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Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Responses to antibody-coated tumor cells are largely mediated by Fcγ receptors (FcγRs), which become activated upon binding to immune complexes. FcγRIIb is an inhibitory FcγR that negatively regulates these responses, and it is expressed on monocytes and macrophages. Therefore, deletion or down-regulation of this receptor may substantially enhance therapeutic outcomes. Here we screened a panel of Toll-like receptor (TLR) agonists and found that those selective for TLR4 and TLR8 could significantly down-regulate the expression of FcγRIIb. Upon further examination, we found that treatment of monocytes with TLR4 agonists could lead to the ubiquitination of FcγRIIb protein. A search of our earlier microarray database of monocytes activated with the TLR7/8 agonist R-848 (in which FcγRIIb was down-regulated) revealed an up-regulation of membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase. Therefore, we tested whether LPS treatment could up-regulate MARCH3 in monocytes and whether this E3 ligase was involved with LPS-mediated FcγRIIb down-regulation. The results showed that LPS activation of TLR4 significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the decrease in FcγRIIb following LPS treatment. These data suggest that activation of TLR4 on monocytes can induce a rapid down-regulation of FcγRIIb protein and that this involves ubiquitination.
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Proteínas de Transporte/metabolismo , Regulação para Baixo/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Receptores de IgG/biossíntese , Receptor 4 Toll-Like/agonistas , Ubiquitinação/efeitos dos fármacos , Regulação para Baixo/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptor 4 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases , Ubiquitinação/fisiologiaRESUMO
FcγRs are critical mediators of mAb cancer therapies, because they drive cytotoxic processes upon binding of effector cells to opsonized targets. Along with NK cells, monocytes are also known to destroy Ab-coated targets via Ab-dependent cellular cytotoxicity (ADCC). However, the precise mechanisms by which monocytes carry out this function have remained elusive. In this article, we show that human monocytes produce the protease granzyme B upon both FcγR and TLR8 activation. Treatment with TLR8 agonists elicited granzyme B and also enhanced FcγR-mediated granzyme B production in an additive fashion. Furthermore, monocyte-mediated ADCC against cetuximab-coated tumor targets was enhanced by TLR8 agonist treatment, and this enhancement of ADCC required granzyme B. Hence we have identified granzyme B as an important mediator of FcγR function in human monocytes and have uncovered another mechanism by which TLR8 agonists may enhance FcγR-based therapies.
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Citotoxicidade Celular Dependente de Anticorpos , Granzimas/metabolismo , Monócitos/metabolismo , Receptor 8 Toll-Like/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Angiotensinogênio/genética , Angiotensinogênio/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Western Blotting , Células Cultivadas , Cetuximab , Análise por Conglomerados , Relação Dose-Resposta a Droga , Granzimas/antagonistas & inibidores , Granzimas/genética , Humanos , Imidazóis/farmacologia , Interleucina-2/genética , Interleucina-2/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Perforina/genética , Perforina/metabolismo , Quinolinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologia , Fatores de Tempo , Receptor 8 Toll-Like/agonistas , Transcriptoma/efeitos dos fármacosRESUMO
BACKGROUND: Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10-20 % of patients. An immunological mechanism of action such as natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells. OBJECTIVE: To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells. METHODS: Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8(+) T cells were isolated and analyzed using (51)Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR853-861 tetramer staining. RESULTS: TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8(+) T cells in the presence of cetuximab. DISCUSSION: VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.
