RESUMO
We recently reported that resistance to PD-1 blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). Thus, we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. In this study, we report that LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist mAb, elicits myeloid inflammation and allogeneic T-cell responses by binding to LAIR1 and blocking collagen engagement. Furthermore, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T-cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic.
Assuntos
Antineoplásicos , Colágeno , Inibidores de Checkpoint Imunológico , Receptores Imunológicos , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Colágeno/metabolismo , Modelos Animais de Doenças , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Antineoplásicos/uso terapêuticoRESUMO
Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.
Assuntos
Antígenos CD , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Glicoproteínas de Membrana , Células Mieloides , Receptores Imunológicos , Microambiente Tumoral , Receptores Imunológicos/metabolismo , Animais , Humanos , Camundongos , Microambiente Tumoral/imunologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Glicoproteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismoRESUMO
Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.
Assuntos
Mieloma Múltiplo , Animais , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Imunoterapia/métodos , Linfócitos T , Plasmócitos/metabolismoRESUMO
Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.
Assuntos
Fibronectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Receptores Imunológicos/metabolismo , Diferenciação Celular , Linhagem Celular , HumanosRESUMO
Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Macrófagos/imunologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/imunologia , Proteínas de Neoplasias/imunologia , Receptor TIE-2/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Macrófagos/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Proteínas de Neoplasias/genética , Oxigênio/imunologia , Receptor TIE-2/genéticaRESUMO
Optimally effective antitumor therapies would not only activate immune effector cells but also engage them at the tumor. Folate conjugated to immunoglobulin (F-IgG) could direct innate immune cells with Fc receptors to folate receptor-expressing cancer cells. F-IgG bound to human KB and HeLa cells, as well as murine L1210JF, a folate receptor (FR)-overexpressing cancer cell line, as determined by flow cytometry. Recognition of F-IgG by natural killer (NK) cell Fc receptors led to phosphorylation of the ERK transcription factor and increased NK cell expression of CD69. Lysis of KB tumor cells by NK cells increased by about 5-fold after treatment with F-IgG, an effect synergistically enhanced by treatment with IL2, IL12, IL15, or IL21 (P< 0.001). F-IgG also enhanced the lysis of chronic lymphocytic leukemia cells by autologous NK cells. NK cells significantly increased production of IFNγ, MIP-1α, and RANTES in response to F-IgG-coated KB target cells in the presence of the NK cell-activating cytokine IL12, and these coculture supernatants induced significant T-cell chemotaxis (P< 0.001). F-IgG-coated targets also stimulated FcR-mediated monocyte effector functions. Studies in a murine leukemia model confirmed the intratumoral localization and antitumor activity of F-IgG, as well as enhancement of its effects by IL12 (P =0.05). The antitumor effect of this combination was dependent on NK cells and led to decreased tumor cell proliferation in vivo Thus, F-IgG can induce an immune response against FR-positive tumor cells that is mediated by NK cells and can be augmented by cytokine therapy.
Assuntos
Citocinas/metabolismo , Citotoxicidade Imunológica , Ácido Fólico/administração & dosagem , Imunoconjugados/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Expressão Gênica , Humanos , Imunoglobulina G/imunologia , Imunomodulação , Interleucina-12/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Neoplasias/genética , Neoplasias/patologia , Carga Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.
Assuntos
Anticorpos Antineoplásicos/farmacologia , MicroRNAs/genética , Neovascularização Patológica , Receptores de IgG/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Matadoras Naturais/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/citologia , Monócitos/metabolismo , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
Under hypoxia, tumor cells, and tumor-associated macrophages produce VEGF (vascular endothelial growth factor), a signaling molecule that induces angiogenesis. The same macrophages, when treated with GM-CSF (granulocyte/macrophage colony-stimulating factor), produce sVEGFR-1 (soluble VEGF receptor-1), a soluble protein that binds with VEGF and inactivates its function. The production of VEGF by macrophages is regulated by HIF-1α (hypoxia inducible factor-1α), and the production of sVEGFR-1 is mediated by HIF-2α. Recent experiments measured the effect of inhibiting tumor growth by GM-CSF treatment in mice with HIF-1α-deficient or HIF-2α-deficient macrophages. In the present paper, we represent these experiments by a mathematical model based on a system of partial differential equations. We show that the model simulations agree with the above experiments. The model can then be used to suggest strategies for inhibiting tumor growth. For example, the model qualitatively predicts the extent to which GM-CSF treatment in combination with a small molecule inhibitor that stabilizes HIF-2α will reduce tumor volume and angiogenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Modelos Biológicos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Simulação por Computador , Macrófagos/metabolismo , Melanoma/irrigação sanguínea , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismoRESUMO
Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.
