RESUMO
CTLA4-Ig is a potent costimulatory blocker that inhibits T cell activation during alloimmune inflammation and increases graft survival and function. CTLA4-Ig-mediated immunosuppression has been demonstrated to support transplant function in various clinical trials and preclinical settings, but its effects on the balance between regulatory T cells (Tregs) and effector T cells (Teffs), as well as complement activation, are less well investigated. In the present study, we proposed to investigate the effects of CTLA4-Ig mediated immunosuppression on the phase of immunotolerance and the subsequent graft microvascular and epithelial repair during the progression of subepithelial fibrosis in a mouse model of orthotopic trachea transplantation. Briefly, CTLA4-Ig treated allografts (2 mg/kg, I.P.), untreated allografts, and syngrafts were serially monitored for peripheral FOXP3+ Tregs, antibody-mediated complement activation (C3d and C4d), tissue oxygenation, donor-recipient microvascular blood flow, and subsequent tissue remodeling following transplantation. Our data demonstrate that CTLA4-Ig mediated immunosuppression significantly results in late increases in both peripheral CD4+/CD8+ FOXP3+ Tregs and serum IL-10, but prevents the microvascular deposition of IgG, complement factor C3d, and epithelial C4d respectively, which proportionally improved blood flow and tissue oxygenation in the graft and, thus, promotes graft repair. Also, it restored the airway lumen, epithelium, and prevented the progression of subepithelial collagen deposition up to 90 days after transplantation. This study demonstrates that CTLA4-Ig-mediated immunosuppression potentially modulates both effector response and a late surge of regulatory activity to preserve graft microvasculature and rescue allograft from sustained hypoxia and ischemia and thereby limits subepithelial fibrosis.
Assuntos
Antígeno CTLA-4 , Rejeição de Enxerto , Sobrevivência de Enxerto , Abatacepte/farmacologia , Abatacepte/uso terapêutico , Animais , Antígeno CTLA-4/administração & dosagem , Antígeno CTLA-4/imunologia , Fibrose , Fatores de Transcrição Forkhead , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologia , Traqueia/transplanteRESUMO
Cytotoxic T-lymphocyte antigen 4 (CTLA4)-FasL, a homo-hexameric signal converter protein, is capable of inducing robust apoptosis in malignant cells of the B-cell lineage expressing its cognate B7 and Fas targets, while sparing nonmalignant ones. This fusion protein's striking proapoptotic efficacy stems from its complementary abilities to coordinately activate apoptotic signals and abrogate antiapoptotic ones. A limiting factor in translating FasL or Fas receptor agonists into the clinic has been lethal hepatotoxicity. Here, we establish CTLA4-FasL's in vivo efficacy in multiple murine and xenograft models, for both systemic and subcutaneous tumors. Significantly, good laboratory practice (GLP) toxicology studies in mice indicate that CTLA4-FasL given repeatedly at doses up to five times the effective dose was well-tolerated and resulted in no significant adverse events. An equivalent single dose of CTLA4-FasL administered to nonhuman primates was also well-tolerated, albeit with a moderate dose-dependent leukopenia that was completely reversible. Interestingly, monkey peripheral blood mononuclear cells were more sensitive to CTLA4-FasL-induced apoptosis when tested in vitro. In both species, there was short-term elevation in serum levels of IL6, IL2, and IFNγ, although this was not associated with clinical signs of proinflammatory cytokine release, and further, this cytokine elevation could be completely prevented by dexamethasone premedication. Liver toxicity was not observed in either species, as confirmed by serum liver enzyme levels and histopathologic assessment. In conclusion, CTLA4-FasL emerges from animal model studies as an effective and safe agent for targeted FasL-mediated treatment of B7-expressing aggressive B-cell lymphomas.
Assuntos
Antígeno CTLA-4/administração & dosagem , Proteína Ligante Fas/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Animais , Antígeno CTLA-4/imunologia , Proteína Ligante Fas/efeitos adversos , Proteína Ligante Fas/imunologia , Proteína Ligante Fas/farmacocinética , Feminino , Humanos , Células Jurkat , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Primatas , Distribuição Aleatória , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The driver and passenger mutations accumulated in the process of malignant transformation offer an adequate spectrum of immune visible alterations to the cellular proteome and resulting peptidome to render these cancers targetable-and, in theory, rejectable-by the host T cell immune response. In addition, cancers often overexpress tissue-specific and developmental antigens to which immune tolerance is incomplete. Sometimes, virally transferred oncogenes drive malignant transformation and remain expressed throughout the cancer. Despite this state of antigenic sufficiency, cancer grows progressively and overcomes all efforts of the host immune system to contain it. While therapeutic cancer vaccination can mobilize high frequencies of tumor-specific T cells, these responses remain subject to intratumoral attenuation. Antibody modulation of T cell function through checkpoint blockade or costimulatory activation can restore survival, proliferation, and effector function to these tumor-infiltrating T cells and convert otherwise subtherapeutic vaccines into potentially curative cancer immunotherapeutics.
Assuntos
Antígeno CTLA-4/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Animais , Antígeno CTLA-4/imunologia , Vacinas Anticâncer/imunologia , Transformação Celular Neoplásica/imunologia , Feminino , Previsões , Humanos , Fatores Imunológicos/administração & dosagem , Masculino , Neoplasias/patologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Medição de Risco , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
BACKGROUND AND OBJECTIVE: Immuno-oncology therapies represent a new treatment opportunity for patients affected by metastatic melanoma. The purpose of this study was to estimate the costs of immune-related adverse events (irAEs) associated with the new anti-PD1 immuno-oncology therapies, with the anti-CTLA-4 immuno-oncology therapy and with the combined therapy (CTLA4 + anti-PD1) in patients affected by metastatic melanoma. MATERIALS AND METHODS: A probabilistic cost-of-illness (COI) model was developed to estimate the management costs of grade ≥ 3 adverse events associated with the new anti-PD1 therapies (pembrolizumab and nivolumab), the anti-CTLA-4 therapy (ipilimumab) and the combined therapy CTLA4 + anti-PD1 (nivolumab + ipilimumab) for the treatment of patients with metastatic melanoma from the National Health Service (NHS) perspective in Italy. Identification of the epidemiological and cost parameters was carried out through a systematic literature review (SLR). Univariate and probabilistic sensitivity analyses were performed to account for uncertainty and variation in the model results. RESULTS: The model estimated a cost associated with the management of grade ≥ 3 immune-related adverse events in patients with metastatic melanoma equal to 176.2 (95% CI 63.5-335.0) for anti-CTLA-4 therapy, 48.6 (95% CI 40.1-58.5) for the new anti-PDI therapies and 276.8 (95% CI 240.4-316.2) for the combined therapy. Among the innovative therapies for the considered metastatic melanoma, the combined therapy was the most expensive innovative treatment in terms of event management of immune-related grade ≥ 3 adverse events. CONCLUSION: This study may represent a useful tool to understand the economic burden associated with the management of irAEs associated with patients affected by metastatic melanoma.
Assuntos
Antineoplásicos Imunológicos/economia , Custos e Análise de Custo/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/economia , Melanoma/economia , Terapias em Estudo/economia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/economia , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Antígeno CTLA-4/administração & dosagem , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Humanos , Ipilimumab/administração & dosagem , Ipilimumab/efeitos adversos , Ipilimumab/economia , Itália/epidemiologia , Melanoma/tratamento farmacológico , Melanoma/epidemiologia , Nivolumabe/administração & dosagem , Nivolumabe/efeitos adversos , Nivolumabe/economia , Terapias em Estudo/efeitos adversosRESUMO
Cytotoxic Tlymphocyte antigen4 (CTLA4) is a critical negative regulator of immune responses. CTLA4 is rapidly upregulated following Tcell activation, and then binds to B7 molecules with a higher affinity than CD28. CTLA4 may abolish the initiation of the responses of T cells by raising the threshold of signals required for full activation of T cells, and it also may terminate ongoing T-cell responses. This regulatory role has led to the development of monoclonal antibodies (mAbs) designed to block CTLA4 activity for enhancing immune responses against cancer. mAbs have several disadvantages including high production cost and unstable behavior. Nanobodies (Nbs) are singledomain antigenbinding fragments derived from the camelid heavychain antibodies, which are highly attractive in cancer immunotherapy due to their small size, high specificity, and stability. We selected CTLA4specific Nbs from a high quality dromedary camel immune library by phage display technology. Four positive colonies were sequenced and classified based on the amino acids sequences in the CDR3 region. These Nbs recognized unique epitopes on CTLA4 and displayed high binding rates when used on PHAstimulated human T cells. Treatment of B16 melanomabearing C57BL/6 mice with antiCTLA4 nanobody 16 (Nb16) delayed melanoma growth and prolonged the survival time of mice. These data indicate that antiCTLA4 Nbs selected from a high quality phage display library may be effective for the treatment of patients with tumors.
Assuntos
Antígeno CTLA-4/antagonistas & inibidores , Vacinas Anticâncer/administração & dosagem , Técnicas de Visualização da Superfície Celular/métodos , Melanoma Experimental/tratamento farmacológico , Anticorpos de Domínio Único/administração & dosagem , Animais , Antígeno CTLA-4/administração & dosagem , Antígeno CTLA-4/química , Camelus , Vacinas Anticâncer/metabolismo , Vacinas Anticâncer/farmacologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunização , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ASP2408 is a next-generation anti-cytotoxic T lymphocyte antigen-4 fusion protein engineered for improved CD86 binding affinity as a treatment for rheumatoid arthritis (RA). In 72 healthy subjects (n = 6/treatment), ASP2408 was administered as single ascending doses intravenously at 0.003 to 10.0 mg/kg or subcutaneously at 0.3 to 3.0 mg/kg. It showed decreased clearance and prolonged half-life with increasing doses, consistent with target-mediated disposition. The apparent bioavailability was 36.3%-56.7% across single subcutaneous doses. Sixteen RA patients (n = 8/treatment) on stable methotrexate received 3 × 3.0 mg/kg subcutaneously every 4 weeks or every 2 weeks. Similar to single-dose treatment, ASP2408 concentrations peaked 2 to 3 days postdose, with a median t1/2 of approximately 8 days. Using CD86 receptor occupancy (RO) as a mechanistic biomarker, ASP2408 demonstrated dose-dependent binding to its target. ASP2408 3.0 mg/kg subcutaneously every 4 weeks and every 2 weeks led to a mean %CD86 RO ≥ 74.7% and ≥ 81.5%, respectively, within each dosing interval. ASP2408 was well tolerated across studies with no evidence of dose-limiting toxicity or clinically significant changes in clinical laboratory test results, vital signs, or 12-lead electrocardiograms. ASP2408 elicited antidrug antibodies in the majority of patients, but with no clinical sequelae.
Assuntos
Antirreumáticos/administração & dosagem , Artrite Reumatoide/tratamento farmacológico , Antígeno CTLA-4/administração & dosagem , Imunoconjugados/administração & dosagem , Imunoglobulina G/administração & dosagem , Linfócitos T/imunologia , Administração Intravenosa , Adulto , Idoso , Anticorpos/imunologia , Antirreumáticos/efeitos adversos , Antirreumáticos/farmacocinética , Antígeno B7-2/metabolismo , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Meia-Vida , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacocinética , Imunoglobulina G/efeitos adversos , Injeções Subcutâneas , Masculino , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Adulto JovemRESUMO
Tumor indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression by direct action on effector T cells and Tregs and through recruitment, expansion and activation of myeloid-derived suppressor cells (MDSCs). Targeting of MDSCs is clinically being explored as a therapeutic strategy, though optimal targeting strategies and biomarkers predictive of response are presently unknown. Maturation and tumor recruitment of MDSCs are dependent on signaling through the receptor tyrosine kinase CSF-1R on myeloid cells. Here, we show that MDSCs are the critical cell population in IDO-expressing B16 tumors in mediating accelerated tumor outgrowth and resistance to immunotherapy. Using a clinically relevant drug, we show that inhibition of CSF-1R signaling can functionally block tumor-infiltrating MDSCs and enhance anti-tumor T cell responses. Furthermore, inhibition of CSF-1R sensitizes IDO-expressing tumors to immunotherapy with T cell checkpoint blockade, and combination of CSF-1R blockade with IDO inhibitors potently elicits tumor regression. These findings provide evidence for a critical and functional role for MDSCs on the in vivo outcome of IDO-expressing tumors.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Imunoterapia/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Melanoma Experimental/tratamento farmacológico , Células Supressoras Mieloides/efeitos dos fármacos , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Antígeno CTLA-4/administração & dosagem , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Melanoma Experimental/metabolismo , Camundongos , Terapia de Alvo Molecular , Receptor de Morte Celular Programada 1/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adenoviruses (Ad) are once potential and promising vectors for gene delivery, but the immunogenicity attenuates its transfer efficiency. Cytotoxic T lymphocyte antigen 4 (CTLA-4) can inhibit T cell immunity. Thus, we aimed to study the effect of CTLA-4 in the process of Ad-mediated gene transfer. The C57BL/6 mice were injected by Ad vectors at twice, and CTLA-4 was administrated after the first Ad injection. Then, the CD3(+)CD4(+) T cells and circulating levels of IL-2, IL-4, and anti-Ad IgG were decreased by CTLA-4, while Ad generated immune responses. The green fluorescence protein (GFP) expressions of tissues were enhanced by CTLA-4 till injection of Ad at twice. Our results indicate that CTLA-4 can inhibit humoral and cellular immunity by adenovirus generation to enhance GFP delivery, and provide a potential way to assist in Ad-mediated gene transfer.
Assuntos
Adenoviridae/imunologia , Antígeno CTLA-4/administração & dosagem , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/administração & dosagem , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Animais , Feminino , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/biossíntese , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Latent reservoirs of HIV-1-infected cells are refractory to antiretroviral therapies (ART) and remain the major barrier to curing HIV-1. Because latently infected cells are long-lived, immunologically invisible, and may undergo homeostatic proliferation, a "shock and kill" approach has been proposed to eradicate this reservoir by combining ART with inducers of viral transcription. However, all attempts to alter the HIV-1 reservoir in vivo have failed to date. Using humanized mice, we show that broadly neutralizing antibodies (bNAbs) can interfere with establishment of a silent reservoir by Fc-FcR-mediated mechanisms. In established infection, bNAbs or bNAbs plus single inducers are ineffective in preventing viral rebound. However, bNAbs plus a combination of inducers that act by independent mechanisms synergize to decrease the reservoir as measured by viral rebound. Thus, combinations of inducers and bNAbs constitute a therapeutic strategy that impacts the establishment and maintenance of the HIV-1 reservoir in humanized mice.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Animais , Fármacos Anti-HIV/uso terapêutico , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígeno CTLA-4/administração & dosagem , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos , Receptores Fc/imunologia , VorinostatRESUMO
In order to overcome limitations of monoclonal antibodies, new protein-based scaffolds have been designed and evaluated pre-clinically, and some of them are in clinical studies for the treatment of cancer. These entities can be placed into two categories: scaffolds which bind ligands via amino acids in exposed loops and those in which ligand binding is mediated by amino acids in secondary structures, such as ß-sheet modules. Accordingly, we discuss adnectins, lipocalins, Kunitz domain-based binders, avimers, knottins, fynomers, atrimers and cytotoxic T-lymphocyte associated protein-4 (CTLA4)-based binders which fall into the first category, while darpins, affibodies, affilins and armadillo repeat protein-based scaffolds are members of the second category. In addition, we also discuss the new molecular entities as imaging tools and outline their unique characteristics in the context of multimeric and multivalent binding.
Assuntos
Aminoácidos/química , Anticorpos Monoclonais/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas/administração & dosagem , Aminoácidos/uso terapêutico , Anticorpos Monoclonais/química , Antígeno CTLA-4/administração & dosagem , Antígeno CTLA-4/química , Humanos , Ligantes , Neoplasias/genética , Neoplasias/imunologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas/químicaRESUMO
Targeted cell therapies are possible through the generation of recombinant fusion proteins that combine a toxin, such as diphtheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the fusion protein to the cell of interest, the diphtheria toxin is internalized which results in protein synthesis inhibition and subsequent cell death. We have recently expressed and purified the recombinant soluble porcine CTLA-4 both with and without N-glycosylation in yeast Pichia pastoris for in vivo use in our preclinical swine model. The glycosylated and non-N-glycosylated versions of this recombinant protein each bind to a porcine CD80 expressing B-cell lymphoma line (LCL13271) with equal affinity (K(D)=13 nM). In this study we have linked each of the glycosylated and non-N-glycosylated soluble porcine CTLA-4 proteins to the truncated diphtheria toxin DT390 through genetic engineering yielding three versions of the porcine CTLA-4 fusion toxins: 1) monovalent glycosylated soluble porcine CTLA-4 fusion toxin; 2) monovalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin and 3) bivalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin. Protein synthesis inhibition analysis demonstrated that while all three fusion toxins are capable of inhibiting protein synthesis in vitro, the non-N-glycosylated porcine CTLA-4 isoforms function most efficiently. Binding analysis using flow cytometry of the porcine CTLA-4 fusion toxins to LCL13271 cells also demonstrated that the non-N-glycosylated porcine CTLA-4 isoforms bind to these cells with higher affinity compared to the glycosylated fusion toxin. The monovalent non-N-glycosylated porcine CTLA-4 fusion toxin was tested in vivo. NSG (NOD/SCID IL-2 receptor γ(-)/(-)) mice were injected with porcine CD80(+) LCL13271 tumor cells. All animals succumbed to tumors and those treated with the monovalent non-N-glycosylated porcine CTLA-4 fusion toxin survived longer based on a symptomatic scoring system compared to the untreated controls. This recombinant protein may therefore provide a novel approach for in vivo depletion of porcine antigen presenting cells (APCs) for studies investigating the induction of transplantation tolerance, autoimmune disease and cancer treatment.