RESUMO
Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients. Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo. Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Camundongos , Animais , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Retinopatia Diabética/tratamento farmacológico , Fatores de Crescimento Endotelial/uso terapêutico , Acuidade Visual , Transtornos da Visão/complicações , Transtornos da Visão/tratamento farmacológico , Cegueira/complicaçõesRESUMO
Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.
Assuntos
Fator de Crescimento Transformador beta2 , Fator de Crescimento Transformador beta3 , Animais , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Camundongos , Isoformas de Proteínas/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/metabolismoRESUMO
A phage-derived human monoclonal antibody against VEGF-C was developed as a potential anti-tumor therapeutic and exhibited fast clearance in preclinical species, with notably faster clearance in serum than in plasma. The purpose of this work was to understand the factors contributing to its fast clearance. In vitro incubations in animal and human blood, plasma, and serum were conducted with radiolabeled anti-VEGF-C to determine potential protein and cell-based interactions with the antibody as well as any matrix-dependent recovery dependent upon the matrix. A tissue distribution study was conducted in mice with and without heparin infusion in order to identify a tissue sink and determine whether heparin could affect antibody recovery from serum and/or plasma. Incubation of radiolabeled anti-VEGF-C in human and animal blood, plasma, or serum revealed that the antibody formed a complex with an endogenous protein, likely VEGF-C. This complex was trapped within the blood clot during serum preparation from blood, but not within the blood cell pellet during plasma preparation. Low level heparin infusion in mice slowed down clot formation during serum preparation and allowed for better recovery of the radiolabeled antibody in serum. No tissue sink was found in mice. Thus, during this characterization, we determined that the blood sampling matrix greatly impacted the amount of antibody recovered in the samples, therefore, altering its derived pharmacokinetic parameters. Target biology should be considered when selecting appropriate sampling matrices for PK analysis.
Assuntos
Anticorpos Monoclonais/farmacocinética , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/sangue , Artefatos , Coagulação Sanguínea , Feminino , Humanos , Macaca fascicularis , Camundongos Nus , Ratos Sprague-Dawley , Distribuição Tecidual , Fator C de Crescimento do Endotélio Vascular/imunologiaRESUMO
IgG antibodies have been used to treat many diseases including cancer. IgG antibody-drug conjugates (ADCs) deliver cytotoxic drugs to target cells for cell elimination, but they have dose limiting toxicity due to target-independent uptake, including pinocytotic uptake. Neonatal Fc receptor (FcRn) recycles pinocytosed IgG in a pH-dependent manner and is the receptor responsible for the long half-life of IgG. Use of IgG variants with stronger FcRn binding at pH 6.0 for ADCs might improve recycling efficiency and reduce toxicity. However, these variants have residual FcRn binding at pH 7.4, which could lead to FcRn-mediated uptake and higher toxicity. Thus, the uptake of such variants at pH 7.4 needs to be evaluated. Here we report a reproducible and quantitative assay using an inducible HM7 colorectal cancer cell line to measure IgG uptake at endogenous and overexpressed FcRn levels. Our assay had comparable reproducibility at pH 6.0, 6.8 and 7.4. The wild type (WT) IgG had similar uptake at endogenous and overexpressed FcRn levels, as expected for pinocytotic uptake. We found similar uptake of a WT IgG and a stronger FcRn binding T307Q/N434A variant (QA variant) at endogenous FcRn levels at pH 7.4, although the QA variant had higher uptake at overexpressed FcRn levels. The QA variant also had higher uptake than the WT IgG at overexpressed FcRn levels at pH 6.8. Our assay can be used to characterize the stronger FcRn binding variants to aid in selection of suitable variants with low uptake at pH 7.4 for use as ADCs.