Assuntos
Antineoplásicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Inibidores do Crescimento/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Antineoplásicos/uso terapêutico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Dioxigenases/antagonistas & inibidores , Dioxigenases/biossíntese , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/uso terapêutico , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Macrófagos/patologia , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Estabilidade Proteica , SolubilidadeRESUMO
BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide. Greater than 90% of SCCHN of the oropharynx overexpress the epidermal growth factor receptor (EGFR or HER1). Cetuximab (Erbitux-TM) is a humanized anti-HER1 monoclonal antibody (mAb) that binds to HER1 overexpressing tumor cells. Cetuximab has a direct effect on HER1-positive cancer cells, but it also can activate immune cells that bear receptors for the Fc (constant portion) of IgG such as natural killer (NK) cells. NK cells have an activating Fc receptor for IgG (FcγRIIIa), which mediates Ab dependent cellular cytotoxicity (ADCC) and enhances production of interferon-γ (IFN-γ) in response to Ab-coated targets. Interleukin-12 (IL-12) is a cytokine produced by antigen-presenting cells that stimulates IFN-γ production from NK cells. We hypothesized that IL-12 would enhance the anti-tumor activity of cetuximab by activating the FcR effector mechanisms of NK cells. METHODS: Expression of HER1 was measured on human papilloma virus (HPV)-positive (UD-SCC2, UM-SCC47) and HPV-negative (Cal27, UM-SCC74B) SCCHN cell lines by immunoblot analysis and flow cytometry. NK cells from normal donors were treated overnight with IL-2 (100 U), IL-12, IL-15, or IL-21 (all 10 ng/mL) and tested for ADCC versus cetuximab-coated cancer cells in a 4 hr (51)Cr assay. Release of cytokines by NK cells in response to cetuximab-coated cells was measured by ELISA. Phosphorylation of the ERK transcription factor in NK cells was measured by flow cytometry. The efficacy of combination therapy with cetuximab plus IL-12 was evaluated in a murine tumor model of head and neck cancer. RESULTS: All cell lines showed >99% expression of HER1 by flow cytometry and immunoblot analysis except UM-SCC74B (73%). Normal NK cells mediated 49.4% lysis of cetuximab-coated SCCHN cell lines as compared to 7.6% lysis of cells treated with control IgG (P = .0002). NK cell lysis of cetuximab-coated SCCHN cells was markedly enhanced by 12 hr pre-treatment of NK cells with IL-12 (71.6% lysis, P = .005 vs cetuximab alone). As a control, IL-12-activated NK cells were tested against IgG-treated cells. ADCC under these conditions was just 21.7%. Similar levels of lysis were noted for both HPV-positive and HPV-negative and cell lines. Other NK cell activating factors such as IL-2, IL-15, and IL-21 were also able to enhance NK cell ADCC. The stimulus of IL-12 and cetuximab-coated tumor cells induced the synergistic production of nanogram levels of IFN-γ (>6-fold increase over controls) (P < .001). A similar effect was seen for NK cell production of the chemokines RANTES, MIP-1α, and IL-8. Phosphorylation of ERK (which is critical for FcR-mediated ADCC and cytokine production) was enhanced in NK cells exposed to IL-12 and IgG as compared to control conditions. The combination of cetuximab plus IL-12 resulted in a reduction in tumor burden when compared to either agent alone in a murine xenograft model of SCCHN. CONCLUSION: Cytokine stimulation of NK cells in the presence of cetuximab-coated head and neck cancer cells leads to enhanced NK cell mediated ADCC and cytokine secretion independent of tumor cell HPV-status. Cytokine administration could be a useful adjuvant in the cetuximab treatment of HER1-positive head and neck cancer.