RESUMEN
Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.
Asunto(s)
Linfocitos T CD8-positivos , Receptor de Muerte Celular Programada 1 , Receptores de Interleucina-2 , Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Anticuerpos Bloqueadores/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Infecciones/tratamiento farmacológico , Infecciones/inmunología , Interleucina-2/inmunología , Interleucina-2/farmacología , Interleucina-2/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2/agonistas , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores de Interleucina-2/agonistasRESUMEN
Although treatment of multiple myeloma (MM) with daratumumab significantly extends the patient's lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/r MM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high-affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38, including those with a lower sensitivity to daratumumab. In a killing assay where multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity toward MM cells compared with daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in 2 therapeutic mouse models, unlike daratumumab. Finally, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/r MM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently being developed in a phase 1 clinical study.
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Anticuerpos Biespecíficos , Mieloma Múltiple , Animales , Ratones , ADP-Ribosil Ciclasa 1/metabolismo , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Linfocitos T/patologíaRESUMEN
T cells are activated by antigen (Ag)-bearing dendritic cells (DCs) in lymph nodes in three phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely correlated with Ag dose. The second phase, characterized by stable DC-T cell contacts, is believed to be necessary for full-fledged T cell activation. Here we have shown that this is not the case. CD8⺠T cells interacting with DCs presenting low-dose, short-lived Ag did not transition to phase 2, whereas higher Ag dose yielded phase 2 transition. Both antigenic constellations promoted T cell proliferation and effector differentiation but yielded different transcriptome signatures at 12 hr and 24 hr. T cells that experienced phase 2 developed long-lived memory, whereas conditions without stable contacts yielded immunological amnesia. Thus, T cells make fate decisions within hours after Ag exposure, resulting in long-term memory or abortive effector responses, correlating with T cell-DCs interaction kinetics.
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Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Memoria Inmunológica/inmunología , Traslado Adoptivo , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/metabolismo , Comunicación Celular , Diferenciación Celular , Células Dendríticas/metabolismo , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transcriptoma/inmunologíaRESUMEN
The skin has a dual function as a barrier and a sensory interface between the body and the environment. To protect against invading pathogens, the skin harbours specialized immune cells, including dermal dendritic cells (DDCs) and interleukin (IL)-17-producing γδ T (γδT17) cells, the aberrant activation of which by IL-23 can provoke psoriasis-like inflammation. The skin is also innervated by a meshwork of peripheral nerves consisting of relatively sparse autonomic and abundant sensory fibres. Interactions between the autonomic nervous system and immune cells in lymphoid organs are known to contribute to systemic immunity, but how peripheral nerves regulate cutaneous immune responses remains unclear. We exposed the skin of mice to imiquimod, which induces IL-23-dependent psoriasis-like inflammation. Here we show that a subset of sensory neurons expressing the ion channels TRPV1 and Nav1.8 is essential to drive this inflammatory response. Imaging of intact skin revealed that a large fraction of DDCs, the principal source of IL-23, is in close contact with these nociceptors. Upon selective pharmacological or genetic ablation of nociceptors, DDCs failed to produce IL-23 in imiquimod-exposed skin. Consequently, the local production of IL-23-dependent inflammatory cytokines by dermal γδT17 cells and the subsequent recruitment of inflammatory cells to the skin were markedly reduced. Intradermal injection of IL-23 bypassed the requirement for nociceptor communication with DDCs and restored the inflammatory response. These findings indicate that TRPV1(+)Nav1.8(+) nociceptors, by interacting with DDCs, regulate the IL-23/IL-17 pathway and control cutaneous immune responses.