Assuntos
Neoplasias Colorretais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/metabolismo , Pinocitose , Receptores Fc/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ligação Proteica , Receptores Fc/genética , Regulação para CimaRESUMO
Transforming growth factor ß (TGFß) signaling has been recently shown to reduce antitumor response to PD-L1 blockade, leading to a renewed enthusiasm in developing anti-TGFß therapies for potential combination with cancer immunotherapy agents. Inhibition of TGFß signaling in nonclinical toxicology species is associated with serious adverse toxicities including cardiac valvulopathies and anemia. Previously, cardiovascular toxicities have been thought to be limited to small molecule inhibitors of TGFß receptor and not considered to be a liability associated with pan-TGFß neutralizing monoclonal antibodies (mAbs). Here, we report the toxicity findings associated with a potent pan-TGFß neutralizing mAb (pan-TGFß mAb; neutralizes TGFß1, 2, and 3) after 5 weekly intravenous doses of 10, 30, and 100 mg/kg, followed by a 4-week recovery period, in mice and cynomolgus monkeys. Mortality was observed due to acute bleeding and cardiovascular toxicity in mice at ≥ 30 mg/kg and prolonged menstruation in female monkeys at 100 mg/kg. Additional findings considered to be on-target exaggerated pharmacology included generalized bleeding and cardiovascular toxicity in mice and monkeys; histopathologic changes in the teeth, tongue, and skin in mice; and abnormal wound healing and microscopic pathology in the bone in monkeys. Importantly, our data indicate that the cardiovascular toxicities associated with the inhibition of TGFß signaling are not limited to small molecule inhibitors but are also observed following administration of a potent pan-TGFß inhibiting mAb.
Assuntos
Anticorpos Monoclonais Humanizados/toxicidade , Anticorpos Neutralizantes/toxicidade , Doenças Cardiovasculares/induzido quimicamente , Coração/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Neutralizantes/sangue , Cardiotoxicidade , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Linhagem Celular , Feminino , Hemorragia/induzido quimicamente , Hemorragia/metabolismo , Humanos , Macaca fascicularis , Masculino , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Medição de Risco , Fatores de Tempo , Testes de Toxicidade , Toxicocinética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.
Assuntos
Anticorpos Monoclonais/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Animais , Antibacterianos/farmacologia , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/imunologia , Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Imunização , Conformação Proteica , Dobramento de Proteína , Transporte Proteico/genética , Transporte Proteico/imunologia , VacinaçãoRESUMO
The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.
Assuntos
Anticorpos Biespecíficos/imunologia , Imunoglobulina G/química , Animais , Formação de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Complemento C1q/imunologia , Cricetinae , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Camundongos , Conformação Proteica , Receptores de IgG/metabolismo , TemperaturaRESUMO
Antigen-binding fragments (Fab) and F(ab')2 antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab')2 C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab')2 in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T225L variant and the natural C-terminal D221 as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab')2 for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.
Assuntos
Autoanticorpos/imunologia , Epitopos de Linfócito B/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Mapeamento de Epitopos , Humanos , Engenharia de ProteínasRESUMO
For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal ("afucosylation"). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcγRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody.
Assuntos
Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/imunologia , Fucose/química , Imunoglobulina G/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Antígenos CD20/genética , Linfócitos B/imunologia , Sangue/imunologia , Células CHO , Cricetinae , Cricetulus , Feminino , Glicosilação , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Linfonodos/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/imunologiaRESUMO
PURPOSE: To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. METHODS: Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship. RESULTS: After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab')2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways. CONCLUSIONS: We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the "ideal therapeutic" by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular half-life and minimizing systemic exposure.
Assuntos
Anticorpos Monoclonais/farmacocinética , Anticorpos/imunologia , Desenho de Fármacos , Oftalmopatias/tratamento farmacológico , Olho/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Afinidade de Anticorpos , Oftalmopatias/imunologia , Oftalmopatias/metabolismo , Injeções Intravítreas , Masculino , Ligação Proteica , CoelhosRESUMO
Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays an important role in physiological and tumor angiogenesis. The human VEGF gene has eight exons. Different VEGF isoforms are expressed via alternative RNA splicing and VEGF121 and VEGF165 are the major isoforms present in human tissues. The exact roles of these different VEGF isoforms are not totally clear. Assays to detect specific VEGF isoforms in biological samples are needed to understand the biological functions of these different VEGF isoforms and to better assess their potential use as predicative biomarkers for anti-angiogenic therapy. Because monoclonal antibodies specific to different VEGF isoforms are lacking, we used antibodies directed to different epitopes on VEGF165 in a set of three enzyme-linked immunosorbent assays (ELISAs) to assess the amount of VEGF121 and VEGF165 as well as VEGF110, which can be generated by plasmin cleavage in vivo. The first ELISA detects VEGF165. The second ELISA detects both VEGF121 and VEGF165. The third ELISA detects VEGF165, VEGF121, and VEGF110. The concentrations of VEGF121 can be assessed from the difference in VEGF concentrations measured by the second and the first ELISAs; the concentrations of VEGF110 can be assessed from the difference in VEGF concentrations measured by the third and the second ELISAs. The same assay strategy may be used to assess the amount of other VEGF isoforms if antibodies directed against the desired amino acids in those isoforms can be obtained.