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/imunologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/imunologia , Fatores Imunológicos/administração & dosagem , Interleucina-12/administração & dosagem , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Animais , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Cetuximab , Técnicas de Cocultura , Citocinas/administração & dosagem , Citocinas/metabolismo , Receptores ErbB/metabolismo , Citometria de Fluxo , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Nus , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Macrophage secretion of vascular endothelial growth factor (VEGF) in response to the hypoxic tumor microenvironment contributes to tumor growth, angiogenesis, and metastasis. We have recently demonstrated that macrophages stimulated with GM-CSF at low O(2) secrete high levels of a soluble form of the VEGF receptor 1 (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using small interfering RNA targeting to deplete hypoxia-inducible factor (HIF)-1α or HIF-2α in murine macrophages, we found that macrophage production of sVEGFR-1 in response to low O(2) was dependent on HIF-2α, whereas HIF-1α specifically regulated VEGF production. In our current report, we evaluated the growth of B16F10 malignant melanoma in mice with a monocyte/macrophage-selective deletion of HIF-1α or HIF-2α (HIF-1α(flox/flox)- or HIF-2α(flox/+)/LysMcre mice). GM-CSF treatment increased intratumoral VEGF and sVEGFR-1 in control mice, an effect that was associated with a decrease in microvessel density. GM-CSF treatment of HIF-1α(flox/flox)/LysMcre mice induced sVEGFR-1 but not VEGF, resulting in an overall greater reduction in tumor growth and angiogenesis compared with control mice. In addition, real-time PCR for melanoma-specific genes revealed a significantly reduced presence of lung micrometastases in HIF-1α(flox/flox)/LysMcre mice treated with GM-CSF. Conversely, GM-CSF treatment induced VEGF but not sVEGFR-1 in HIF-2α(flox/+)/LysMcre mice, and, correspondingly, GM-CSF did not decrease tumor growth, angiogenesis, or lung metastasis in these mice. This study reveals opposing roles for the HIFs in the regulation of angiogenesis by tumor-associated macrophages and suggests that administration of GM-CSF might be an effective means of inducing sVEGFR-1 and inhibiting tumor growth and angiogenesis in patients with melanoma.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Neoplasias Pulmonares/imunologia , Melanoma/imunologia , Proteínas de Neoplasias/imunologia , Neovascularização Patológica/imunologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Knockout , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Oxigênio/imunologia , Oxigênio/metabolismo , Consumo de Oxigênio/genética , Consumo de Oxigênio/imunologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 µg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.
Assuntos
Adenocarcinoma/terapia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias do Colo/terapia , Interferon gama/biossíntese , Interleucina-12/uso terapêutico , Células Matadoras Naturais/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais Humanizados , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Testes Imunológicos de Citotoxicidade , Feminino , Interferon gama/fisiologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Receptor ErbB-2/imunologia , Trastuzumab , Regulação para Cima/imunologiaRESUMO
Macrophages contribute to tumor growth through the secretion of the proangiogenic molecule vascular endothelial growth factor (VEGF). We previously observed that monocytes treated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produce a soluble form of the VEGF receptor-1 (sVEGFR-1), which neutralizes VEGF biologic activity. The VEGF and VEGFR-1 promoters both contain a hypoxia regulatory element, which binds the hypoxia-inducible factor (HIF) transcription factors under hypoxic conditions. Based on this observation, we examined VEGF and sVEGFR-1 production from monocytes cultured at various O(2) concentrations. The amount of sVEGFR-1 production observed from GM-CSF-treated monocytes increased with decreasing levels of O(2). This sVEGFR-1 was biologically active and sequestered VEGF. To evaluate the role of the HIFs in sVEGFR-1 production, we used macrophages with a genetic deletion of HIF-1α. HIF-1α(-/-) macrophages cultured with GM-CSF at hypoxia secreted diminished amounts of VEGF compared with HIF-1α(+/+) macrophages, whereas sVEGFR-1 secretion was unaffected. In contrast, siRNA-mediated knockdown of HIF-2α inhibited the production of sVEGFR-1 in response to GM-CSF and low O(2), whereas VEGF production was unaffected. These studies suggest that hypoxia, generally thought to promote angiogenesis, can induce antiangiogenic behavior from macrophages within a GM-CSF-rich environment. Furthermore, these results suggest specific and independent roles for HIF-1α and HIF-2α in hypoxic macrophages.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Endotélio Vascular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Hipóxia/metabolismo , Sistema Fagocitário Mononuclear/metabolismo , Neovascularização Fisiológica/fisiologia , Animais , Western Blotting , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5 , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
CD19 is a B cell-specific antigen expressed on chronic lymphocytic leukemia (CLL) cells but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified constant fragment (Fc)-domain designed to enhance binding of FcgammaRIIIa. Herein, we demonstrate that XmAb5574 mediates potent antibody-dependent cellular cytotoxicity (ADCC), modest direct cytotoxicity, and antibody-dependent cellular phagocytosis but not complement-mediated cytotoxicity against CLL cells. Interestingly, XmAb5574 mediates significantly higher ADCC compared with both the humanized anti-CD19 nonengineered antibody it is derived from and also rituximab, a therapeutic antibody widely used in the treatment of CLL. The XmAb5574-dependent ADCC is mediated by natural killer (NK) cells through a granzyme B-dependent mechanism. The NK cell-mediated cytolytic and secretory function with XmAb5574 compared with the nonengineered antibody is associated with enhanced NK-cell activation, interferon production, extracellular signal-regulated kinase phosphorylation downstream of Fcgamma receptor, and no increased NK-cell apoptosis. Notably, enhanced NK cell-mediated ADCC with XmAb5574 was enhanced further by lenalidomide. These findings provide strong support for further clinical development of XmAb5574 as both a monotherapy and in combination with lenalidomide for the therapy of CLL and related CD19(+) B-cell malignancies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Antígenos CD19/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Apoptose , Western Blotting , Citotoxicidade Imunológica/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Granzimas/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Leucemia de Células B/genética , Leucemia de Células B/imunologia , Leucemia de Células B/terapia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Fagocitose , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Our preclinical work showed a dramatic synergy between interleukin-12 (IL-12) and trastuzumab for stimulation of natural killer cell cytokine secretion. We aimed to determine the safety profile of IL-12 when given in combination with trastuzumab and paclitaxel to patients with metastatic HER2-overexpressing cancers. Paclitaxel was given i.v. at 175 mg/m(2) every 3 weeks. Trastuzumab was given on day 1 each week (4 mg/kg initially and 2 mg/kg thereafter) in combination with injections of IL-12 on days 2 and 5 starting in cycle 2. This trial accrued 21 patients with metastatic HER2-positive tumors (breast, 7; colon, 6; esophagus, 4; stomach, 2; pancreas, 