RESUMO
Pericytes are the major source of scar-producing myofibroblasts following kidney injury; however, the mechanisms of this transition are unclear. To clarify this, we examined Collagen 1 (α1)-green fluorescent protein (GFP) reporter mice (pericytes and myofibroblasts express GFP) following ureteral obstruction or ischemia-reperfusion injury and focused on the role of platelet-derived growth factor (PDGF)-receptor (PDGFR) signaling in these two different injury models. Pericyte proliferation was noted after injury with reactivation of α-smooth muscle actin expression, a marker of the myofibroblast phenotype. PDGF expression increased in injured tubules, endothelium, and macrophages after injury, whereas PDGFR subunits α and ß were expressed exclusively in interstitial GFP-labeled pericytes and myofibroblasts. When PDGFRα or PDGFRß activation was inhibited by receptor-specific antibody following injury, proliferation and differentiation of pericytes decreased. The antibodies also blunted the injury-induced transcription of PDGF, transforming growth factor ß1, and chemokine CCL2. They also reduced macrophage infiltration and fibrosis. Imatinib, a PDGFR tyrosine kinase inhibitor, attenuated pericyte proliferation and kidney fibrosis in both fibrogenic models. Thus, PDGFR signaling is involved in pericyte activation, proliferation, and differentiation into myofibroblasts during progressive kidney injury. Hence, pericytes may be a novel target to prevent kidney fibrosis by means of PDGFR signaling blockade.
Assuntos
Fibrose/etiologia , Nefropatias/patologia , Miofibroblastos/patologia , Pericitos/patologia , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Traumatismo por Reperfusão/patologia , Obstrução Ureteral/patologia , Animais , Diferenciação Celular , Proliferação de Células , Nefropatias/etiologia , Camundongos , Transdução de Sinais/fisiologiaRESUMO
The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/988/F1.large.jpg.
Assuntos
Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Ativação Linfocitária/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Apoptose , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fresenius Biotech GmbH (licensed from TRION Pharma GmbH) is developing catumaxomab, a rat/murine hybrid, trifunctional, bispecific (anti-epithelial cell adhesion molecule and anti-CD3) mAb for the potential intravenous and intraperitoneal treatment of ovarian cancer, gastric cancer, malignant pleural effusion and other cancers, and also for malignant ascites associated with various cancers. In 2007, following the successful completion of a phase II/III clinical trial in patients with malignant ascites from various cancers, Fresenius filed for European approval. Catumaxomab is currently undergoing phase II clinical trials in patients with malignant ascites, ovarian and stomach cancer.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/biossíntese , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/farmacocinética , Ensaios Clínicos como Assunto , Contraindicações , Avaliação Pré-Clínica de Medicamentos , Camundongos , Ratos , Relação Estrutura-AtividadeRESUMO
Bispecific antibodies (BsAb) have been traditionally utilized to redirect cytotoxic effector cells and agents to kill tumor cells expressing the target antigens. Recently a new concept is emerging to develop BsAb that simultaneously block the functions of two tumor-associated targets, eg., growth factor receptors, for enhanced antitumor efficacies. Broad clinical applications of BsAb have been, and still are, significantly hampered by the difficulty in producing the materials in sufficient quantity and quality by traditional approaches. Here we describe a recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, using a single variable domain (sVD) antibody as a versatile building block. In this method, a sVD of a defined specificity is genetically fused to either the N-terminus of the light chain or the C-terminus of the heavy chain of a functional IgG antibody of a different specificity. A model BsAb was constructed using a sVD to mouse platelet derived growth factor receptor alpha and a conventional IgG antibody to mouse platelet derived growth factor receptor beta. The BsAb were expressed in mammalian cells and purified to homogeneity by a one-step Protein A affinity chromatography. Further, the BsAb retained the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. Importantly, the BsAb inhibited the activation of both its target receptors in tumor cells stimulated by both platelet derived growth factor AA and BB, whereas the parent monospecific antibody only inhibited the activation of a single receptor stimulated by its cognate ligand. This format of BsAb should be readily applicable to the production of other BsAb recognizing any pairs of antigens.
Assuntos
Anticorpos Biespecíficos/imunologia , Região Variável de Imunoglobulina/genética , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Becaplermina , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/genética , Humanos , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/imunologia , Cinética , Camundongos , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/química , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/imunologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/imunologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , TransfecçãoRESUMO
Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Anticorpos Monoclonais/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Adenocarcinoma de Pulmão , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Bronquíolos/patologia , Proliferação de Células/efeitos dos fármacos , Fator 9 de Crescimento de Fibroblastos/metabolismo , Humanos , Ligantes , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Alvéolos Pulmonares/patologiaRESUMO
Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Neutralizantes/farmacologia , Receptores de Somatomedina/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Nus , Microscopia Confocal , Neoplasias Experimentais/tratamento farmacológico , Estabilidade Proteica , Receptor IGF Tipo 1 , Ressonância de Plasmônio de Superfície , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Platelet-derived growth factor receptor beta (PDGFRbeta) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRbeta from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRbeta and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRbeta antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor beta de Fator de Crescimento Derivado de Plaquetas/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Citometria de Fluxo , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Neoplasias/patologia , Biblioteca de Peptídeos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We generated three fully human monoclonal antibody antagonists against fibroblast growth factor receptor-1 (FGFR1) that potently block FGF signaling. We found that antibodies targeting the c-splice form of the receptor (FGFR1c) were anorexigenic when administered intraperitoneally three times weekly to mice, resulting in rapid, dose-dependent weight loss that plateaued (for doses>4 mg/kg) at 35-40% in 2 wk. Animals appeared healthy during treatment and regained their normal body weights and growth trajectories upon clearance of the antibodies from the bloodstream. Measurements of food consumption and energy expenditure indicated that the rapid weight loss was induced primarily by decreased energy intake and not by increased energy expenditure or cachexia and was accompanied by a greater reduction in fat than lean body mass. Hypophagia was not caused through malaise or illness, as indicated by absence of conditioned taste aversion, pica behavior, and decreased need-induced salt intake in rats. In support of a hypothalamic site of action, we found that, after intraperitoneal injections, anti-FGFR1c (IMC-A1), but not a control antibody, accumulated in the median eminence and adjacent mediobasal hypothalamus and that FGFR1c is enriched in the hypothalamus of mice. Furthermore, a single intracerebroventricular administration of 3 microg of IMC-A1 via the 3rd ventricle to mice caused an approximately 36% reduction in food intake and an approximately 6% weight loss within the ensuing 24 h. Our data suggest that FGF signaling through FGFR1c may play a physiological role in hypothalamic feeding circuit and that blocking it leads to hypophagia and weight loss.
Assuntos
Anticorpos Monoclonais/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/imunologia , Redução de Peso/efeitos dos fármacos , Animais , Anticorpos Monoclonais/efeitos adversos , Especificidade de Anticorpos , Composição Corporal/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Feminino , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/imunologia , Ratos , Ratos Sprague-DawleyRESUMO
Platelet-derived growth factor (PDGF) and its receptors (PDGFR) play important roles in tumorigenesis through stimulating tumor growth and promoting angiogenesis via enhancing pericyte recruitment and vessel maturation. Here we produced a neutralizing antibody, 1B3, directed against mouse PDGFRbeta. 1B3 binds to PDGFRbeta with high affinity (9x10(-11)M) and blocks PDGF-BB from binding to the receptor with an IC(50) of approximately 1.2 nM. The antibody also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules, including Akt and MAPK p42/44, in tumor cells. In animal studies, 1B3 significantly enhanced the antitumor and the anti-angiogenic activities of DC101, an antibody directed against mouse vascular endothelial growth factor receptor 2, in a pancreatic (BxPC-3) and a non-small cell lung (NCI-H460) tumor xenograft models. Treatment with the combination of 1B3 and DC101 in BxPC-3 xenograft-bearing mice resulted in tumor regression in 58% of mice compared to that in 18% of mice treated with DC101 alone. Taken together, these results lend great support to use PDGFRbeta antagonists in combinations with other antitumor and/or anti-angiogenic agents in the treatment of a variety of cancers.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Neoplasias/imunologia , Neovascularização Patológica/imunologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologiaRESUMO
Both laboratory and early clinical studies to date have demonstrated that bispecific antibodies (BsAb) may have potentially significant application in cancer therapy. The clinical development of BsAb as therapeutics has been hampered, however, by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods has been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. Here we describe a novel recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a single variable domain antibody to the N terminus of the light chain of a functional IgG antibody of different specificity. A model BsAb was constructed using a single variable domain antibody to mouse platelet-derived growth factor receptor alpha and a conventional IgG antibody to mouse vascular endothelial growth factor receptor 2. The BsAb was expressed in mammalian cells and purified to homogeneity by one-step protein A affinity chromatography. Furthermore, the BsAb retains the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. This design and expression of Fc domain-containing, IgG-like BsAb should be applicable to the construction of similar BsAb from antibodies recognizing any pair of antigens.
Assuntos
Anticorpos Biespecíficos/química , Imunoglobulina G/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sítios de Ligação , Cromatografia , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Fragmentos de Imunoglobulinas , Técnicas Imunológicas , Concentração Inibidora 50 , Cinética , Camundongos , Dados de Sequência Molecular , Neoplasias/imunologia , Neoplasias/metabolismo , Biblioteca de Peptídeos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original naïve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides also cross-react with clone 1121, but none of the clone 1121-specific peptides binds to 2C6, indicating that clone 1121 retained part of the original binding epitope(s) of 2C6 while gaining new binding specificity. Taken together, our observation suggests that clone 1121 may have great clinical potential in anti-angiogenesis therapy. It further underscores the efforts to identify antibodies of high affinity for enhanced biological activities.