Assuntos
Ensaio de Imunoadsorção Enzimática , Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Isoformas de Proteínas , Fatores de Crescimento do Endotélio Vascular/sangueRESUMO
Neuropilin-1 (NRP1) is a single spanning transmembrane glycoprotein that acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factors. Naturally occurring soluble NRP1 isoforms containing partial extracellular domain (ECD) have been reported. In addition to soluble NRP1, full-length NRP1 ECD has also been identified in human and animal sera. Here, we describe primate and rodent NRP1 ELISAs that measure total circulating NRP1 including soluble NPR1 and NRP1 ECD in human, monkey, mouse, and rat sera.
Assuntos
Ensaio de Imunoadsorção Enzimática , Neuropilina-1/sangue , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Haplorrinos , Humanos , Camundongos , RatosRESUMO
Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/ßKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/ßKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/ßKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/ßKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/ßKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.
Assuntos
Tecido Adiposo Marrom/metabolismo , Anticorpos Biespecíficos/farmacologia , Insulina/farmacologia , Proteínas de Membrana/agonistas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Adiponectina/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Animais , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Células HEK293 , Humanos , Proteínas Klotho , Macaca fascicularis , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Obesos , Ligação Proteica/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Termogênese/efeitos dos fármacos , Redução de Peso/efeitos dos fármacosRESUMO
Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacocinética , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/genética , Área Sob a Curva , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Ensaio de Imunoadsorção Enzimática , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Macaca fascicularis , Taxa de Depuração Metabólica , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Ratos Sprague-Dawley , Receptores Fc/imunologia , Receptores Fc/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.
Assuntos
Fatores de Crescimento Endotelial/análise , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/análise , Anticorpos/química , Anticorpos/imunologia , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Biotina/química , Biotina/metabolismo , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Família de Proteínas EGF , Fatores de Crescimento Endotelial/sangue , Fatores de Crescimento Endotelial/imunologia , Feminino , Células HEK293 , Humanos , Masculino , Estreptavidina/química , Estreptavidina/metabolismo , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2-1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Modelos Animais de Doenças , Cobaias , Células HEK293 , HumanosRESUMO
Although angiogenesis inhibitors have provided substantial clinical benefit as cancer therapeutics, their use is limited by resistance to their therapeutic effects. While ample evidence indicates that such resistance can be influenced by the tumor microenvironment, the underlying mechanisms remain incompletely understood. Here, we have uncovered a paracrine signaling network between the adaptive and innate immune systems that is associated with resistance in multiple tumor models: lymphoma, lung and colon. Tumor-infiltrating T helper type 17 (T(H)17) cells and interleukin-17 (IL-17) induced the expression of granulocyte colony-stimulating factor (G-CSF) through nuclear factor κB (NF-κB) and extracellular-related kinase (ERK) signaling, leading to immature myeloid-cell mobilization and recruitment into the tumor microenvironment. The occurrence of T(H)17 cells and Bv8-positive granulocytes was also observed in clinical tumor specimens. Tumors resistant to treatment with antibodies to VEGF were rendered sensitive in IL-17 receptor (IL-17R)-knockout hosts deficient in T(H)17 effector function. Furthermore, pharmacological blockade of T(H)17 cell function sensitized resistant tumors to therapy with antibodies to VEGF. These findings indicate that IL-17 promotes tumor resistance to VEGF inhibition, suggesting that immunomodulatory strategies could improve the efficacy of anti-angiogenic therapy.
Assuntos
Inibidores da Angiogênese/farmacologia , Resistencia a Medicamentos Antineoplásicos , Interleucina-17/metabolismo , Neoplasias/tratamento farmacológico , Neovascularização Patológica/imunologia , Células Th17/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos/imunologia , Antígeno CD11b/metabolismo , Neoplasias Colorretais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Hormônios Gastrointestinais/metabolismo , Fator Estimulador de Colônias de Granulócitos/biossíntese , Granulócitos/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Linfoma/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , NF-kappa B/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Neuropeptídeos/metabolismo , Comunicação Parácrina , Microambiente Tumoral/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b(+)Gr1(+) myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b(+)Ly6G(+) neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies.
Assuntos
Fator Estimulador de Colônias de Granulócitos/metabolismo , Sistema de Sinalização das MAP Quinases , Neutrófilos/citologia , Proteína Proto-Oncogênica c-ets-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neoplasias/metabolismo , Neovascularização Patológica , Infiltração de Neutrófilos , Proteínas Tirosina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK.