RESUMO
BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.
Assuntos
Neoplasias da Mama , Linfócitos T CD8-Positivos , Células Matadoras Naturais , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Humanos , Animais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Camundongos , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Feminino , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Receptor ErbB-2/imunologia , Linhagem Celular Tumoral , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismoRESUMO
Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.
Assuntos
Proteínas de Transporte/fisiologia , Endotélio Vascular/embriologia , Endotélio Vascular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Junções Intercelulares/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neovascularização Fisiológica , Gravidez , Interferência de RNA , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Neuropilin-1 (NRP1) guides the development of the nervous and vascular systems. Binding to either semaphorins or VEGF, NRP1 acts with plexins to regulate neuronal guidance, or with VEGFR2 to mediate vascular development. We have generated two monoclonal antibodies that bind to the Sema- and VEGF-binding domains of NRP1, respectively. Both antibodies reduce angiogenesis and vascular remodeling, while having little effect on other VEGFR2-mediated events. Importantly, anti-NRP1 antibodies have an additive effect with anti-VEGF therapy in reducing tumor growth. Vessels from tumors treated with anti-VEGF show a close association with pericytes, while tumors treated with both anti-NRP1 and anti-VEGF lack this organization. We propose that blocking NRP1 function inhibits vascular remodeling, rendering vessels more susceptible to anti-VEGF therapy.
Assuntos
Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica/metabolismo , Neuropilina-1/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Monoclonais , Movimento Celular , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Neurônios/metabolismo , Ratos , Semaforina-3A/imunologiaRESUMO
Neuropilin (NRP)-1 is a co-receptor for vascular endothelial growth factor (VEGF). Preclinical data suggest that blockade of NRP1 suppresses tumour growth by inhibiting angiogenesis, in addition to directly inhibiting tumour cell proliferation in certain models. A humanized monoclonal antibody to NRP1 is currently being evaluated as a potential anti-cancer therapy in clinical trials. However, the expression of NRP1 in cancer and physiological angiogenesis has yet to be systematically described. Here we characterize the in situ expression of NRP1 in human cancer and during mammalian development. A monoclonal antibody to human NRP1 was generated and validated for immunohistochemistry by western blotting, use of formalin-fixed cell pellets transfected with NRP1, immunofluorescence, and comparison with in situ hybridization. NRP1 expression was assessed in whole sections of 65 primary breast carcinomas, 95 primary colorectal adenocarcinomas, and 90 primary lung carcinomas. An additional 59 human metastases, 16 xenografts, and three genetically engineered mouse tumour models were also evaluated. Immunoreactivity for NRP1 was seen in vessels from normal tissues adjacent to cancer and in 98-100% of carcinomas. Tumour cell expression of NRP1 was also observed in 36% of primary lung carcinomas and 6% of primary breast carcinomas, but no colorectal adenocarcinomas. NRP1 was evaluated in mouse embryos, where expression was limited to the nervous system, endocardium, vascular smooth muscle, and, focally, endothelium on subsets of vessels. Moreover, in a model of VEGF-dependent angiogenesis in the postnatal mouse trachea, blockade of NRP1 signalling resulted in defective angiogenesis and recapitulated the effects of anti-VEGF treatment. These observations confirm NRP1 as a valid anti-angiogenic target in malignancy, and as a potential direct anti-tumour target in a subset of cancers. The data also confirm a role for NRP1 in physiological, VEGF-mediated angiogenesis.
Assuntos
Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/fisiologia , Neuropilina-1/biossíntese , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting , Progressão da Doença , Imunofluorescência , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
Antiangiogenic therapies that target the VEGF pathway have been used clinically to combat cancer for over a decade. Beyond having a direct impact on blood vessel development and tumor perfusion, accumulating evidence indicates that these agents also affect antitumor immune responses. Numerous clinical trials combining antiangiogenic drugs with immunotherapies for the treatment of cancer are ongoing, but a mechanistic understanding of how disruption of tumor angiogenesis may impact immunity is not fully discerned. Here, we reveal that blockade of VEGF-A with a mAb to VEGF augments activation of CD8+ T cells within tumors and potentiates their capacity to produce cytokines. We demonstrate that this phenomenon relies on the disruption of VEGFR2 signaling in the tumor microenvironment but does not affect CD8+ T cells directly. Instead, the augmented functional capacity of CD8+ T cells stems from increased tumor hypoxia that initiates a hypoxia-inducible factor-1α program within CD8+ T cells that directly enhances cytokine production. Finally, combinatorial administration of anti-VEGF with an immunotherapeutic antibody, anti-OX40, improved antitumor activity over single-agent treatments. Our findings illustrate that anti-VEGF treatment enhances CD8+ T-cell effector function and provides a mechanistic rationale for combining antiangiogenic and immunotherapeutic drugs for cancer treatment.