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Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Receptor 8 Toll-Like/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Benzazepinas/imunologia , Benzazepinas/farmacologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Cetuximab , Técnicas de Cocultura , Apresentação Cruzada/imunologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Citometria de Fluxo , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Subunidade p40 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/metabolismo , Células Matadoras Naturais/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Antígeno CD83RESUMO
Myeloid-derived suppressor cells (MDSCs) accumulate in tumors and the peripheral blood of cancer patients and demonstrate cancer-promoting activity across multiple tumor types. A limited number of agents are known to impact MDSC activity. TLR8 is expressed in myeloid cells. We investigated expression of TLR8 on MDSC and the effect of a TLR8 agonist, motolimod, on MDSC survival and function. TLR8 was highly expressed in monocytic MDSC (mMDSC) but absent in granulocytic MDSC (gMDSC). Treatment of human PBMC with motolimod reduced the levels of mMDSC in volunteers and cancer donors versus control (P < 0.001). Motolimod did not impact levels of gMDSC. The reduction of mMDSC was due to induced cell death by TLR8 ligation. Pretreatment of PBMC with a FAS neutralizing antibody inhibited motolimod-induced reduction of mMDSC (P < 0.001). Finally, we demonstrated that mMDSC impeded IL-2 secretion by CD3/CD28-activated T cells; IL-2 secretion was partially restored when cells were cocultured with motolimod (142 ± 36 pg/ml vs. 59 ± 13 pg/ml; P = 0.03). There is increasing evidence that MDSCs contribute to the progression of cancer by inhibiting tumor-directed T cells. TLR8 agonists may synergize with cancer immunotherapeutic approaches to enhance the antitumor effects of the adaptive immune response.
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Apoptose , Leucócitos Mononucleares/patologia , Monócitos/patologia , Células Supressoras Mieloides/patologia , Neoplasias/patologia , Receptor 8 Toll-Like/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Monócitos/imunologia , Monócitos/metabolismo , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismoRESUMO
Importance: Immunotherapy for recurrent and/or metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN) is promising. The toll-like receptor 8 (TLR8) agonist motolimod may stimulate innate and adaptive immunity. Objective: To determine whether motolimod improves outcomes for R/M SCCHN when combined with standard therapy. Design, Setting, and Participants: The Active8 study was a multicenter, randomized, double-blind, placebo-controlled clinical trial enrolling adult patients (age ≥18 years) with histologically confirmed R/M SCCHN of the oral cavity, oropharynx, hypopharynx, or larynx between October 2013 and August 2015. Follow-up ended September 2016. Analysis for the present report was conducted between June 2016 and December 2017. Interventions: Combination treatment with platinum (carboplatin or cisplatin), fluorouracil, cetuximab (the EXTREME regimen), and either placebo or motolimod, each administered intravenously every 3 weeks. Patients received a maximum of 6 chemotherapy cycles, after which patients received weekly cetuximab with either placebo or motolimod every 4 weeks. Main Outcomes and Measures: Progression-free survival (PFS) as determined by independent central review using immune-related RECIST (Response Evaluation Criteria in Solid Tumors). Key secondary end points included overall survival (OS) and safety. Results: Of 195 patients enrolled, 85% were men (n = 166); 82% were white (n = 159); median age was 58 years (range 23-81 years). Median PFS was 6.1 vs 5.9 months (hazard ratio [HR], 0.99; 1-sided 90% CI, 0.00-1.22; P = .47), and median OS was 13.5 vs 11.3 months (HR, 0.95; 1-sided 90% CI, 0.00-1.22; P = .40) for motolimod vs placebo. Increased incidence of injection site reactions, pyrexia, chills, anemia, and acneiform rash were noted with motolimod. Of 83 cases oropharyngeal cancer, 52 (63%) were human papillomavirus (HPV) positive. In a prespecified subgroup analysis of HPV-positive participants, motolimod vs placebo resulted in significantly longer PFS (7.8 vs 5.9 months; HR, 0.58; 1-sided 90% CI, 0.00-0.90; P = .046) and OS (15.2 vs 12.6 months; HR, 0.41; 1-sided 90% CI, 0.00-0.77; P = .03). In an exploratory analysis, patients with injection site reactions had longer PFS and OS (median PFS, 7.1 vs 5.9 months; HR, 0.69; 1-sided 90% CI, 0.00-0.93; P = .06; and median OS, 18.7 vs 12.6; HR, 0.56; 1-sided 90% CI, 0.00-0.81; P = .02). Conclusions and Relevance: Adding motolimod to the EXTREME regimen was well tolerated but did not improve PFS or OS in the intent-to-treat population. Significant benefit was observed in HPV-positive patients and those with injection site reactions, suggesting that TLR8 stimulation may benefit subset- and biomarker-selected patients. Trial Registration: ClinicalTrials.gov identifier: NCT01836029.
