Inmunológicos , Linfocitos T Reguladores , Animales , Humanos , Ratones , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Quimioterapia Combinada , Inhibidores de Puntos de Control Inmunológico/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Activación de Macrófagos , Células Mieloides/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Receptores de IgG/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/inmunología
6.
Eur J Pharm Sci ; 186: 106450, 2023 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-37084985

RESUMEN

XmAb24306 is a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein currently under clinical investigation as an immunotherapeutic agent for cancer treatment. XmAb24306 contains mutations in IL-15 that attenuate its affinity to the heterodimeric IL-15 receptor ßγ (IL-15R). We observe substantially prolonged pharmacokinetics (PK) (half-life ∼ 2.5 to 4.5 days) in single- and repeat-dose cynomolgus monkey (cyno) studies compared to wild-type IL-15 (half-life ∼ 1 hour), leading to increased exposure and enhanced and durable expansion of NK cells, CD8+ T cells and CD4-CD8- (double negative [DN]) T cells. Drug clearance varied with dose level and time post-dose, and PK exposure decreased upon repeated dosing, which we attribute to increased target-mediated drug disposition (TMDD) resulting from drug-induced lymphocyte expansion (i.e., pharmacodynamic (PD)-enhanced TMDD). We developed a quantitative systems pharmacology (QSP) model to quantify the complex PKPD behaviors due to the interactions of XmAb24306 with multiple cell types (CD8+, CD4+, DN T cells, and NK cells) in the peripheral blood (PB) and lymphoid tissues. The model, which includes nonspecific drug clearance, binding to and TMDD by IL15R differentially expressed on lymphocyte subsets, and resultant lymphocyte margination/migration out of PB, expansion in lymphoid tissues, and redistribution to the blood, successfully describes the systemic PK and lymphocyte kinetics observed in the cyno studies. Results suggest that after 3 doses of every-two-week (Q2W) doses up to 70 days, the relative contributions of each elimination pathway to XmAb24306 clearance are: DN T cells > NK cells > CD8+ T cells > nonspecific clearance > CD4+ T cells. Modeling suggests that observed cellular expansion in blood results from the influx of cells expanded by the drug in lymphoid tissues. The model is used to predict lymphoid tissue expansion and to simulate PK-PD for different dose regimens. Thus, the model provides insight into the mechanisms underlying the observed PK-PD behavior of an engineered cytokine and can serve as a framework for the rapid integration and analysis of data that emerges from ongoing clinical studies in cancer patients as single-agent or given in combination.


Asunto(s)
Antineoplásicos , Interleucina-15 , Animales , Macaca fascicularis/metabolismo , Interleucina-15/metabolismo , Farmacología en Red , Linfocitos/metabolismo , Factores Inmunológicos , Receptores de Interleucina-15
8.
Eur J Nucl Med Mol Imaging ; 50(2): 287-301, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36271158

RESUMEN

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.


Asunto(s)
Neoplasias , Tomografía de Emisión de Positrones , Adulto , Humanos , Ratones , Ratas , Animales , Tomografía de Emisión de Positrones/métodos , Indicadores y Reactivos/uso terapéutico , Distribución Tisular , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Inmunoterapia/métodos , Circonio/química , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral
9.
J Immunother Cancer ; 10(4)2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35379739

RESUMEN

Recent advances in understanding the roles of immune checkpoints in allowing tumors to circumvent the immune system have led to successful therapeutic strategies that have fundamentally changed oncology practice. Thus far, immunotherapies against only two checkpoint targets have been approved, CTLA-4 and PD-L1/PD-1. Antibody blockade of these targets enhances the function of antitumor T cells at least in part by relieving inhibition of the T cell costimulatory receptor CD28. These successes have stimulated considerable interest in identifying other pathways that may bte targeted alone or together with existing immunotherapies. One such immune checkpoint axis is comprised of members of the PVR/nectin family that includes the inhibitory receptor T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT). Interestingly, TIGIT acts to regulate the activity of a second costimulatory receptor CD226 that works in parallel to CD28. There are currently over two dozen TIGIT-directed blocking antibodies in various phases of clinical development, testament to the promise of modulating this pathway to enhance antitumor immune responses. In this review, we discuss the role of TIGIT as a checkpoint inhibitor, its interplay with the activating counter-receptor CD226, and its status as the next advance in cancer immunotherapy.


Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Neoplasias , Antígenos de Diferenciación de Linfocitos T , Humanos , Inmunoterapia , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Receptores Inmunológicos , Receptores Virales , Linfocitos T
10.
Immunity ; 55(3): 512-526.e9, 2022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35263569

RESUMEN

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.


Asunto(s)
Linfocitos T CD8-positivos , Neoplasias , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos CD28/metabolismo , Humanos , Neoplasias/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Inmunológicos/metabolismo
11.
Nature ; 579(7798): 274-278, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32103181

RESUMEN

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.


Asunto(s)
Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/patología , Variantes Farmacogenómicas , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Células Clonales , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Linfocitos T/metabolismo , Transcriptoma
12.
Eur J Immunol ; 50(6): 891-902, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32043568

RESUMEN

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.


Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas/inmunología , Modelos Inmunológicos , Animales , Antígenos CD/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados
13.
Mol Cell Proteomics ; 18(11): 2310-2323, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31308249

RESUMEN

Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implement a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins. Using this method to study key immune receptors, we identify and further validate the interleukin receptor IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Further, KIR2DL5, a natural killer cell protein that had remained orphan, is uncovered as a functional binding partner for the poliovirus receptor (PVR). This interaction is characterized using orthogonal assays, which demonstrate that PVR specifically engages KIR2DL5 on natural killer cells leading to inhibition of cytotoxicity. Altogether, these results reveal unappreciated links between protein families that may importantly influence receptor-driven functions during disease. Applicable to any target of interest, this technology represents a versatile and powerful approach for elucidation of receptor-ligand interactomes, which is essential to understand basic aspects of the biology of the plasma membrane proteins and ultimately inform the development of novel therapeutic strategies.


Asunto(s)
Antígenos B7/metabolismo , Matriz Extracelular/metabolismo , Células Asesinas Naturales/metabolismo , Receptores de Interleucina/metabolismo , Receptores KIR2DL5/metabolismo , Receptores Virales/metabolismo , Comunicación Celular , Células HEK293 , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ligandos , Unión Proteica , Mapas de Interacción de Proteínas
14.
Cell Rep ; 27(1): 269-281.e4, 2019 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-30943407

RESUMEN

Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, in vivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs in vitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity.


Asunto(s)
Carcinogénesis/metabolismo , Histonas/metabolismo , Células Mieloides/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Arginasa/genética , Arginasa/metabolismo , Línea Celular Tumoral , Células Cultivadas , Elementos de Facilitación Genéticos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Regiones Promotoras Genéticas , Dominios Proteicos , Factores de Transcripción STAT/metabolismo , Factores de Transcripción p300-CBP/química
15.
Proc Natl Acad Sci U S A ; 115(50): E11731-E11740, 2018 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-30504141

RESUMEN

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226, with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells, with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore, inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors.


Asunto(s)
Antígenos de Diferenciación de Linfocitos T/metabolismo , Proteína Forkhead Box O1/antagonistas & inhibidores , Proteína Forkhead Box O1/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Línea Celular Tumoral , Citotoxicidad Inmunológica , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Ratones Noqueados , Nectinas/metabolismo , Fosforilación , Receptores Virales/metabolismo , Transducción de Señal/inmunología
16.
PLoS One ; 12(7): e0180154, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28683073

RESUMEN

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Potenciales de la Membrana/fisiología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Técnicas de Placa-Clamp/métodos , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Animales , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Expresión Génica , Ensayos Analíticos de Alto Rendimiento/instrumentación , Canal de Potasio Kv1.3/deficiencia , Canal de Potasio Kv1.3/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.3/genética , Canal de Sodio Activado por Voltaje NAV1.3/metabolismo , Canal de Sodio Activado por Voltaje NAV1.4/genética , Canal de Sodio Activado por Voltaje NAV1.4/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Técnicas de Placa-Clamp/instrumentación , Cultivo Primario de Células , Ratas , Canales de Sodio/genética , Canales de Sodio/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Transgenes
17.
Nat Commun ; 8: 14644, 2017 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-28248292

RESUMEN

Voltage-gated Kv1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of Kv1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether Kv1.3 is required for their induction and function is unclear. Here we show, using Kv1.3-deficient rats, that Kv1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in Kv1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of Kv1.3 under these specific settings. However, experiments with Kv1.3 KO rats and Kv1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both Kv1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on Kv1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.


Asunto(s)
Epítopos/inmunología , Memoria Inmunológica , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canal de Potasio Kv1.3/metabolismo , Linfocitos T/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Activación de Linfocitos/inmunología , Fenotipo , Bloqueadores de los Canales de Potasio/farmacología , ARN Interferente Pequeño/metabolismo , Ratas , Linfocitos T/efectos de los fármacos
18.
Trends Immunol ; 38(1): 20-28, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27793572

RESUMEN

Immunotherapies that harness the activity of the immune system against tumors are proving to be an effective therapeutic approach in multiple malignancies. Indeed, through accumulation of genetic mutations, many tumors express antigens that can potentially elicit specific tumor immunity. However, tumors can also suppress these responses by activating negative regulatory pathways and checkpoints such as PD-1/PD-L1 and CTLA-4. Blocking these checkpoints on T cells has provided dramatic clinical benefit, but only a subset of patients exhibit clear and durable responses, suggesting that other mechanisms must be limiting the immune response. We discuss here the role of TIGIT, an inhibitory receptor expressed by lymphocytes, in limiting antitumor responses and we review its mechanisms of action during the cancer immunity cycle.


Asunto(s)
Inmunidad Celular , Inmunoterapia/métodos , Neoplasias/inmunología , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Receptores Coestimuladores e Inhibidores de Linfocitos T/inmunología , Receptores Coestimuladores e Inhibidores de Linfocitos T/metabolismo , Humanos , Neoplasias/terapia , Receptores Inmunológicos/inmunología
19.
J Biol Chem ; 291(25): 13014-27, 2016 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-27056325

RESUMEN

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.


Asunto(s)
Proteína de Unión a CREB/antagonistas & inhibidores , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Acetilación/efectos de los fármacos , Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/metabolismo , Factores de Transcripción Forkhead/metabolismo , Histonas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Transcriptoma/efectos de los fármacos
20.
J Biol Chem ; 290(50): 29732-41, 2015 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-26491012

RESUMEN

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.


Asunto(s)
Región Variable de Inmunoglobulina/inmunología , Farmacocinética , Animales , Ensayo de Inmunoadsorción Enzimática , Región Variable de Inmunoglobulina/química , Macaca fascicularis , Medición de Riesgo
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