1; thyroid, 1). The IL-12 component was dose-escalated in cohorts of three patients. The dose-limiting toxicity was grade 3 fatigue at the 300 ng/kg dose level in two patients. The recommended phase II dose was 200 ng/kg administered s.c. There was one complete response in a patient with breast cancer, partial responses in 4 patients (breast, 2; esophageal, 2), and stabilization of disease lasting 3 months or greater (SD) in 6 other patients. All but one response occurred in patients with HER2 3+ disease. Two SD patients completed 1 year of therapy. Ten patients had progressive disease. There was increased activation of extracellular signal-regulated kinase in peripheral blood mononuclear cells and increased levels of IFN-gamma and several chemokines in patients with clinical benefit (complete response, partial response, or SD), but not in patients with progressive disease. IL-12 in combination with trastuzumab and paclitaxel therefore exhibits an acceptable toxicity profile and has activity in patients with HER2-overexpressing cancers.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias/tratamento farmacológico , Receptor ErbB-2/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiocina CXCL10/sangue , Quimiocina CXCL9/sangue , Estudos de Coortes , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-12/administração & dosagem , Interleucina-12/efeitos adversos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias/enzimologia , Neoplasias/imunologia , Neoplasias/patologia , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Fosforilação , Trastuzumab , Resultado do TratamentoRESUMO
Trastuzumab mediates the lysis of HER2-expressing breast cancer cell lines by interleukin-2 (IL-2) primed natural killer (NK) cells. We hypothesized that IL-2 would augment the anti-tumor effects of trastuzumab in MBC in patients who had progressed on or within 12 months of receiving a trastuzumab-containing regimen. Secondary objectives were to measure antibody-directed cellular cytotoxicity (ADCC) against HER2 over-expressing target cells, and to measure serum cytokines. Patients received trastuzumab (4 mg/kg intravenously (IV)) every 2 weeks in combination with daily low-dose IL-2 (1 million IU/m(2) subcutaneously (SC)) and pulsed intermediate-dose IL-2 (12 million IU/m(2) SC). Samples were analyzed for NK cell expansion and ADCC against a HER2-positive breast cancer cell line. In addition, interferon-gamma (IFN-gamma), mRNA expression in peripheral blood mononuclear cells (PBMC) and the following serum cytokines were measured: IFN-gamma, monokine-induced by IFN-gamma (MIG), and interferon-inducible protein ten (IP-10). The median number of treatment cycles was four (range 1-23) and the treatment was well tolerated. There were no objective responses. NK cells were not expanded and ADCC was not enhanced. Eight (62%) patients had a twofold or higher increase in mRNA transcript for IFN-gamma, two (15%) patients had elevated serum levels of IFN-gamma and 12 (92%) had increases angiogenic MIG and IP-10. In trastuzumab-refractory patients adding IL-2 did not produce responses and did not result in NK cell expansion. However, these patients had the ability to respond to IL-2 as evidenced by increases in IFN-gamma transcripts and chemokines. The lack of NK cell expansion may explain the absence of clinical benefit.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Citotoxicidade Imunológica/efeitos dos fármacos , Terapia de Salvação/métodos , Adulto , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/imunologia , Quimiocina CXCL10/sangue , Quimiocina CXCL9/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-2/administração & dosagem , Interleucina-2/efeitos adversos , Interleucina-2/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Pessoa de Meia-Idade , RNA Mensageiro/análise , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TrastuzumabRESUMO
Interleukin-21 (IL-21) is a recently identified gamma-chain receptor cytokine family member that promotes B-cell apoptosis as well as activation of innate immune system. Based on this, we hypothesized that IL-21 might enhance the apoptosis induced by fludarabine and rituximab and also play a role in augmenting immune-mediated clearance of the chronic lymphocytic leukemia (CLL) cells. Our studies demonstrate that the majority of CLL patients have surface IL-21 receptor-alpha, and its expression correlates with apoptosis, tyrosine phosphorylation of STAT1, and up-regulation of the proapoptotic BH3 domain protein BIM. IL-21-induced BIM up-regulation is critical for apoptosis because inhibition of BIM expression using small interfering RNA prevented IL-21-induced apoptosis. IL-21 treatment of CLL cells but not normal T cells with fludarabine or rituximab additively enhanced the direct cytotoxic effect of these therapies. In addition to its proapoptotic effect, IL-21 promoted STAT1 and STAT5 phosphorylation in natural killer cells with concurrent enhanced antibody-dependent cellular cytotoxicity against rituximab-coated CLL cells in vitro. These data provide justification for combination studies of IL-21 with fludarabine and rituximab in CLL and suggest that BIM up-regulation might serve as relevant pharmacodynamic end point to measure biologic effect of this cytokine in vivo.