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Antineoplásicos Imunológicos/uso terapêutico , Benzazepinas/uso terapêutico , Cetuximab/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/farmacologia , Benzazepinas/farmacologia , Cetuximab/farmacologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Adulto JovemRESUMO
Purpose: As Toll-like receptors (TLR) are key mediators of immune responses, TLR agonists may be important for augmenting the efficacy of therapies for squamous cell carcinoma of the head and neck (SCCHN). Motolimod (VTX-2337), a selective small-molecule agonist of TLR8, stimulates natural killer (NK) cells, dendritic cells, and monocytes. A phase Ib clinical trial assessed the safety and antitumor activity of motolimod in combination with cetuximab in patients with SCCHN. Correlative biomarkers of immune activity were explored.Experimental Design: Thirteen patients with recurrent or metastatic SCCHN were enrolled in this open-label, dose-escalation study using a standard 3 + 3 design. Doses of motolimod (2.5, 3.0, or 3.5 mg/m2) were given on days 1, 8, and 15, in combination with fixed weekly doses of cetuximab in 28-day cycles.Results: There were no protocol-defined dose-limiting toxicities, drug-related deaths, or evidence of synergistic toxicities between motolimod and cetuximab. Clinical tolerability at the 3.5 mg/m2 dose level was not optimal for repeated dosing and 3.0 mg/m2 was identified as the MTD. Two patients achieved partial responses for an overall response rate of 15%. Five patients had disease stabilization equating to a disease control rate of 54%. Statistically significant increases in plasma cytokines and in the frequency and activation of circulating NK cells were observed.Conclusions: Motolimod can be safely administered in combination with cetuximab with an acceptable toxicity profile. Encouraging antitumor activity and robust pharmacodynamic responses were observed. Motolimod is being further investigated in a phase II trial in patients with SCCHN (ClinicalTrials.gov ID: NCT01836029). Clin Cancer Res; 23(10); 2442-50. ©2016 AACR.
Assuntos
Benzazepinas/administração & dosagem , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Receptor 8 Toll-Like/genética , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Benzazepinas/efeitos adversos , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/genética , Cetuximab/administração & dosagem , Intervalo Livre de Doença , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/classificação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/genética , Humanos , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/patologia , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
A novel approach to immunotherapy is the activation of toll-like receptor 8 (TLR8). Motolimod, a selective TLR8 agonist can act in concert with approved immunotherapies to sensitize T cells and augment natural killer (NK) cell function. Despite treatment with chemotherapeutic agents and advance disease, cancer patients remain sensitive to motolimod.
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VTX-1463 is a selective toll-like receptor (TLR) 8 agonist that activates a subset of innate immune cells to produce a unique cytokine profile. Delivery of VTX-1463 via nasal spray may modulate the nasal response in allergic rhinitis. The aim of this study was to determine the effects of VTX-1463 on the nasal response in a dog model of allergic rhinitis. Ragweed (RW)-sensitized dogs were pretreated with increasing doses of VTX-1463 1 day prior to RW challenge or with two doses (4 or 8 days and 1 day prior to RW). Changes in nasal cavity volume (NV) were determined by acoustic rhinometry and nasal lavage fluid was assessed for histamine, lipid mediators, and cellular infiltrates at sequential times following RW challenge. VTX-1463 pretreatment significantly preserved NV during the acute response to RW challenge for all doses tested. The area under the curve (AUC) for NV over the 1.5 h assessment period in RW challenged vehicle controls averaged 51.5% (SEM: ±2.12%) of the baseline NV over all studies. A single 100 µg dose of VTX-1463 given 1 day prior to RW yielded an AUC for NV of 69.3% (±6.59%) of baseline, while a 1000 µg dose administered twice (8 days and 1 day prior to RW) resulted in an AUC for NV of 85.4% (±4.74%, P < 0.05) of baseline. For a single 1000 µg VTX-1463 dose 1 day prior to RW, multiple mediators produced by mast cells, including histamine, PGE2, PGD2, and cysteinyl LTs, were significantly reduced relative to the vehicle control. The selective TLR8 agonist, VTX-1463, preserved NV in a dose-dependent manner in the acute phase of a nasal allergic response. The therapeutic effect appears to result from attenuated mast cell mediator release. Modulating the local cytokine response via TLR8 agonism appears to have a therapeutic effect on the acute allergic nasal response.
