RESUMEN
Harnessing innate immunity is emerging as a promising therapeutic approach in cancer. We report here the design of tetraspecific molecules engaging natural killer (NK) cell-activating receptors NKp46 and CD16a, the ß-chain of the interleukin-2 receptor (IL-2R), and a tumor-associated antigen (TAA). In vitro, these tetraspecific antibody-based natural killer cell engager therapeutics (ANKETs) induce a preferential activation and proliferation of NK cells, and the binding to the targeted TAA triggers NK cell cytotoxicity and cytokine and chemokine production. In vivo, tetraspecific ANKETs induce NK cell proliferation and their accumulation at the tumor bed, as well as the control of local and disseminated tumors. Treatment of non-human primates with CD20-directed tetraspecific ANKET leads to CD20+ circulating B cell depletion, with minimal systemic cytokine release and no sign of toxicity. Tetraspecific ANKETs, thus, constitute a technological platform for harnessing NK cells as next-generation cancer immunotherapies.
Asunto(s)
Interleucina-2 , Neoplasias , Animales , Interleucina-2/genética , Células Asesinas Naturales , Receptores de Interleucina-2/metabolismo , Citocinas , Neoplasias/genética , Quimiocinas/metabolismoRESUMEN
Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.
Asunto(s)
5'-Nucleotidasa/metabolismo , Inmunidad Adaptativa/fisiología , Adenosina/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Inmunoterapia/tendencias , Neoplasias/terapia , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/inmunología , Adenosina/análogos & derivados , Adenosina/antagonistas & inhibidores , Adenosina/inmunología , Antígenos CD/inmunología , Apirasa/antagonistas & inhibidores , Apirasa/inmunología , Humanos , Inmunoterapia/métodos , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Neoplasias/inmunología , Transducción de Señal/inmunologíaRESUMEN
Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer.
Asunto(s)
5'-Nucleotidasa/inmunología , Anticuerpos Bloqueadores/inmunología , Antígenos CD/inmunología , Apirasa/inmunología , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Anticuerpos Bloqueadores/uso terapéutico , Antígenos CD/genética , Antineoplásicos/uso terapéutico , Apirasa/deficiencia , Apirasa/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Melanoma/mortalidad , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxaliplatino/uso terapéutico , Tasa de Supervivencia , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Microambiente TumoralRESUMEN
Dendritic cells (DCs) cross-present antigen (Ag) to initiate T-cell immunity against most infections and tumors. Natural killer (NK) cells are innate cytolytic lymphocytes that have emerged as key modulators of multiple DC functions. Here, we show that human NK cells promote cross-presentation of tumor cell-derived Ag by DC leading to Ag-specific CD8(+) T-cell activation. Surprisingly, cytotoxic function of NK cells was not required. Instead, we highlight a critical and nonredundant role for IFN-γ and TNF-α production by NK cells to enhance cross-presentation by DC using two different Ag models. Importantly, we observed that NK cells promote cell-associated Ag cross-presentation selectively by monocytes-derived DC (Mo-DC) and CD34-derived CD11b(neg) CD141(high) DC subsets but not by myeloid CD11b(+) DC. Moreover, we demonstrate that triggering NK cell activation by monoclonal antibodies (mAbs)-coated tumor cells leads to efficient DC cross-presentation, supporting the concept that NK cells can contribute to therapeutic mAbs efficiency by inducing downstream adaptive immunity. Taken together, our findings point toward a novel role of human NK cells bridging innate and adaptive immunity through selective induction of cell-associated Ag cross-presentation by CD141(high) DC, a process that could be exploited to better harness Ag-specific cellular immunity in immunotherapy.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Inmunidad Celular/inmunología , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Células Dendríticas/patología , Humanos , Células Asesinas Naturales/patología , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1(+) DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1(+) human DC. Assessment of the immunoactivation potential of XCR1(+) human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1(+) and XCR1(-) human DC in CD34(+) progenitor cultures (CD34-DC). Gene expression profiling, phenotypic characterization, and functional studies demonstrated that XCR1(-) CD34-DC are similar to canonical MoDC, whereas XCR1(+) CD34-DC resemble XCR1(+) blood DC (bDC). XCR1(+) DC were strongly activated by polyinosinic-polycytidylic acid but not LPS, and conversely for MoDC. XCR1(+) DC and MoDC expressed strikingly different patterns of molecules involved in inflammation and in cross-talk with NK or T cells. XCR1(+) CD34-DC but not MoDC efficiently cross-presented a cell-associated Ag upon stimulation by polyinosinic-polycytidylic acid or R848, likewise to what was reported for XCR1(+) bDC. Hence, it is feasible to generate high numbers of bona fide XCR1(+) human DC in vitro as a model to decipher the functions of XCR1(+) bDC and as a potential source of XCR1(+) DC for clinical use.
