RESUMO
We report the generation of a novel anti-LAG-3/TIGIT bispecific IgG4 antibody, ZGGS15, and evaluated its anti-tumor efficacy in mouse models as monotherapy or in combination with a PD-1 antibody. ZGGS15 exhibited strong affinities for human LAG-3 and TIGIT, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 has EC50s of 0.69 nM and 1.87 nM for binding to human LAG-3 and TIGIT on CHO-K1 cells, respectively. ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50 = 0.24 nM). ZGGS15 does not induce ADCC, CDC, or obvious cytokine production. In vivo results showed that ZGGS15 had better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agents and demonstrated a synergistic effect when combined with nivolumab, with a significantly higher tumor growth inhibition of 95.80% (p = 0.001). The tumor volume inhibition rate for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% (p < 0.001). Our data reveal that ZGGS15 exhibits potent anti-tumor efficacy without eliciting ADCC or CDC or causing cytokine production, therefore having a safe profile.
Assuntos
Anticorpos Biespecíficos , Proteína do Gene 3 de Ativação de Linfócitos , Receptor de Morte Celular Programada 1 , Receptores Imunológicos , Animais , Feminino , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD/imunologia , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Células CHO , Cricetulus , Modelos Animais de Doenças , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores Imunológicos/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.
Assuntos
Anemia Ferropriva/tratamento farmacológico , Anticorpos Neutralizantes/farmacologia , Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Ferro/metabolismo , Anemia Ferropriva/genética , Anemia Ferropriva/imunologia , Anemia Ferropriva/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Brucella abortus , Modelos Animais de Doenças , Eritropoese/efeitos dos fármacos , Eritropoese/genética , Hepcidinas , Humanos , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.
Assuntos
Anticorpos Monoclonais/imunologia , Colesterol/sangue , Testes de Neutralização , Serina Endopeptidases/imunologia , Animais , Colesterol/imunologia , Cristalografia por Raios X , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Receptores de LDL/genética , Receptores de LDL/fisiologiaRESUMO
Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, but vector manufacturing remains a challenge. New methods are needed to increase rAAV yields and reduce costs. Past efforts to improve rAAV production have focused on optimizing a single variable at a time, but this approach does not account for the interactions of multiple factors that contribute to vector generation. Here, we utilized a design-of-experiment (DOE) methodology to optimize rAAV production in a HEK293T suspension cell system. We simultaneously varied the transgene, packaging, and helper plasmid ratios, the total DNA concentration, and the cell density to systematically evaluate the impact of each variable across 52 conditions. The results revealed a unique set of parameters with a lower concentration of transgene plasmid, a higher concentration of packaging plasmid, and a higher cell density than previously described protocols. Using this DOE-optimized protocol, we achieved unpurified yields approaching 3 × 1014 viral genomes (VGs)/L of cell culture. Additionally, we incorporated polyethylene glycol (PEG)-based virus precipitation, pH-mediated protein removal, and affinity chromatography to our downstream processing, enabling average purified yields of >1 × 1014 VGs/L for rAAV-EGFPs across 13 serotypes and capsid variants.
RESUMO
Mammalian expression systems are used routinely for the production of recombinant proteins as therapeutic molecules as well as research tools. Transient expression has become increasingly popular in recent years due to its rapid timeline and improvements in expression level. While improvements to transient expression systems have focused mainly on the level of protein expression, the aspect of protein quality has received little attention. The removal of undesirable products, such as aggregation, depends primarily on purification, requiring additional cumbersome steps, which can lead to a lower product yield and longer timelines. In this study, we show that reducing the level of transcription by transfecting at a lower gene dose improves the quality of secreted molecules prone to aggregation. For gene dosing to have this effect, it is critical for the carrier DNA to be an empty vector containing the same elements as the gene containing plasmid. This approach can be used in combination with a temperature shift to hypothermic conditions during production to enhance the effect. The observed improvements not only minimized aggregation levels, but also generated products with overall superior quality, including more homogeneous signal peptide cleavage and N-linked glycosylation profiles. These techniques have produced a similar improvement in product quality with a variety of other molecules, suggesting that this may be a general approach to enhance product quality from transient expression systems.
