RESUMEN
BACKGROUND: B7-H3 protein is an important regulator of the adaptive immune response in human tumorigenesis. 4-1BB is a co-stimulatory receptor expressed on activated CD8+ T cells, and regulates T cell immunity. Here, we investigated the role of B7-H3 in the growth and invasion of nasopharyngeal carcinoma (NPC) and the effect of its interaction with 4-1BB on tumor immunity. METHODS: Short hairpin (sh) RNA was designed to knock down B7-H3 expression in NPC cells. NPC cells with stable knockdown of B7-H3 were established and injected into nude mice. The effects of B7-H3 on cell proliferation, apoptosis, and epithelial-to-mesenchymal transition (EMT) were detected by the CCK8 assay, flow cytometry, TUNEL assay, and western blot analysis. The migration and invasion abilities were determined using the Transwell assay and scratch assay. Co-immunoprecipitation (Co-IP) assays were performed to study the interaction between B7-H3 and 4-1BB. Anti-4-1BB antibody was used in a co-culture system and xenograft mice to study the effect of 4-1BB on NPC development. RESULTS: NPC cells transfected with sh-B7-H3 showed a higher rate of apoptosis, slower growth rate, impaired migration, and less EMT in vitro. Xenograft mice with stable knockout of B7-H3 had lower tumor burdens, and the stripped tumors had lower rates of cell proliferation, higher rates of apoptosis, and less EMT in vivo. Additionally, decreased B7-H3 expression was positively correlated with interferon-γ, tumor necrosis factor-α, and 4-1BB+CD8+ tumor-infiltrating lymphocytes. Co-IP studies showed that B7-H3 interacts with 4-1BB. Also, the inhibitory effects of sh-B7-H3 on NPC tumor growth, invasion, and tumor immunity could be alleviated by the anti-4-1BB antibody both in vivo and in vitro. CONCLUSION: Our findings suggest that B7-H3 may accelerate tumor growth, tumor cell invasion, and EMT, and interact with 4-1BB to produce CD8+ T cell exhaustion that inhibits tumor immunity. B7-H3 might serve as a novel target for treating NPC.
Asunto(s)
Antígenos B7 , Linfocitos T CD8-positivos , Proliferación Celular , Transición Epitelial-Mesenquimal , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Antígenos B7/genética , Antígenos B7/metabolismo , Antígenos B7/inmunología , Animales , Humanos , Carcinoma Nasofaríngeo/inmunología , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Linfocitos T CD8-positivos/inmunología , Ratones , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/inmunología , Ratones Desnudos , Apoptosis , Progresión de la Enfermedad , Movimiento Celular , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Agotamiento de Células TRESUMEN
Cervical cancer (CC) poses a significant health burden, particularly in low- and middle-income countries. NK cells play a crucial role against CC; however, they can become exhausted and lose their cytotoxic capacity. This work explores the expression of costimulatory receptors (ICOS, 4-1BB, OX-40) in exhausted NK cells from CC patients. Peripheral blood and tumor biopsies were collected, and flow cytometry was used to evaluate the expression of costimulatory receptors in exhausted NK cells. There is an increase of peripheral exhausted NK cells (PD-1+TIGIT+) in CC patients; this subpopulation has a selectively increased expression of the costimulatory receptors ICOS and 4-1BB. An exhausted population is also highly increased in tumor-infiltrating NK cells, and it shows a dramatically increased expression of the costimulatory receptors ICOS (>15×) and 4-1BB (>10×) compared to peripheral NK cells. The exhausted cells, both in the periphery and in the tumor infiltrating lymphocytes (TILs), are also more likely than non-exhausted NK cell populations (PD-1-TIGIT-) to express these costimulatory receptors; increases ranging from 2.0× ICOS, 2.4× 4-1BB, and 2.6× OX-40 in CD56dim PBMCs to 1.5× ICOS, 5× 4-1BB, and 10× OX-40 in TILs were found. Our study demonstrates for the first time the increased expression of the costimulatory receptors ICOS, 4-1BB, and OX-40 in peripheral CD56dim, CD56bright, and tumor-infiltrating NK cells in CC. Targeting these receptors for stimulation could reverse exhaustion and be a promising immunotherapy strategy.
Asunto(s)
Proteína Coestimuladora de Linfocitos T Inducibles , Células Asesinas Naturales , Linfocitos Infiltrantes de Tumor , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Neoplasias del Cuello Uterino , Humanos , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Persona de Mediana Edad , Adulto , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Ligando OX40/metabolismoRESUMEN
Adoptive cell transfer (ACT) is a therapeutic strategy to augment antitumor immunity. Here, we report that ex vivo treatment of mouse CD8+ T cells with dimethyloxalylglycine (DMOG), a stabilizer of hypoxia-inducible factors (HIFs), induced HIF binding to the genes encoding the costimulatory receptors CD81, GITR, OX40, and 4-1BB, leading to increased expression. DMOG treatment increased T cell killing of melanoma cells, which was further augmented by agonist antibodies targeting each costimulatory receptor. In tumor-bearing mice, ACT using T cells treated ex vivo with DMOG and agonist antibodies resulted in decreased tumor growth compared to ACT using control T cells and increased intratumoral markers of CD8+ T cells (CD7, CD8A, and CD8B1), natural killer cells (NCR1 and KLRK1), and cytolytic activity (perforin-1 and tumor necrosis factor-α). Costimulatory receptor gene expression was also induced when CD8+ T cells were treated with three highly selective HIF stabilizers that are currently in clinical use.
Asunto(s)
Linfocitos T CD8-positivos , Inmunoterapia Adoptiva , Animales , Ratones , Inmunoterapia Adoptiva/métodos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Aminoácidos Dicarboxílicos/farmacología , Línea Celular Tumoral , Receptores OX40/metabolismo , Proteína Relacionada con TNFR Inducida por Glucocorticoide/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Ratones Endogámicos C57BL , Melanoma Experimental/terapia , Melanoma Experimental/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Citotoxicidad Inmunológica/efectos de los fármacosRESUMEN
The Hodgkin and Reed - Sternberg (HRS) cells in classical Hodgkin Lymphoma (cHL) actively modify the immune tumor microenvironment (TME) attracting immunosuppressive cells and expressing inhibitory molecules. A high frequency of myeloid cells in the TME is correlated with an unfavorable prognosis, but more specific and rare cell populations lack precise markers. Myeloid-derived suppressor cells (MDSCs) have been identified in the peripheral blood of cHL patients, where they appear to be correlated with disease aggressiveness. TNFRSF9 (CD137) is a T cell co-stimulator expressed by monocytic and dendritic cells. Its expression has also been described in HRS cells, where it is thought to play a role in reducing antitumor responses. Here, we perform qualitative and quantitative analyses of lymphocytic and MDSC subtypes and determine the CD137 cell distribution in cHL primary tumors using multiplex immunofluorescence and automated multispectral imaging. The results were correlated with patients' clinical features. Cells were stained with specific panels of immune checkpoint markers (PD-1, PD-L1, CD137), tumor-infiltrating T lymphocytes (CD3, PD-1), and monocytic cells/MDSCs (CD68, CD14, CD33, Arg-1, CD11b). This approach allowed us to identify distinct phenotypes and to analyze spatial interactions between immune subpopulations and tumor cells. The results confirm CD137 expression by T, monocytic and HRS cells. In addition, the expression of CD137, T exhausted cells, and monocytic MDSCs (m-MDSCs) in the vicinity of malignant HRS cells were associated with a worse prognosis. Our findings reveal new elements of the TME that mediate immune escape, and confirm CD137 as a candidate target for immunotherapy in cHL.
CD137-expressing immune cells and HRS cells are more abundant and in closer proximity in refractory patients than in responders.Monocytic myeloid-derived suppressor cells (m-MDSCs) are associated with unfavorable outcomes and relapse in cHL, unlike granulocytic MDSCs (g-MDSCs), which are located far from HRS cells in non-responders.The cHL tumor microenvironment promotes immune escape in refractory patients by holistically driving polarization and/or recruitment of several cell types with increased expression of CD137 and PD-L1 checkpoints.
Asunto(s)
Enfermedad de Hodgkin , Células Supresoras de Origen Mieloide , Células de Reed-Sternberg , Microambiente Tumoral , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Humanos , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Microambiente Tumoral/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/patología , Femenino , Masculino , Adulto , Persona de Mediana Edad , Células de Reed-Sternberg/patología , Células de Reed-Sternberg/metabolismo , Anciano , Análisis Espacial , Adulto Joven , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Adolescente , Pronóstico , Biomarcadores de Tumor/metabolismoRESUMEN
Biomarkers for cancer immunotherapy are an unmet medical need. The group of Daniela Thommen at the NKI recently reported on novel methodologies based on short-term cultures of patient-derived tumor fragments whose cytokine concentrations in the supernatants and activation markers on infiltrating T cells were associated with clinical response to PD-1 blockade. We set up a similar culture technology with tumor-derived fragments using mouse tumors transplanted into syngeneic immunocompetent mice to test an agonist anti-CD137 mAb and its combinations with anti-PD-1 and/or anti-TGF-ß. Increases in IFNγ concentrations in the tissue culture supernatants were detected upon in-culture activation with the anti-CD137 and anti-PD-1 mAb combinations or concanavalin A as a positive control. No other cytokine from a wide array was informative of stimulation with these mAbs. Interestingly, increases in Ki67 and other activation markers were substantiated in lymphocytes from cell suspensions gathered at the end of 72 h cultures. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137 + anti-PD-1 treatment to perform the fragment culture evaluation, no association was found between IFNγ production from the fragments and the in vivo therapeutic outcome in the non-resected contralateral tumors. The experimental system permitted freezing and thawing of the fragments with similar functional outcomes. Using a series of patient-derived tumor fragments from excised solid malignancies, we showed IFNγ production in a fraction of the studied cases, that was conserved in frozen/thawed fragments. The small tumor fragment culture technique seems suitable to preclinically explore immunotherapy combinations.
Asunto(s)
Inmunoterapia , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Animales , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Ratones , Humanos , Inmunoterapia/métodos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Femenino , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Interferón gamma/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Células Tumorales Cultivadas , Ratones Endogámicos C57BL , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéuticoRESUMEN
Co-activation signal that induces/sustains pleiotropic effector functions of antigen-specific γδ T cells remains unknown. Here, Mycobacteria tuberculosis (Mtb) tuberculin administration during tuberculosis (TB) skin test resulted in rapid expression of co-activation signal molecules CD137 and CD107a by fast-acting Vγ2Vδ2 T cells in TB-resistant subjects (Resisters), but not patients with active TB. And, anti-CD137 agonistic antibody treatment experiments showed that CD137 signaling enabled Vγ2Vδ2 T cells to produce more effector cytokines and inhibit intracellular Mtb growth in macrophages (Mɸ). Consistently, Mtb antigen (Ag) HMBPP stimulation induced sustainable high-level CD137 expression in fresh and activated Vγ2Vδ2 T cells from uninfected subjects, but not TB patients. CD137+Vγ2Vδ2 T-cell subtype predominantly displayed central memory phenotype and mounted better proliferative responses than CD137-Vγ2Vδ2 T-cells. In response to HMBPP, CD137+Vγ2Vδ2 T-cell subtype rapidly differentiated into greater numbers of pleiotropic effector cells producing anti-Mtb cytokines compared to CD137-Vγ2Vδ2 T subtype, with the non-canonical NF-κB pathway involved. CD137 expression in Vγ2Vδ2 T cells appeared to signal anti-Mtb effector functions leading to intracellular Mtb growth inhibition in Mɸ, and active TB disrupted such CD137-driven anti-Mtb effector functions. CD137+Vγ2Vδ2 T-cells subtype exhibited an epigenetic-driven high-level expression of GM-CSF and de novo production of GM-CSF critical for Vγ2Vδ2 T-cell controlling of Mtb growth in MÏ. Concurrently, exosomes produced by CD137+Vγ2Vδ2 T cells potently inhibited intracellular mycobacterial growth. Furthermore, adoptive transfer of human CD137+Vγ2Vδ2 T cells to Mtb-infected SCID mice conferred protective immunity against Mtb infection. Thus, our data suggest that CD137 expression/signaling drives pleiotropic γδ T-cell effector functions that inhibit intracellular Mtb growth.
Asunto(s)
Mycobacterium tuberculosis , Transducción de Señal , Tuberculosis , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Humanos , Mycobacterium tuberculosis/inmunología , Transducción de Señal/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiología , Ratones , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Femenino , Animales , Macrófagos/inmunología , Masculino , Adulto , Antígenos Bacterianos/inmunología , Citocinas/metabolismo , Citocinas/inmunología , Activación de Linfocitos/inmunología , Ratones SCIDRESUMEN
CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling. Mechanistic studies illustrate that 4-1BB sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity, cell aggregation via ICAM-1 overexpression, and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway. Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1BB by deleting the TRAF-binding motifs of 4-1BB rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.
Asunto(s)
Muerte Celular , Receptores Quiméricos de Antígenos , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Humanos , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Necroptosis , Apoptosis , Transducción de Señal , Ratones , FN-kappa B/metabolismo , Línea Celular Tumoral , Ubiquitina/metabolismoRESUMEN
Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.
Asunto(s)
Linfocitos T CD8-positivos , Virus de la Hepatitis B , Hepatitis B Crónica , Humanos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Hepatitis B Crónica/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Animales , Receptores OX40/metabolismo , Ratones , Receptor de Muerte Celular Programada 1/metabolismo , Antígenos CD/metabolismoRESUMEN
Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.
Asunto(s)
Complejo CD3 , Neoplasias Colorrectales , Molécula de Adhesión Celular Epitelial , Animales , Humanos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/terapia , Molécula de Adhesión Celular Epitelial/metabolismo , Complejo CD3/inmunología , Ratones , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Células HCT116 , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Femenino , Línea Celular Tumoral , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Ratones Endogámicos BALB C , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Inmunoterapia/métodosRESUMEN
Multiple myeloma (MM) progression is closely dependent on cells in the bone marrow (BM) microenvironment, including fibroblasts (FBs) and immune cells. In their BM niche, MM cells adhere to FBs sustaining immune evasion, drug resistance and the undetectable endurance of tumor cells known as minimal residual disease (MRD). Here, we describe the novel bi-specific designed ankyrin repeat protein (DARPin) α-FAPx4-1BB (MP0310) with FAP-dependent 4-1BB agonistic activity. The α-FAPx4-1BB DARPin simultaneously binds to FAP and 4-1BB overexpressed by activated FBs and immune cells, respectively. Although flow cytometry analysis showed that T and NK cells from MM patients were not activated and did not express 4-1BB, stimulation with daratumumab or elotuzumab, monoclonal antibodies (mAbs) currently used for the treatment of MM, significantly upregulated 4-1BB both in vitro and in MM patients following mAb-based therapy. The mAb-induced 4-1BB overexpression allowed the engagement of α-FAPx4-1BB that acted as a bridge between FAP+FBs and 4-1BB+NK cells. Therefore, α-FAPx4-1BB enhanced both the adhesion of daratumumab-treated NK cells on FBs as well as their activation by improving release of CD107a and perforin, hence MM cell killing via antibody-mediated cell cytotoxicity (ADCC). Interestingly, α-FAPx4-1BB significantly potentiated daratumumab-mediated ADCC in the presence of FBs, suggesting that it may overcome the BM FBs' immunosuppressive effect. Overall, we speculate that treatment with α-FAPx4-1BB may represent a valuable strategy to improve mAb-induced NK cell activity fostering MRD negativity in MM patients through the eradication of latent MRD cells.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales , Células Asesinas Naturales , Mieloma Múltiple , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Humanos , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/agonistas , EndopeptidasasRESUMEN