Assuntos
Bevacizumab/farmacologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/terapia , Hipóxia/patologia , Ativação Linfocitária/imunologia , Melanoma Experimental/terapia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Apoptose , Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imunoterapia , Ativação Linfocitária/efeitos dos fármacos , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.
Assuntos
Homeostase/imunologia , Inflamação/imunologia , Interleucinas/imunologia , Fator Estimulador de Colônias de Macrófagos/imunologia , Macrófagos/imunologia , Neoplasias/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Homeostase/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos NZB , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMO
We report that Slit proteins, a family of secreted chemorepellents, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 and, even more so, mice deficient in both Slit1 and Slit2 show significant axon guidance errors in a variety of pathways, including corticofugal, callosal, and thalamocortical tracts. Analysis of multiple pathways suggests several generalizations regarding the functions of Slit proteins in the brain, which appear to contribute to (1) the maintenance of dorsal position by prevention of axonal growth into ventral regions, (2) the prevention of axonal extension toward and across the midline, and (3) the channeling of axons toward particular regions.
Assuntos
Axônios/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Prosencéfalo/embriologia , Vias Aferentes/embriologia , Animais , Córtex Cerebral/embriologia , Corpo Caloso/embriologia , Dopamina/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Mesencéfalo/embriologia , Camundongos , Camundongos Mutantes , Mutação/fisiologia , Fibras Nervosas/fisiologia , Proteínas do Tecido Nervoso/genética , Vias Neurais/embriologia , Receptores Imunológicos/metabolismo , Serotonina/fisiologia , Transmissão Sináptica/fisiologia , Telencéfalo/embriologia , Tálamo/embriologia , Proteínas RoundaboutRESUMO
PURPOSE: The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell-dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice. Efficacy and immunologic effects of anti-HER2/CD3 TDB were investigated in mammary tumor model with very few T cells prior treatment. We further describe the mechanism for TDB-induced T-cell recruitment to tumors. EXPERIMENTAL DESIGN: The immunologic effects and the mechanism of CD3-bispecific antibody-induced T-cell recruitment into spontaneous HER2-overexpressing mammary tumors was studied using human HER2 transgenic, immunocompetent mouse models. RESULTS: Anti-HER2/CD3 TDB treatment induced an inflammatory response in tumors converting them from poorly infiltrated to an inflamed, T-cell abundant, phenotype. Multiple mechanisms accounted for the TDB-induced increase in T cells within tumors. TDB treatment induced CD8+ T-cell proliferation. T cells were also actively recruited post-TDB treatment by IFNγ-dependent T-cell chemokines mediated via CXCR3. This active T-cell recruitment by TDB-induced chemokine signaling was the dominant mechanism and necessary for the therapeutic activity of anti-HER2/CD3 TDB. CONCLUSIONS: In summary, we demonstrate that the activity of anti-HER2/CD3 TDB was not dependent on high-level baseline T-cell infiltration. Our results suggest that anti-HER2/CD3 TDB may be efficacious in patients and indications that respond poorly to checkpoint inhibitors. An active T-cell recruitment mediated by TDB-induced chemokine signaling was the major mechanism for T-cell recruitment.
Assuntos
Anticorpos Biespecíficos/farmacologia , Complexo CD3/antagonistas & inibidores , Quimiocinas/metabolismo , Interferon gama/metabolismo , Neoplasias/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptores CXCR3/metabolismo , Linfócitos T/metabolismo , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/patologia , Transdução de Sinais , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Neuropilina-2/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Forma Celular , Células Cultivadas , Feminino , Linfangiogênese , Vasos Linfáticos/embriologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Neuropilina-2/genética , Ligação Proteica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.