Asunto(s)
Antígenos CD34/inmunología , Células Sanguíneas/inmunología , Células Dendríticas/inmunología , Monocitos/inmunología , Receptores Acoplados a Proteínas G/inmunología , Adyuvantes Inmunológicos/farmacología , Presentación de Antígeno/inmunología , Técnicas de Cultivo de Célula , Diferenciación Celular/inmunología , Línea Celular , Reactividad Cruzada/inmunología , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Imidazoles/inmunología , Células Asesinas Naturales/inmunología , Lipopolisacáridos/inmunología , Fenotipo , Poli I-C/inmunología , Linfocitos T/inmunología , Receptor Toll-Like 3 , Receptor Toll-Like 4RESUMEN
Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Células Dendríticas/efectos de los fármacos , Interferón gamma/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Receptor Toll-Like 3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Células Cultivadas , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Células Dendríticas/citología , Células Dendríticas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Helicasa Inducida por Interferón IFIH1 , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/citología , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Poli A-U/farmacología , Poli I-C/farmacología , ARN Bicatenario/farmacología , Receptores Inmunológicos , Receptor Toll-Like 3/genética , TransfecciónRESUMEN
TRIF is an adaptor protein associated with the signaling by Toll-like receptor (TLR)3 and TLR4 for the induction of type I IFNs. Here, we demonstrate a mechanism by which TLR signaling controls cell proliferation and survival. We show that TLR3 and TLR4 can induce cell cycle entry via TRIF, which targets the cell cycle inhibitor p27(kip1) for relocalization, phosphorylation by cyclin/cdk complexes, and proteasome degradation. These events are antagonized by type I IFN induced by the TRIF pathway. Furthermore, in human dendritic cells treated with TLR3, TLR4, or TLR5 ligands, we demonstrate that IFN signaling modulates p27(kip1) degradation and apoptosis, identifying an immunoregulatory "switching" function of type I IFNs. These findings reveal a previously uncharacterized function of TLR signaling in cell proliferation and survival.
Asunto(s)
Interferón Tipo I/fisiología , Transducción de Señal/fisiología , Receptor Toll-Like 3/fisiología , Receptor Toll-Like 4/fisiología , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Células Dendríticas/metabolismo , Humanos , Lipopolisacáridos/farmacología , FN-kappa B/genética , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/fisiología , RatasRESUMEN
The efficacy of immune response to control human cancer remains controversial. It is particularly debated whether and to what extent the capacity of tumor-infiltrating dendritic cells (DC) to drive immunization can be turned off by transformed cells, leading to tumor-specific tolerance rather than immunization. To address this issue, we have characterized the DC isolated from human non-small cell lung cancer (NSCLC). These biopsy specimens contained CD11c(high) myeloid DC (mDC), but also CD11c(-) plasmacytoid DC (pDC) and a third DC subset expressing intermediate level of CD11c. Compared with peripheral blood, CD11c(high) tumor-infiltrating DC (TIDC) displayed a "semi-mature" phenotype, and TLR4 or TLR8 stimulation drove them to mature partially and to secrete limited amounts of cytokines. In contrast, most tumor-infiltrating pDC were immature but underwent partial maturation after TLR7 activation, whereas TLR9 ligation triggered low secretion of IFN-alpha. CD11c(int) mDC represented approximately 25% of total DC in tumoral and peritumoral tissues and expressed low levels of costimulatory molecules contrasting with high levels of the immunoinhibitory molecule B7-H1. Finally, the poor APC function of total TIDC even after TLR stimulation and the migratory response of both tumor-infiltrating mDC and pDC toward CCL21 and SDF-1 in vitro suggested their ability to compromise the tumor-specific immune response in draining lymph nodes in vivo. Further studies will be required to establish the specific role of the three TIDC subsets in tumor immunity and to draw conclusions for the design of therapeutic strategies.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/inmunología , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Células Dendríticas/inmunología , Células Mieloides/inmunología , Escape del Tumor , Antígeno CD11c/inmunología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Quimiocina CCL21 , Quimiocina CXCL12 , Quimiocinas CC/inmunología , Quimiocinas CXC/inmunología , Humanos , Receptores Toll-Like/inmunología , Células Tumorales Cultivadas , VacunaciónRESUMEN
Human CD4(+)CD25(+) regulatory T cells (Treg) play an essential role in the prevention of autoimmune diseases. However, the mechanisms of immune suppression and the spectrum of cells they target in vivo remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they have been shown to inhibit the Ag-presenting functions of macrophage- and monocyte-derived dendritic cells (DC). We have now studied the maturation of human blood-derived myeloid DC and plasmacytoid DC activated with TLR ligands in the presence of Treg. Preactivated Treg suppressed strongly TLR-triggered myeloid DC maturation, as judged by the blocking of costimulatory molecule up-regulation and the inhibition of proinflammatory cytokines secretion that resulted in poor Ag presentation capacity. Although IL-10 played a prominent role in inhibiting cytokines secretion, suppression of phenotypic maturation required cell-cell contact and was independent of TGF-beta and CTLA-4. In contrast, the acquisition of maturation markers and production of cytokines by plasmacytoid DC triggered with TLR ligands were insensitive to regulatory T cells. Therefore, human Treg may enlist myeloid, but not plasmacytoid DC for the initiation and the amplification of tolerance in vivo by restraining their maturation after TLR stimulation.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Mieloides/inmunología , Células Mieloides/metabolismo , Receptores de Interleucina-2/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Presentación de Antígeno , Comunicación Celular , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Fenotipo , Linfocitos T Reguladores/citología , Receptores Toll-Like/metabolismoRESUMEN
This work aims to further characterize the newly described leukemic plasmacytoid dendritic cells (LPDC), for which we had previously demonstrated their normal, PDC-like ability to produce IFN-alpha. In addition, LPDC also express the specific antigens BDCA-2 and BDCA-4. Importantly, they become fully competent antigen-presenting cells (APC) after a short maturation induced by IL-3 + CD40L or virus, exhibiting a characteristic APC phenotype (high expression of CD83 and of the costimulatory molecules CD40, CD80, CD86). Whereas IL-3 + CD40L-activated LPDC prime naive CD4(+) T cells towards a Th2 pathway (IL-4-secreting T cells), virus-activated LPDC drive a Th1 profile (IFN-gamma-secreting T cells). Moreover, we show in one case that LPDC are able to capture, process and present exogenous antigens, leading to the activation of both CD4(+) and CD8(+) T cell clones in an antigen-specific manner. This study further characterizes the phenotype and immunological functions of LPDC.