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
Asunto(s)
Complejo CD3 , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares , Proteínas de la Membrana , Carcinoma Pulmonar de Células Pequeñas , Linfocitos T , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Carcinoma Pulmonar de Células Pequeñas/inmunología , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/terapia , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Complejo CD3/inmunología , Animales , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Línea Celular Tumoral , Activación de Linfocitos/inmunología , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The cellular source of positive signals that reinvigorate T cells within the tumor microenvironment (TME) for the therapeutic efficacy of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade has not been clearly defined. We now show that Batf3-lineage dendritic cells (DCs) are essential in this process. Flow cytometric analysis, gene-targeted mice, and blocking antibody studies revealed that 4-1BBL is a major positive co-stimulatory signal provided by these DCs within the TME that translates to CD8+ T cell functional reinvigoration and tumor regression. Immunofluorescence and spatial transcriptomics on human tumor samples revealed clustering of Batf3+ DCs and CD8+ T cells, which correlates with anti-PD-1 efficacy. In addition, proximity to Batf3+ DCs within the TME is associated with CD8+ T cell transcriptional states linked to anti-PD-1 response. Our results demonstrate that Batf3+ DCs within the TME are critical for PD-1/PD-L1 blockade efficacy and indicate a major role for the 4-1BB/4-1BB ligand (4-1BBL) axis during this process.
Asunto(s)
Antígeno B7-H1 , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Linfocitos T CD8-positivos , Células Dendríticas , Receptor de Muerte Celular Programada 1 , Proteínas Represoras , Microambiente Tumoral , Animales , Humanos , Ratones , Ligando 4-1BB/metabolismo , Ligando 4-1BB/genética , Antígeno B7-H1/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Células Dendríticas/metabolismo , Células Dendríticas/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Transducción de Señal , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.
Asunto(s)
Epítopos , Humanos , Epítopos/inmunología , Epítopos/química , Animales , Receptores del Factor de Necrosis Tumoral/agonistas , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidores , Receptores OX40/agonistas , Receptores OX40/inmunología , Receptores OX40/metabolismo , Receptores OX40/antagonistas & inhibidores , Anticuerpos/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/farmacología , RatonesRESUMEN
BACKGROUND AIMS: Vγ9Vδ2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy as the result of their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vδ2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain. METHODS: Specifically, we compared the anti-tumor activity of Vδ2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium. RESULTS: We found that HPL supported greater expansion of CAR-Vδ2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vδ2 T cells. HPL-expanded CAR-Vδ2 T cells showed enhanced in vivo anti-tumor activity with longer T-cell persistence compared with FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vδ2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vδ2 T cells and increased telomerase activity. CONCLUSIONS: This study supports enhancement of therapeutic potency of CAR-Vδ2 T cells through a manufacturing improvement.
Asunto(s)
Apoptosis , Plaquetas , Senescencia Celular , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Animales , Inmunoterapia Adoptiva/métodos , Ratones , Plaquetas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Línea Celular Tumoral , Antígenos CD28/metabolismo , Antígenos CD28/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
We demonstrate an optimized oligonucleotide library-based approach for the identification of virus-reactive T-cell receptors using Epstein-Barr virus as an example. HEK293T served as antigen-presenting cells and were co-cultured with human T cells that were transduced with T-cell receptors in question. T-cell activation was detected by CD137 expression.
Asunto(s)
Biblioteca de Genes , Herpesvirus Humano 4 , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T , Humanos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/genética , Células HEK293 , Herpesvirus Humano 4/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Técnicas de CocultivoRESUMEN
Altered mitochondrial dynamics affect pulmonary artery endothelial cells (PAECs) proliferation, contributing to the development of pulmonary hypertension. CD137 signaling promotes mitochondrial fission. We hypothesize CD137 signaling is involved in the excessive proliferation of PAECs. The levels of CD137 protein were increased in the lung tissue of hypoxic mice and hypoxic-stimulated PAECs. Activation of CD137 signal in hypoxic-PAECs upregulated the levels of hypoxia-inducible factor-2α (HIF-2α), glucose transporters type 4, the lactate transporter monocarboxylate transporter 4, key glycolysis rate-limiting enzymes and promoted mitochondrial division; moreover, increased glucose uptake, lactic acid and ATP production and proliferative cells were observed in these PAECs. Whereas, knockdown HIF-2α reversed CD137 signal-mediated effects in PAECs mentioned above. Compared with wild-type mice, the proliferation of PAECs and the percentage of vascular lateral wall thickness decreased in CD137 knockout mice. Together, CD137 signal participated in pulmonary vascular remodeling through the regulation of mitochondrial dynamics dependent on HIF-2α in PAECs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proliferación Celular , Células Endoteliales , Ratones Endogámicos C57BL , Ratones Noqueados , Dinámicas Mitocondriales , Arteria Pulmonar , Transducción de Señal , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Animales , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Cultivadas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Remodelación Vascular , Masculino , Modelos Animales de Enfermedad , Hipoxia de la Célula , Mitocondrias/metabolismo , Mitocondrias/patologíaRESUMEN
ABSTRACT: Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart after MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared with wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings because CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. In addition, the administration of CD137L recombinant protein further promoted alpha-smooth muscle actin expression and collagen I synthesis, suggesting a profibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the profibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function.
Asunto(s)
Modelos Animales de Enfermedad , Fibrosis , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Remodelación Ventricular , Animales , Infarto del Miocardio/patología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/genética , Infarto del Miocardio/enzimología , Infarto del Miocardio/fisiopatología , Remodelación Ventricular/efectos de los fármacos , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Masculino , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Miofibroblastos/metabolismo , Miofibroblastos/patología , Miofibroblastos/enzimología , Sistema de Señalización de MAP Quinasas , Miocardio/patología , Miocardio/metabolismo , Miocardio/enzimología , Ligando 4-1BB/metabolismo , Ligando 4-1BB/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Actinas/metabolismo , Células Cultivadas , Transducción de Señal , Movimiento Celular , Ratones , Función Ventricular Izquierda , Diferenciación Celular , Miocitos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/efectos de los fármacosRESUMEN
OBJECTIVE: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancy in the United States, and biomarkers of patient outcomes are limited. Data using immunohistochemical (IHC) analysis are mixed regarding whether and which tumor infiltrating lymphocytes (TILs) impact survival, and IHC does not adequately quantify rare cell populations, including CD137+ (4-1BB) tumor-reactive TILs. Our study investigates if a higher percentage of CD3+ CD137+ TILs is associated with improved overall survival (OS) in OC. METHODS: Flow cytometry was performed on viably banked OC digests. Chart review and statistical analysis were performed. Forty-seven patients were included, 40 of whom were diagnosed with high-grade serous ovarian carcinoma (HGSOC), papillary serous carcinoma, or undifferentiated histology. RESULTS: A high percentage of CD3+ CD137+ TILs correlated with improved OS (n = 40, r = 0.48, P = 0.0016). Subjects were divided into CD3+ CD137+ TIL high and low groups by the median. Subjects with high CD3+CD137+ TIL frequencies (>9.6%) had longer OS (Wilcoxon rank-sum test; P = 0.0032) and improved OS (logrank test; P = 0.007). Differences in CD3+ or CD3+ CD8+ TILs did not impact survival. CD3+ CD137+ TILs were predictive of OS regardless of germline mutation or debulking status. Analysis of subgroups including late stage HGSOC and late stage HGSOC with primary optimal cytoreduction indicated CD3+ CD137+ TILs correlated with improved OS after adjusting for age and PARP inhibitor use (P = 0.034 and P = 0.016, respectively). CONCLUSIONS: Prevalence of CD3+ CD137+ TILs in digested OC specimens is associated with improved OS, while general TIL markers are not. CD137 has the potential to be a novel biomarker for survival in OC.