Assuntos
Técnicas de Cultura de Células/métodos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos , Dosagem de Genes , Glicosilação , Células HEK293 , Humanos , Agregados Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , TemperaturaRESUMO
Osteoporosis is a chronic condition that is typically treated by the long-term repeated administration of antiresorptive agents. Gene therapy has the potential to deliver protein-based antiresorptive agents without the need for repeated administration. Osteoprotegerin (OPG) is a naturally occuring protein that prevents bone resorption by inhibiting osteoclast formation, function and survival. We tested whether adeno-associated virus (AAV) could deliver OPG at levels that are sufficient to reverse established osteopenia in ovariectomized (OVX) mice without causing liver toxicity. Tibial bone mineral density (BMD) was measured by peripheral quantitative computed tomography (pQCT) in 12-week-old CDF1 mice prior to OVX or sham surgery. Six weeks later, BMD was significantly reduced in OVX mice compared to sham controls or pre-surgery values. Sham and OVX mice were then injected once IV with an AAV vector carrying cDNA for recombinant hOPG (AAV-OPG) or beta-galactosidase (AAV-betaGal). BMD and bone histomorphometry were assessed 10 weeks after treatment. A single injection of AAV-OPG led to the appearance of human OPG (hOPG) in the serum of mice within 7 days, and high serum levels of hOPG were maintained for the duration of the 10-week study. At the end of the study, OVX mice given AAV-OPG had significantly greater tibial BMD compared to age-matched OVX animals given AAV-betaGal. In sham-operated mice, AAV-OPG also significantly increased tibial BMD compared to AAV-betaGal. The increased BMD in AAV-OPG animals was accompanied by significantly increased bone volume and significantly reduced osteoclast surfaces in the proximal tibial metaphysis. Liver histology was normal, and circulating activities of hepatocyte cytosolic enzymes were unaffected by AAV exposure. In an accompanying experiment, young (3-4 weeks) C57BL/6 mice treated once IV with AAV-OPG maintained pharmacologically active levels of OPG in serum for at least 16 months. In summary, a single AAV-OPG treatment reversed established osteopenia in OVX mice without evidence of liver toxicity. AAV delivery appears to be a safe and effective method for producing sustained systemic exposure to OPG.
Assuntos
Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/terapia , Terapia Genética , Glicoproteínas/genética , Glicoproteínas/uso terapêutico , Ovariectomia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/uso terapêutico , Animais , Densidade Óssea/fisiologia , Doenças Ósseas Metabólicas/patologia , Reabsorção Óssea/genética , Reabsorção Óssea/prevenção & controle , Reabsorção Óssea/terapia , Dependovirus/genética , Dependovirus/fisiologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Glicoproteínas/efeitos adversos , Glicoproteínas/metabolismo , Humanos , Fígado/efeitos dos fármacos , Camundongos , Osteoprotegerina , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Tíbia/fisiopatologia , Fatores de TempoRESUMO
Three fibroblast growth factor (FGF) molecules, FGF19, FGF21, and FGF23, form a unique subfamily that functions as endocrine hormones. FGF19 and FGF21 can regulate glucose, lipid, and energy metabolism, while FGF23 regulates phosphate homeostasis. The FGF receptors and co-receptors for these three FGF molecules have been identified, and domains important for receptor interaction and specificity determination are beginning to be elucidated. However, a number of questions remain unanswered, such as the identification of fibroblast growth factor receptor responsible for glucose regulation. Here, we have generated a variant of FGF23: FGF23-21c, where the C-terminal domain of FGF23 was replaced with the corresponding regions from FGF21. FGF23-21c showed a number of interesting and unexpected properties in vitro. In contrast to wild-type FGF23, FGF23-21c gained the ability to activate FGFR1c and FGFR2c in the presence of ßKlotho and was able to stimulate glucose uptake into adipocytes in vitro and lower glucose levels in ob/ob diabetic mice model to similar extent as FGF21 in vivo. These results suggest that ßKlotho/FGFR1c or FGFR2c receptor complexes are sufficient for glucose regulation. Interestingly, without the FGF23 C-terminal domain, FGF23-21c was still able to activate fibroblast growth factor receptors in the presence of αKlotho. This suggests not only that sequences outside of the C-terminal region may also contribute to the interaction with co-receptors but also that FGF23-21c may be able to regulate both glucose and phosphate metabolisms. This raises an interesting concept of designing an FGF molecule that may be able to address multiple diseases simultaneously. Further understanding of FGF/receptor interactions may allow the development of exciting opportunities for novel therapeutic discovery.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/metabolismo , Proteínas de Membrana/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Adipócitos/metabolismo , Animais , Glicemia/efeitos dos fármacos , Linhagem Celular , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/farmacologia , Glucose/metabolismo , Proteínas Klotho , Masculino , Camundongos , Camundongos ObesosRESUMO
Diabetes and associated metabolic conditions have reached pandemic proportions worldwide, and there is a clear unmet medical need for new therapies that are both effective and safe. FGF19 and FGF21 are distinctive members of the FGF family that function as endocrine hormones. Both have potent effects on normalizing glucose, lipid, and energy homeostasis, and therefore, represent attractive potential next generation therapies for combating the growing epidemics of type 2 diabetes and obesity. The mechanism responsible for these impressive metabolic effects remains unknown. While both FGF19 and FGF21 can activate FGFRs 1c, 2c, and 3c in the presence of co-receptor ßKlotho in vitro, which receptor is responsible for the metabolic activities observed in vivo remains unknown. Here we have generated a variant of FGF19, FGF19-7, that has altered receptor specificity with a strong bias toward FGFR1c. We show that FGF19-7 is equally efficacious as wild type FGF19 in regulating glucose, lipid, and energy metabolism in both diet-induced obesity and leptin-deficient mouse models. These results are the first direct demonstration of the central role of the ßKlotho/FGFR1c receptor complex in glucose and lipid regulation, and also strongly suggest that activation of this receptor complex alone might be sufficient to achieve all the metabolic functions of endocrine FGF molecules.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Glucose/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Animais , Western Blotting , Linhagem Celular , Dependovirus/genética , Fatores de Crescimento de Fibroblastos/genética , Vetores Genéticos , Teste de Tolerância a Glucose , Fígado/metabolismo , Masculino , Camundongos , Ligação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on lipid, body weight, and glucose metabolism and has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to native FGF21, we have developed a monoclonal antibody, mimAb1, that binds to ßKlotho with high affinity and specifically activates signaling from the ßKlotho/FGFR1c (FGF receptor 1c) receptor complex. In obese cynomolgus monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose during tolerance testing. Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements in glucose metabolism and body weight. These results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated the essential role of FGFR1c in FGF21 function and suggest fat as a critical target tissue for the cytokine and antibody. Because mimAb1 depends on ßKlotho to activate FGFR1c, it is not expected to induce side effects caused by activating FGFR1c alone. The unexpected finding of an antibody that can activate FGF21-like signaling through cell surface receptors provided preclinical validation for an innovative therapeutic approach to diabetes and obesity.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Diabetes Mellitus/tratamento farmacológico , Fatores de Crescimento de Fibroblastos/imunologia , Glucuronidase/metabolismo , Obesidade/tratamento farmacológico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Anticorpos Monoclonais/farmacologia , Peso Corporal/genética , Diabetes Mellitus/sangue , Epitopos/química , Glucose/metabolismo , Glucuronidase/imunologia , Humanos , Proteínas Klotho , Macaca fascicularis , Camundongos , Obesidade/sangue , Obesidade/complicações , Fosfatos/sangue , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/sangueRESUMO
The physiological role of Dickkopf-1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1-Ab) that blocked Dkk1 binding to both low density lipoprotein receptor-related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1-Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.
Assuntos
Envelhecimento/metabolismo , Osso e Ossos/lesões , Osso e Ossos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteogênese/fisiologia , Envelhecimento/efeitos dos fármacos , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/farmacologia , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/sangue , Doenças Ósseas Metabólicas/fisiopatologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Linhagem Celular , Estrogênios/deficiência , Feminino , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Fêmur/patologia , Consolidação da Fratura/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Masculino , Camundongos , Osteogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
Adiponectin is an adipocyte-derived hormone that has been shown to play important roles in the regulation of glucose and energy homeostasis. It exists as homotrimers or complexes containing multiple homotrimer units in plasma. The recombinant adiponectin proteins have been difficult to produce, making it challenging for both research as well as potential therapeutic development. Here, we show a novel approach for the generation of globular adiponectin that involves linking three monomer sequences together in tandem to generate one continuous polypeptide, which we have termed single-chain globular adiponectin (sc-gAd). To improve the pharmacokinetic properties of sc-gAd further, we fused it to an Fc fragment. The combined effects of single-chain and Fc fusion improved the plasma half-life from less than 2 h to close to 2 weeks. Using adeno-associated virus as a delivery method, we demonstrate that Fc-sc-gAd improved both fasting glucose levels and the tolerance to a glucose challenge in ob/ob mice without changes in body weight. Therefore, our results demonstrated the feasibility of generating globular adiponectin trimers from a single polypeptide and a long-acting globular adiponectin that could serve as a starting point for adiponectin-based therapeutics. This novel approach could also be applied to other complement factor C1q family members; in particular, this opens the possibility to study the biological functions of precisely defined heterotrimers of various family members that had not been previously possible.