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Interleucina-23/inmunología , Nociceptores/metabolismo , Psoriasis/inmunología , Psoriasis/patología , Células Receptoras Sensoriales/metabolismo , Piel/inervación , Piel/patología , Aminoquinolinas , Animales , Modelos Animales de Enfermedad , Femenino , Imiquimod , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Interleucina-23/biosíntesis , Interleucinas/biosíntesis , Interleucinas/inmunología , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Nociceptores/efectos de los fármacos , Psoriasis/inducido químicamente , Células Receptoras Sensoriales/efectos de los fármacos , Piel/citología , Piel/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Canales Catiónicos TRPV/metabolismo , Interleucina-22RESUMEN
Vascular inflammation is a major contributor to the severity of acute kidney injury. In the context of vasospasm-independent reperfusion injury we studied the potential anti-inflammatory role of the Gα-related RGS protein, RGS4. Transgenic RGS4 mice were resistant to 25 min injury, although post-ischemic renal arteriolar diameter was equal to the wild type early after injury. A 10 min unilateral injury was performed to study reperfusion without vasospasm. Eighteen hours after injury, blood flow was decreased in the inner cortex of wild-type mice with preservation of tubular architecture. Angiotensin II levels in the kidneys of wild-type and transgenic mice were elevated in a sub-vasoconstrictive range 12 and 18 h after injury. Angiotensin II stimulated pre-glomerular vascular smooth muscle cells (VSMCs) to secrete the macrophage chemoattractant RANTES, a process decreased by angiotensin II R2 (AT2) inhibition. However, RANTES increased when RGS4 expression was suppressed implicating Gα protein activation in an AT2-RGS4-dependent pathway. RGS4 function, specific to VSMC, was tested in a conditional VSMC-specific RGS4 knockout showing high macrophage density by T2 MRI compared with transgenic and non-transgenic mice after the 10 min injury. Arteriolar diameter of this knockout was unchanged at successive time points after injury. Thus, RGS4 expression, specific to renal VSMC, inhibits angiotensin II-mediated cytokine signaling and macrophage recruitment during reperfusion, distinct from vasomotor regulation.
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Angiotensina II/metabolismo , Corteza Renal/irrigación sanguínea , Miocitos del Músculo Liso/metabolismo , Proteínas RGS/metabolismo , Daño por Reperfusión/metabolismo , Vasoconstricción , Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 2 de Angiotensina II/farmacología , Animales , Aorta/citología , Arteriolas/fisiopatología , Células Cultivadas , Quimiocina CCL5/metabolismo , Humanos , Corteza Renal/metabolismo , Macrófagos , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas RGS/genética , Receptor de Angiotensina Tipo 2/metabolismo , Circulación Renal , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Transducción de SeñalRESUMEN
Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells.
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Linfocitos T/citología , Linfocitos T/inmunología , Timo/inmunología , Proteínas WT1/inmunología , Animales , Línea Celular , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre/inmunología , Células Madre/metabolismo , Vacunación , Proteínas WT1/genética , Tumor de Wilms/inmunologíaRESUMEN
BACKGROUND: The molecular cause of inflammatory bowel disease is largely unknown. METHODS: We performed genetic-linkage analysis and candidate-gene sequencing on samples from two unrelated consanguineous families with children who were affected by early-onset inflammatory bowel disease. We screened six additional patients with early-onset colitis for mutations in two candidate genes and carried out functional assays in patients' peripheral-blood mononuclear cells. We performed an allogeneic hematopoietic stem-cell transplantation in one patient. RESULTS: In four of nine patients with early-onset colitis, we identified three distinct homozygous mutations in genes IL10RA and IL10RB, encoding the IL10R1 and IL10R2 proteins, respectively, which form a heterotetramer to make up the interleukin-10 receptor. The mutations abrogate interleukin-10-induced signaling, as shown by deficient STAT3 (signal transducer and activator of transcription 3) phosphorylation on stimulation with interleukin-10. Consistent with this observation was the increased secretion of tumor necrosis factor alpha and other proinflammatory cytokines from peripheral-blood mononuclear cells from patients who were deficient in IL10R subunit proteins, suggesting that interleukin-10-dependent "negative feedback" regulation is disrupted in these cells. The allogeneic stem-cell transplantation performed in one patient was successful. CONCLUSIONS: Mutations in genes encoding the IL10R subunit proteins were found in patients with early-onset enterocolitis, involving hyperinflammatory immune responses in the intestine. Allogeneic stem-cell transplantation resulted in disease remission in one patient.
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Enfermedades Inflamatorias del Intestino/genética , Subunidad alfa del Receptor de Interleucina-10/genética , Subunidad beta del Receptor de Interleucina-10/genética , Mutación Missense , Edad de Inicio , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 21 , Femenino , Ligamiento Genético , Humanos , Lactante , Enfermedades Inflamatorias del Intestino/terapia , Interleucina-10/metabolismo , Subunidad alfa del Receptor de Interleucina-10/química , Subunidad beta del Receptor de Interleucina-10/química , Masculino , Linaje , Polimorfismo de Nucleótido Simple , Inducción de Remisión , Análisis de Secuencia de ADN , Trasplante de Células Madre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: The hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by infections of the lung and skin, elevated serum IgE, and involvement of the soft and bony tissues. Recently, HIES has been associated with heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3) and severe reductions of T(H)17 cells. OBJECTIVE: To determine whether there is a correlation between the genotype and the phenotype of patients with HIES and to establish diagnostic criteria to distinguish between STAT3 mutated and STAT3 wild-type patients. METHODS: We collected clinical data, determined T(H)17 cell numbers, and sequenced STAT3 in 100 patients with a strong clinical suspicion of HIES and serum IgE >1000 IU/mL. We explored diagnostic criteria by using a machine-learning approach to identify which features best predict a STAT3 mutation. RESULTS: In 64 patients, we identified 31 different STAT3 mutations, 18 of which were novel. These included mutations at splice sites and outside the previously implicated DNA-binding and Src homology 2 domains. A combination of 5 clinical features predicted STAT3 mutations with 85% accuracy. T(H)17 cells were profoundly reduced in patients harboring STAT3 mutations, whereas 10 of 13 patients without mutations had low (<1%) T(H)17 cells but were distinct by markedly reduced IFN-gamma-producing CD4(+)T cells. CONCLUSION: We propose the following diagnostic guidelines for STAT3-deficient HIES. Possible: IgE >1000IU/mL plus a weighted score of clinical features >30 based on recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic face, and high palate. Probable: These characteristics plus lack of T(H)17 cells or a family history for definitive HIES. Definitive: These characteristics plus a dominant-negative heterozygous mutation in STAT3.
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Síndrome de Job/diagnóstico , Síndrome de Job/genética , Factor de Transcripción STAT3/genética , Adolescente , Adulto , Separación Celular , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina E/sangre , Lactante , Interleucina-17/inmunología , Síndrome de Job/inmunología , Masculino , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa , Guías de Práctica Clínica como Asunto , Linfocitos T Colaboradores-Inductores/inmunología , Adulto JovenRESUMEN
Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors. While it is recognized that TMEs play a pivotal role in disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical cancer models, the MC38 colorectal and KPC pancreatic murine tumors engineered to express human carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed tumors or after immunotherapy with a CEA T cell bispecific (CEA-TCB) surrogate antibody and anti-PD-L1 treatment. CEA-TCB mono and combination immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1-PD1+ effector T cells and with more minor increases in TCF1+PD1+ resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII+ macrophages, but largely avoided the deeper tumor nest regions composed of cancer cells and non-activated macrophages. These myeloid cell - T cell aggregates were found in close proximity to tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after CEA-TCB therapy, with its relative abundance positively associated with response to therapy. Together, these studies demonstrate the utility of advanced spatial analysis in cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.
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Neoplasias/inmunología , Microambiente Tumoral/inmunología , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Macrófagos/inmunología , Masculino , Ratones Endogámicos BALB C , Ratones Transgénicos , Microscopía Confocal , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Linfocitos T/inmunologíaRESUMEN
Diffuse large B cell lymphomas (DLBCL) are a highly heterogeneous subtype of Non Hodgkin Lymphoma (NHL), accounting for about 25% of NHL. Despite an increased progression-free survival upon therapy, 40-50% of patients develop relapse/refractory disease, therefore there remains an important medical need. T cell recruiting therapies, such as the CD20xCD3 T cell bi-specific antibody CD20-TCB (RG6026 or glofitamab), represent a novel approach to target all stages of DLBCL, especially those that fail to respond to multiple lines of treatment. We aimed for a better understanding of the molecular features related to the mode of action (MoA) of CD20-TCB in inducing Target/T cell synapse formation and human T cell recruitment to the tumor. To directly evaluate the correlation between synapse, cytokine production and anti-tumor efficacy using CD20-TCB, we developed an innovative preclinical human DLBCL in vivo model that allowed tracking in vivo human T cell dynamics by multiphoton intravital microscopy (MP-IVM). By ex vivo and in vivo approaches, we revealed that CD20-TCB is inducing strong and stable synapses between human T cell and tumor cells, which are dependent on the dose of CD20-TCB and on LFA-1 activity but not on FAS-L. Moreover, despite CD20-TCB being a large molecule (194.342 kDa), we observed that intra-tumor CD20-TCB-mediated human T cell-tumor cell synapses occur within 1 hour upon CD20-TCB administration. These tight interactions, observed for at least 72 hours post TCB administration, result in tumor cell cytotoxicity, resident T cell proliferation and peripheral blood T cell recruitment into tumor. By blocking the IFNγ-CXCL10 axis, the recruitment of peripheral T cells was abrogated, partially affecting the efficacy of CD20-TCB treatment which rely only on resident T cell proliferation. Altogether these data reveal that CD20-TCB's anti-tumor activity relies on a triple effect: i) fast formation of stable T cell-tumor cell synapses which induce tumor cytotoxicity and cytokine production, ii) resident T cell proliferation and iii) recruitment of fresh peripheral T cells to the tumor core to allow a positive enhancement of the anti-tumor effect.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígenos CD20/inmunología , Antineoplásicos Inmunológicos/farmacología , Quimiocina CXCL10/inmunología , Interferón gamma/inmunología , Linfoma de Células B Grandes Difuso/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/inmunología , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Ratones , Neoplasias Experimentales/tratamiento farmacológicoRESUMEN
OBJECTIVE: Depleting pathogenic B cells could treat systemic lupus erythematosus (SLE). However, depleting B cells in an inflammatory setting such as lupus is difficult. This study was undertaken to investigate whether a type II anti-CD20 monoclonal antibody (mAb) with a different mechanism of action, obinutuzumab (GA101), is more effective than a type I anti-CD20 mAb, rituximab (RTX), in B cell depletion in lupus, and whether efficient B cell depletion results in amelioration of disease. METHODS: We treated lupus-prone MRL/lpr mice expressing human CD20 on B cells (hCD20 MRL/lpr mice) with either RTX or GA101 and measured B cell depletion under various conditions, as well as multiple clinical end points. RESULTS: A single dose of GA101 was markedly more effective than RTX in depleting B cells in diseased MRL/lpr mice (P < 0.05). RTX overcame resistance to B cell depletion in diseased MRL/lpr mice with continuous treatments. GA101 was more effective in treating hCD20 MRL/lpr mice with early disease, as GA101-treated mice had reduced glomerulonephritis (P < 0.05), lower anti-RNA autoantibody titers (P < 0.05), and fewer activated CD4+ T cells (P < 0.0001) compared to RTX-treated mice. GA101 also treated advanced disease, and continual treatment prolonged survival. Using variants of GA101, we also elucidated B cell depletion mechanisms in vivo in mice with lupus. CONCLUSION: Albeit both anti-CD20 antibodies ameliorated early disease, GA101 was more effective than RTX in important parameters, such as glomerulonephritis score. GA101 proved beneficial in an advanced disease model, where it prolonged survival. These data support clinical testing of GA101 in SLE and lupus nephritis.
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Anticuerpos Monoclonales Humanizados/farmacología , Linfocitos B/efectos de los fármacos , Factores Inmunológicos/farmacología , Riñón/efectos de los fármacos , Lupus Eritematoso Sistémico/inmunología , Rituximab/farmacología , Piel/efectos de los fármacos , Animales , Linfocitos B/inmunología , Citometría de Flujo , Riñón/patología , Lupus Eritematoso Sistémico/patología , Depleción Linfocítica , Ratones , Ratones Endogámicos MRL lpr , Piel/patologíaRESUMEN
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RESUMEN
PURPOSE: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. EXPERIMENTAL DESIGN: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. RESULTS: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. CONCLUSIONS: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.
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Antineoplásicos Inmunológicos/administración & dosificación , Antígenos CD40/agonistas , Endopeptidasas/efectos de los fármacos , Inmunoterapia/métodos , Proteínas de la Membrana/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Ratones , Células Tumorales CultivadasRESUMEN
Leukocyte function-associated antigen 1 (LFA-1) is the most widely expressed member of the ß2 integrin family of cell-cell adhesion molecules. Although LFA-1 is thought to regulate multiple aspects of T cell immunity, its role in the response of CD8+ T cells to viral infections remains unclear. Indeed, compelling clinical evidence shows that loss of LFA-1 function predisposes to infection in humans but animal models show limited to no susceptibility to infection. Here, we addressed this conundrum in a mouse model of infection with lymphocytic choriomeningitis virus (LCMV), where CD8+ T cells are necessary and sufficient to confer protection. To this end, we followed the fate and function of wild-type and LFA-1 deficient virus-specific CD8+ T cells and assessed the effect of blocking anti-LFA-1 monoclonal antibody in the outcome of infection. Our analysis of viral clearance and T cell responses using transcriptome profiling reveals a role for LFA-1 as a gatekeeper of effector T cell survival and dysfunction that when defective can predispose to LCMV infection.
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Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/prevención & control , Virus de la Coriomeningitis Linfocítica/patogenicidad , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/fisiología , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Activación de Linfocitos , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
T-cell Bispeciï¬c Antibodies (TCBs) elicit anti-tumor responses by cross-linking T-cells to tumor cells and mediate polyclonal T-cell expansion that is independent of T-cell receptor specificity. TCBs thus offer great promise for patients who lack antigen-speciï¬c T-cells or have non-inflamed tumors, which are parameters known to limit the response of checkpoint inhibitors. The current study deepens the understanding of TCB mode of action and elaborates on one of the adaptive resistance mechanisms following its treatment in vivo in humanized mice and syngeneic pre-clinical tumor models. Single-agent TCB treatment reduced tumor growth compared with controls and led to a 2-10-fold increase in tumor-infiltrating T-cells, regardless of the baseline tumor immune cell infiltration. TCB treatment strongly induced the secretion of CXCL10 and increased the frequency of intra-tumor CXCR3+ T-cells pointing to the potential role of the CXCL10-CXCR3 pathway as one of the mechanisms for T-cell recruitment to tumors upon TCB treatment. Tumor-infiltrating T-cells displayed a highly activated and proliferating phenotype, resulting in the generation of a highly inflamed tumor microenvironment. A molecular signature of TCB treatment was determined (CD8, PD-1, MIP-a, CXCL10, CXCL13) to identify parameters that most robustly characterize TCB activity. Parallel to T-cell activation, TCB treatment also led to a clear upregulation of PD-1 on T-cells and PD-L1 on tumor cells and T-cells. Combining TCB treatment with anti-PD-L1 blocking antibody improved anti-tumor efficacy compared to either agent given as monotherapy, increasing the frequency of intra-tumoral T-cells. Together, the data of the current study expand our knowledge of the molecular and cellular features associated with TCB activity and provide evidence that the PD-1/PD-L1 axis is one of the adaptive resistance mechanisms associated with TCB activity. This mechanism can be managed by the combination of TCB with anti-PD-L1 blocking antibody translating into more efficacious anti-tumor activity and prolonged control of the tumor outgrowth. The elucidation of additional resistance mechanisms beyond the PD-1/PD-L1 axis will constitute an important milestone for our understanding of factors determining tumor escape and deepening of TCB anti-tumor responses in both solid tumors and hematological disorders.
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Recently developed approaches for highly multiplexed imaging have revealed complex patterns of cellular positioning and cell-cell interactions with important roles in both cellular- and tissue-level physiology. However, tools to quantitatively study cellular patterning and tissue architecture are currently lacking. Here, we develop a spatial analysis toolbox, the histo-cytometric multidimensional analysis pipeline (CytoMAP), which incorporates data clustering, positional correlation, dimensionality reduction, and 2D/3D region reconstruction to identify localized cellular networks and reveal features of tissue organization. We apply CytoMAP to study the microanatomy of innate immune subsets in murine lymph nodes (LNs) and reveal mutually exclusive segregation of migratory dendritic cells (DCs), regionalized compartmentalization of SIRPα- dermal DCs, and preferential association of resident DCs with select LN vasculature. The findings provide insights into the organization of myeloid cells in LNs and demonstrate that CytoMAP is a comprehensive analytics toolbox for revealing features of tissue organization in imaging datasets.
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Tejido Linfoide/metabolismo , Células Mieloides/metabolismo , Animales , Ratones , Análisis EspacialRESUMEN
PD-L1/PD-1 blocking antibodies have demonstrated therapeutic efficacy across a range of human cancers. Extending this benefit to a greater number of patients, however, will require a better understanding of how these therapies instigate anticancer immunity. Although the PD-L1/PD-1 axis is typically associated with T cell function, we demonstrate here that dendritic cells (DCs) are an important target of PD-L1 blocking antibody. PD-L1 binds two receptors, PD-1 and B7.1 (CD80). PD-L1 is expressed much more abundantly than B7.1 on peripheral and tumor-associated DCs in patients with cancer. Blocking PD-L1 on DCs relieves B7.1 sequestration in cis by PD-L1, which allows the B7.1/CD28 interaction to enhance T cell priming. In line with this, in patients with renal cell carcinoma or non-small cell lung cancer treated with atezolizumab (PD-L1 blockade), a DC gene signature is strongly associated with improved overall survival. These data suggest that PD-L1 blockade reinvigorates DC function to generate potent anticancer T cell immunity.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas , Inmunoterapia , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Células Dendríticas , Humanos , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.
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Anticuerpos Biespecíficos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Anticuerpos Biespecíficos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Proliferación Celular/fisiología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Humanos , Inmunoterapia , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Neoplasias/inmunología , Neoplasias/terapiaRESUMEN
The Fumarase (Fumarate Hydratase, FH) is a tumor suppressor gene whose germline heterozygous mutations predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). The FH gene encodes an enzyme of the Krebs cycle, functioning as a homotetramer and catalyzing the hydration of fumarate to malate. Among the numerous FH mutations reported so far, the R190H missense mutation is the most frequent in HLRCC patients. Here we show the functional analyses of the R190H, in comparison to the better characterized E319Q mutation. We first expressed wild-type and mutated proteins in FH deficient human skin fibroblasts, using lentiviral vectors. The wild-type transgene was able to restore the FH enzymatic activity in cells, while the R190H- and E319Q-FH were not. More interestingly, when the same transgenes were expressed in normal, FH-proficient cells, only the R190H-FH reduced the endogenous FH enzymatic activity. By enforcing the expression of equal amount of wild-type and R190H-FH in the same cell, we showed that the mutated FH protein directly inhibited enzymatic activity by nearly abrogating the FH homotetramer formation. These data demonstrate the dominant negative effect of the R190H missense mutation in the FH gene and suggest that the FH tumor-suppressing activity might be impaired in cells carrying a heterozygous mutation.
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Fibroblastos/metabolismo , Fumarato Hidratasa/genética , Fumarato Hidratasa/metabolismo , Genes Dominantes , Mutación Missense/genética , Piel/metabolismo , Western Blotting , Células Cultivadas , Fibroblastos/citología , Humanos , Lentivirus/genética , Mitocondrias/metabolismo , Mutagénesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citologíaRESUMEN
Purpose: Despite promising clinical activity, T-cell-engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug.Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug.Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell-binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD.Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785-97. ©2018 AACR See related commentary by Prakash and Diefenbach, p. 4631.