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UNLABELLED: VTX-2337 (USAN: motolimod) is a selective toll-like receptor 8 (TLR8) agonist, which is in clinical development as an immunotherapy for multiple oncology indications, including squamous cell carcinoma of the head and neck (SCCHN). Activation of TLR8 enhances natural killer cell activation, increases antibody-dependent cell-mediated cytotoxicity, and induces Th1 polarizing cytokines. Here, we show that VTX-2337 stimulates the release of mature IL-1ß and IL-18 from monocytic cells through coordinated actions on both TLR8 and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome complex. In vitro, VTX-2337 primed monocytic cells to produce pro-IL-1ß, pro-IL-18, and caspase-1, and also activated the NLRP3 inflammasome, thereby mediating the release of mature IL-1ß family cytokines. Inhibition of caspase-1 blocked VTX-2337-mediated NLRP3 inflammasome activation, but had little impact on production of other TLR8-induced mediators such as TNFα. IL-18 activated natural killer cells and complemented other stimulatory pathways, including FcγRIII and NKG2D, resulting in IFNγ production and expression of CD107a. NLRP3 activation in vivo was confirmed by a dose-related increase in plasma IL-1ß and IL-18 levels in cynomolgus monkeys administered VTX-2337. These results are highly relevant to clinical studies of combination VTX-2337/cetuximab treatment. Cetuximab, a clinically approved, epidermal growth factor receptor-specific monoclonal antibody, activates NK cells through interactions with FcγRIII and facilitates ADCC of tumor cells. Our preliminary findings from a Phase I open-label, dose-escalation, trial that enrolled 13 patients with recurrent or metastatic SCCHN show that patient NK cells become more responsive to stimulation by NKG2D or FcγRIII following VTX-2337 treatment. Together, these results indicate that TLR8 stimulation and inflammasome activation by VTX-2337 can complement FcγRIII engagement and may augment clinical responses in SCCHN patients treated with cetuximab. TRIAL REGISTRATION: ClinicalTrials.gov NCT01334177.
Assuntos
Benzazepinas/farmacologia , Proteínas de Transporte/agonistas , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Receptor 8 Toll-Like/agonistas , Animais , Benzazepinas/uso terapêutico , Caspase 1/metabolismo , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Inflamassomos/metabolismo , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Células K562 , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Macaca fascicularis , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR , Neoplasias/genética , Receptores de IgG/metabolismoRESUMO
PURPOSE: Immunotherapy as a treatment for cancer holds the promise of complete and durable tumor remission, yet the immunosuppressive environment created by many tumors, advanced patient age, and previous treatments with cytotoxic agents may limit the approach. The activity of motolimod (VTX-2337), a potent and selective Toll-like receptor 8 (TLR8) agonist, was therefore assessed in the context of advanced, late-stage cancer patients. EXPERIMENTAL DESIGN: The repertoire of mediators induced from human peripheral blood mononuclear cells in response to motolimod was characterized. Translational studies in cynomolgus monkeys elucidated the activity of motolimod on an intact immune system, identified biomarkers of TLR8 activation, and defined the relationship between the pharmacokinetic and pharmacodynamic (PK/PD) response. The PK/PD relationship for motolimod in cancer patients was assessed, compared with preclinical findings, and contrasted with activity in healthy volunteers. RESULTS: In late-stage cancer patients, plasma levels of multiple biomarkers, including IL6, G-CSF, MCP-1, and MIP1-ß, increased with increasing motolimod dose. The magnitude and breadth of the biomarker response closely aligned with the response seen in preclinical studies, demonstrating that advanced cancer patients remained responsive to TLR8 activation. In addition, the PK/PD response in cancer patients closely aligned with the activity of motolimod seen in healthy volunteers. CONCLUSIONS: Late-stage cancer patients are highly sensitive to TLR8 activation by motolimod. Tumor burden, advanced age, and prior treatment history with cytotoxic agents did not moderate or modify the response predicted by nonclinical studies and confirmed in healthy volunteers. Clin Cancer Res; 21(24); 5445-52. ©2015 AACR.