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Proteínas Reguladoras de Apoptose/genética , Apoptose , Citotoxicidade Imunológica , Interleucinas/fisiologia , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Proteína 11 Semelhante a Bcl-2 , Humanos , Rituximab , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT5/metabolismo , Regulação para Cima/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Natural killer (NK) cells express an activating receptor for the Fc portion of IgG (FcgammaRIIIa) that mediates interferon (IFN)-gamma production in response to antibody (Ab)-coated targets. We have previously demonstrated that NK cells activated with interleukin-12 (IL-12) in the presence of immobilized IgG secrete 10-fold or more higher levels of IFN-gamma as compared with stimulation with either agent alone. We examined the intracellular signaling pathways responsible for this synergistic IFN-gamma production. NK cells costimulated via the FcR and the IL-12 receptor (IL-12R) exhibited enhanced levels of activated STAT4 and Syk as compared with NK cells stimulated through either receptor alone. Extracellular signal-regulated kinase (ERK) was also synergistically activated under these conditions. Studies with specific chemical inhibitors revealed that the activation of ERK was dependent on the activation of PI3-K, whose activation was dependent on Syk, and that sequential activation of these molecules was required for NK cell IFN-gamma production in response to FcR and IL-12 stimulation. Retroviral transfection of ERK1 into primary human NK cells substantially increased IFN-gamma production in response to immobilized IgG and IL-12, while transfection of human NK cells with a dominant-negative ERK1 abrogated IFN-gamma production. Confocal microscopy and cellular fractionation experiments revealed that FcgammaRIIIa and the IL-12R colocalized to areas of lipid raft microdomains in response to costimulation with IgG and IL-12. Chemical disruption of lipid rafts inhibited ERK signaling in response to costimulation and significantly inhibited IFN-gamma production. These data suggest that dual recruitment of FcgammaRIIIa and the IL-12R to lipid raft microdomains allows for enhanced activation of downstream signaling events that lead to IFN-gamma production.
Assuntos
Interferon gama/biossíntese , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Microdomínios da Membrana/enzimologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de IgG/imunologia , Receptores de Interleucina-12/imunologia , Colesterol/deficiência , Ativação Enzimática/efeitos dos fármacos , Humanos , Imunoglobulina G/farmacologia , Interleucina-12/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT4/metabolismo , Quinase Syk , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: Natural killer (NK) cells express an activating Fc receptor (FcgammaRIIIa) that mediates antibody-dependent cellular cytotoxicity (ADCC) and production of immune modulatory cytokines in response to antibody-coated targets. Cetuximab is a therapeutic monoclonal antibody directed against the HER1 antigen. We hypothesized that the NK cell response to cetuximab-coated tumor cells could be enhanced by the administration of NK cell-stimulatory cytokines. EXPERIMENTAL DESIGN: Human NK cells stimulated with cetuximab-coated tumor cells and interleukin-2 (IL-2), IL-12, or IL-21 were assessed for ADCC and secretion of IFN-gamma and T cell-recruiting chemokines. IL-21 and cetuximab were given to nude mice bearing HER1-positive xenografts. RESULTS: Stimulation of human NK cells with cetuximab-coated tumor cells and IL-2, IL-12, or IL-21 resulted in 3-fold to 10-fold higher IFN-gamma production than was observed with either agent alone. NK cell-derived IFN-gamma significantly enhanced monocyte ADCC against cetuximab-coated tumor cells. Costimulated NK cells also secreted elevated levels of chemokines (IL-8, macrophage inflammatory protein-1alpha, and RANTES) that could direct the migration of naive and activated T cells. IL-2, IL-12, and IL-21 enhanced NK cell ADCC against tumor cells treated with cetuximab. The combination of cetuximab, trastuzumab (an anti-HER2 monoclonal antibody), and IL-21 mediated greater NK cell cytokine secretion and ADCC than any agent alone. Furthermore, administration of IL-21 enhanced the effects of cetuximab in a murine tumor model. CONCLUSIONS: These results show that cetuximab-mediated NK cell activity can be significantly enhanced in the presence of NK cell-stimulatory cytokines. These factors, therefore, may be effective adjuvants to administer, in combination with cetuximab, to patients with HER1-positive malignancies.