Asunto(s)
Linfocitos Infiltrantes de Tumor , Neoplasias Ováricas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Humanos , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/análisis , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/mortalidad , Persona de Mediana Edad , Anciano , Complejo CD3/análisis , Adulto , Cistadenocarcinoma Seroso/patología , Cistadenocarcinoma Seroso/inmunología , Cistadenocarcinoma Seroso/mortalidad , Anciano de 80 o más AñosRESUMEN
Since its initial description 35 years ago as an inducible molecule expressed in cytotoxic and helper T cells, 4-1BB has emerged as a crucial receptor in T-cell-mediated immune functions. Numerous studies have demonstrated the involvement of 4-1BB in infection and tumor immunity. However, the clinical development of 4-1BB agonist antibodies has been impeded by the occurrence of strong adverse events, notably hepatotoxicity, even though these antibodies have exhibited tremendous promise in in vivo tumor models. Efforts are currently underway to develop a new generation of agonist antibodies and recombinant proteins with modified effector functions that can harness the potent T-cell modulation properties of 4-1BB while mitigating adverse effects. In this review, we briefly examine the role of 4-1BB in T-cell biology, explore its clinical applications, and discuss future prospects in the field of 4-1BB agonist immunotherapy.
Asunto(s)
Neoplasias , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Humanos , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Neoplasias/tratamiento farmacológico , Inmunoterapia , AnticuerposRESUMEN
Objective: The role of T cells in the pathogenesis of systemic lupus erythematosus (SLE) has recently gained attention. Costimulatory molecules are membrane proteins strictly associated with T-cell receptor (TCR), acting by activating or inhibiting T cells and antigen-presenting cells (APC) through direct and reverse signaling, thus becoming responsible for the development of effector T cells or regulatory T cells. The primary objective of the present case-control study was to evaluate the cell membrane expression of CD137 on T cells and the serum concentration of CD137 (sCD137) in a SLE cohort. Materials: We enrolled SLE patients and sex/age-matched healthy subjects (HS). Disease activity was assessed by SLEDAI-2K. By application of flow cytometry, we evaluated the expression of CD137 on CD4+ and CD8+ lymphocytes. ELISA test was performed to evaluate serum levels of sCD137. Results: Twenty-one SLE patients (M/F 1/20; median age 48 years (IQR 17); median disease duration 144 months (IQR 204)) were evaluated. SLE patients showed %CD3+CD137+ cells significantly higher compared to HS (median 5.32 (IQR 6.11) versus 3.3 (IQR 1.8), p = 0.001). In SLE patients, %CD4+CD137+ cells positively correlated with SLEDAI-2K (p = 0.0082, r = 0.58, CI (0.15-0.82); indeed, %CD4+CD137+ cells were significantly lower in SLE patients with a remission status compared to those not reaching this condition (median 1.07 (IQR 0.91) versus 1.58 (IQR 2.42), p = 0.013). Accordingly, sCD137 levels were significantly lower in remission status (31.30 pg/mL (IQR 102.2 versus median 122.8 pg/mL (IQR 536); p = 0.03) and correlated with %CD4+CD137+ cells (p = 0.012, r = 0.60, CI (0.15-0.84)). Conclusion: Our results suggest a possible involvement of CD137-CD137L axis in SLE pathogenesis, as demonstrated by higher expression of CD137 on CD4+ cells in SLE compared with HS. Furthermore, the positive correlation between SLEDAI-2K and membrane CD137 expression on CD4+ cells, as well as soluble CD137, indicates a possible use as biomarkers for disease activity.