Assuntos
Antineoplásicos/uso terapêutico , Benzazepinas/uso terapêutico , Imunomodulação/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor 8 Toll-Like/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Benzazepinas/farmacologia , Estudos de Casos e Controles , Terapia Combinada , Citocinas/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Imunoterapia , Contagem de Leucócitos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 8 Toll-Like/metabolismo , Resultado do TratamentoRESUMO
OBJECT: Adhesion of leukocytes and their migration into the periadventitial space may be critical in the pathophysiology of vasospasm following subarachnoid hemorrhage (SAH). The cell adhesion molecules involved in this process are lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/CD18), which are present on neutrophils/macrophages, and intercellular adhesion molecule-1 (CD54), which is present in endothelial cells. A humanized monoclonal antibody (mAb), Hu23F2G, targets CD11/CD18 and prevents leukocyte adhesion to endothelial cells. In this study, systemic administration of Hu23F2G prevented vasospasm in the rabbit model of SAH. METHODS: Twenty-six New Zealand White rabbits were injected with autologous blood into the cisterna magna to induce SAH, after which they were randomized to receive injections of either Hu23F2G (10 animals) or a placebo at 30 minutes and 24 and 48 hours after SAH (six animals). Control animals underwent sham operations (four animals) or SAH alone (six animals). The animals were killed 72 hours after SAH, their bodies perfused and fixed, and their basilar arteries processed for morphometric analysis. Peripheral white blood cells (WBCs) were counted at 72 hours. The percentages of lumen patency were compared using the Student t-test. The presence of neutrophils and macrophages was confirmed by immunohistochemical analysis in which a rat anti-rabbit anti-CD18 mAb and cresyl violet were used. Treatment with Hu23F2G resulted in the significant prevention of vasospasm. Animals treated with Hu23F2G had 90 +/- 7% lumen patency compared with 65 +/- 7% in the placebo group (p = 0.025). The percentage of lumen patency in the SAH-only group was 59 +/- 10%. The mean WBC count was 16,300 +/- 2710/microl in the treatment group, compared with 7000 +/- 386/microl in the control group (p = 0.02). Administration of Hu23F2G produced increased numbers of WBCs in 70% of the animals treated. CONCLUSIONS: This study supports the concept that leukocyte-endothelial cell interactions play an important role in the pathophysiology of chronic vasospasm after SAH. Systemic therapy with an anti-CD11/CD18 mAb prevents vasospasm after SAH by inhibiting adhesion of neutrophils and macrophages and their migration into the periadventitial space.
Assuntos
Anticorpos Monoclonais/farmacologia , Hemorragia Subaracnóidea/complicações , Grau de Desobstrução Vascular/efeitos dos fármacos , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/prevenção & controle , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Artéria Basilar/efeitos dos fármacos , Modelos Animais de Doenças , Contagem de Leucócitos , Coelhos , Hemorragia Subaracnóidea/sangue , Vasoespasmo Intracraniano/sangueRESUMO
OBJECT: Leukocyte-endothelial cell interactions occurring in the first hours after subarachnoid hemorrhage (SAH) initiate changes in the endothelium and vessel wall that lead to an influx of leukocytes and the development of chronic vasospasm days later. Upregulation of intercellular adhesion molecule-1 (ICAM-1), also called CD54, appears to be a crucial step in this process. There is increasing experimental evidence that blocking the interaction between ICAM-1, which is expressed on endothelium, and integrins such as lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (complement receptor 3, CD11b/CD18), which are expressed on the surface of leukocytes,prevents not only inflammation of vessel walls but also chronic vasospasm. The authors extend their previous work with monoclonal antibody (mAb) blockade of leukocyte migration to a nonhuman primate model of chronic, posthemorrhagic cerebral vasospasm. METHODS: Before surgery was performed, six young adult male cynomolgus monkeys underwent baseline selective biplane common carotid and vertebrobasilar artery cerebral angiography via a transfemoral route. On Day 0, a right frontosphenotemporal craniectomy was performed with arachnoid microdissection and placement of 2 to 3 ml of clotted autologous blood in the ipsilateral basal cisterns. The animals were given daily intravenous infusions of 2 mg/kg of either a humanized anti-CD11/CD18 or a placebo mAb beginning 30 to 60 minutes postoperatively. The monkeys were killed on Day 7 after a repeated selective cerebral angiogram was obtained. The area of contrast-containing vessels observed in each hemisphere on anteroposterior angiographic views was calculated for the angiograms obtained on Day 7 and expressed as a percentage of the area on baseline angiograms (percent control areal fraction). Review of flow cytometry and enzyme immunoassay data confirmed the presence of the anti-CD11/CD18 antibody in the serum and bound to leukocytes in the peripheral blood of treated animals. Comparisons of the groups revealed 53 +/- 4.8% control vascular areal fraction in the placebo group (two animals) and 95.8 +/- 9.4% in the anti-CD11/CD18-treated group (three animals), a statistically significant difference (p = 0.043, t-test). CONCLUSIONS: These results show that blockade of leukocyte migration into the subarachnoid space by an anti-CD11/CD18 mAb is effective in preventing experimental cerebral vasospasm in nonhuman primates, despite the unaltered presence of hemoglobin in the subarachnoid space. These experimental data support the hypothesis that inflammation plays a role in cerebral vasospasm after SAH.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos CD11/imunologia , Antígenos CD18/imunologia , Hemorragia Subaracnóidea/complicações , Regulação para Cima/efeitos dos fármacos , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/prevenção & controle , Animais , Movimento Celular/efeitos dos fármacos , Angiografia Cerebral , Endotélio Vascular/metabolismo , Citometria de Fluxo/métodos , Molécula 1 de Adesão Intercelular/metabolismo , Macaca fascicularis , Masculino , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/metabolismo , Fatores de Tempo , Vasoespasmo Intracraniano/diagnóstico por imagemRESUMO
OBJECT: The authors have previously shown that a monoclonal antibody (mAb) that recognizes intercellular adhesion molecule-1 (ICAM-1), also known as CD54, when administered systemically inhibits experimental vasospasm in a rat femoral artery model, suggesting that ICAM-1 and leukocyte-endothelial adhesion play a crucial role in the molecular chain of events leading to posthemorrhagic vasospasm. In this report the authors confirm this hypothesis with mAbs directed against lymphocyte function-associated antigen-1 ([LFA-1] CD11a/CD18), the molecule on the surface of leukocytes that interacts with ICAM-1. METHODS: Femoral arteries in 38 Sprague-Dawley rats were isolated and exposed to autologous blood. Twenty-nine animals were then randomized into three groups and received intraperitoneal injections of anti-LFA-1 mAb (10 rats), anti-ICAM-1 mAb (10 rats), or an isotype-matched control mAb (nine rats). Injections were administered at 3 hours and 3, 6, and 9 days after surgery. Before their deaths, six animals underwent spleen harvest, and splenocytes were used in fluorescence-activated cell sorter (FACS) analysis to verify saturation of appropriate binding sites. Animals were killed at 12 days and vessels were harvested for histological study and measurement of the luminal cross-sectional area. Nine animals were randomized as earlier, killed 24 hours after a single injection of mAb, and evaluated for periadventitial infiltration of granulocytes and macrophages. Results of FACS analysis demonstrated saturation of both LFA-1 and ICAM-1 binding sites in animals treated with the respective mAb. The mean ratios of blood-exposed to saline-exposed luminal cross-sectional areas (expressed as the percentage of lumen patency) were 90.1 +/- 5.8% (mean +/- standard error of the mean) for animals treated with the anti-LFA-1 mAb (p = 0.0218), 94.2 +/- 3.3% for animals treated with the anti-ICAM-1 mAb (p = 0.0067), and 62 +/- 7.4% for animals treated with the isotype-matched control mAb. Macrophage and granulocyte counts in the periadventitial region were 39.5 +/- 3.2/hpf for animals treated with anti-LFA-1 mAb (p = 0.001), 42 +/- 3.7/hpf for animals treated with anti-ICAM-1 mAb (p = 0.003), and 72.2 +/- 6.2/hpf for control animals. CONCLUSIONS: The systemic administration of anti-LFA-1 or anti-ICAM-1 mAb initiated 3 hours after exposure to autologous blood inhibits the development of delayed chronic vasospasm at 12 days in a rat femoral artery model and leads to a significant reduction in periadventitial inflammatory cells at 24 hours. The authors conclude that blocking the migration of inflammatory cells across the endothelial surface of an artery after adventitial exposure to blood prevents the initiation of biological cascades necessary for the subsequent development of chronic vasospasm.
Assuntos
Anticorpos Monoclonais/farmacologia , Artéria Femoral/fisiologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Vasoconstrição/imunologia , Vasoespasmo Intracraniano/terapia , Animais , Proteínas Sanguíneas/farmacologia , Modelos Animais de Doenças , Citometria de Fluxo , Granulócitos/imunologia , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Macrófagos/imunologia , Masculino , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem , Hemorragia Subaracnóidea/imunologia , Hemorragia Subaracnóidea/terapia , Vasoespasmo Intracraniano/imunologiaRESUMO
PURPOSE: This phase I, open-label, uncontrolled, ascending-dose study explored the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacology of the TLR8 agonist VTX-2337 in subjects with advanced solid tumors or lymphoma. EXPERIMENTAL DESIGN: VTX-2337 doses (0.1-3.9 mg/m(2)) were administered subcutaneously on days 1, 8, and 15 of each 28-day cycle. Safety/tolerability assessments included adverse events (AE); physical, ophthalmologic, and laboratory evaluations; and electrocardiograms. Dose-limiting toxicities (DLT) were evaluated during the first cycle. Pharmacokinetics were evaluated after the first dose. Plasma samples were quantitatively assessed for chemokines, cytokines, and other inflammatory mediators. Antitumor activity was assessed. RESULTS: Thirty-three subjects were enrolled in 8 cohorts and received an average of 2 treatment cycles (range, 1-8 cycles). Most AEs were grades 1 to 2; the most common drug-related AEs were injection site reactions, chills, pyrexia, and influenza-like illness. One DLT was reported: grade 3 hypotension (3.9 mg/m(2)). The MTD was considered the highest dose administered. Peak drug plasma levels and total systemic exposure were generally dose proportional. At doses ≥0.4 mg/m(2), increases above baseline levels were observed for plasma levels of G-CSF, monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, and TNFα. Eight subjects (24.2%) had a best response of stable disease (median duration, 54.5 days). CONCLUSIONS: VTX-2337 is clinically well tolerated and biologically active with a predictable pharmacokinetic profile. Suitable doses for testing in combination studies were identified. Phase II placebo-controlled studies of VTX-2337 in combination with doxorubicin in ovarian cancer, and in combination with platinum chemotherapy, 5 FU, and cetuximab in head and neck cancer have been initiated (NCT #01666444 and NCT#01836029).
Assuntos
Antineoplásicos/uso terapêutico , Benzazepinas/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Linfoma/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Benzazepinas/farmacocinética , Benzazepinas/toxicidade , Neoplasias Colorretais/patologia , Feminino , Humanos , Injeções Subcutâneas , Linfoma/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Receptor 8 Toll-Like/agonistas , Resultado do TratamentoRESUMO
BACKGROUND: Newborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed. METHODS: TLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-κB reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry). RESULTS: VTX-294 was ≈ 100x more active on TLR8- than TLR7-transfected HEK cells (EC50, ≈ 50 nM vs. ≈ 5700 nM). VTX-294-induced TNF and IL-1ß production were comparable in newborn cord and adult peripheral blood, while VTX-294 was 1 log more potent in inducing TNF and IL-1ß production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1ß responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs. CONCLUSIONS: VTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood.
Assuntos
Benzazepinas/farmacologia , Leucócitos/efeitos dos fármacos , Receptor 8 Toll-Like/agonistas , Adulto , Análise de Variância , Células Dendríticas/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Imidazóis/farmacologia , Recém-Nascido , Leucócitos/fisiologia , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Quinolinas/farmacologia , Tiazóis/farmacologiaRESUMO
PURPOSE: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody-based immunotherapy that includes the activation of natural killer (NK) cells. EXPERIMENTAL DESIGN: HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibody-dependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcγR3A single-nucleotide polymorphism (SNP). RESULTS: VTX-2337 selectively activates TLR8 with an EC(50) of about 100 nmol/L and stimulates production of TNFα and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNγ production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNγ production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcγR3A genotypes (V/V, V/F, and F/F at position 158). CONCLUSIONS: VTX-2337 is a novel small-molecule TLR8 agonist that activates monocytes, DCs, and NK cells. Through the activation of NK cells, it has the potential to augment the effectiveness of monoclonal antibody treatments where a polymorphism in FcγR3A limits clinical efficacy.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Benzazepinas/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Receptor 8 Toll-Like/agonistas , Aminoquinolinas/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Sinergismo Farmacológico , Células HEK293 , Humanos , Imiquimode , Mediadores da Inflamação/metabolismo , Interferon gama/biossíntese , Interleucina-12/biossíntese , Interleucina-12/metabolismo , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , NF-kappa B/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Polimorfismo de Nucleotídeo Único , Receptores de IgG/genética , Receptores de IgG/metabolismo , Rituximab , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The primary toxicity associated with repeated oral administration of the PDE4 inhibitor IC542 to the rat is an inflammatory response leading to tissue damage primarily in the gastrointestinal tract and mesentery. Although necrotizing vasculitis is frequently seen with other PDE4 inhibitors, blood vessel injury was rare following IC542 administration and was always associated with inflammation in the surrounding tissue. The incidence and severity of the histologic changes in these studies correlated with elevated peripheral blood leukocytes, serum IL-6, haptoglobin, and fibrinogen, and with decreased serum albumin. By monitoring haptoglobin, fibrinogen and serum albumin changes in IC542-treated rats, it was possible to identify rats with early histologic changes that were reversible. Since PDE4 inhibition is generally associated with anti-inflammatory activity, extensive inflammation in multiple tissues was unexpected with IC542. Co-administration of dexamethasone completely blocked IC542-induced clinical and histologic changes in the rat, confirming the toxicity resulted from inflammatory response. In addition, IC542 augmented release of the proinflammatory cytokine IL-6 in LPS-activated whole blood from rats but not monkeys or humans. The effect of IC542 on IL-6 release from rat leukocytes in vitro is consistent with the proinflammatory response observed in vivo and demonstrates species differences to PDE4 inhibition.