RESUMO
MEDI4893 is an investigational immunoglobulin G1(κ) monoclonal antibody that specifically binds to and neutralizes alpha-toxin, a key Staphylococcus aureus virulence factor. A triple-amino-acid substitution, M252Y/S254T/T256E, was engineered into the MEDI4893 Fc region to extend its serum half-life. A phase 1, double-blind, dose escalation study was designed to evaluate the safety, tolerability, pharmacokinetics, anti-alpha-toxin-neutralizing activity, and antidrug antibody (ADA) response of MEDI4893 following a single intravenous infusion in healthy adults 18 to 65 years of age. Thirty-three subjects were randomly assigned to receive MEDI4893 at 225 mg (n = 3), 750 mg (n = 3), 2,250 mg (n = 8), or 5,000 mg (n = 12) or placebo (n = 7) and were followed for 360 days. Adverse events were mild or moderate in severity; none were serious. The MEDI4893 peak serum concentration increased dose proportionally from 77.2 µg/ml (225-mg dose) to 1,784 µg/ml (5,000-mg dose). The area under the concentration-time curve from 0 to 360 days also increased dose proportionally, from 4,840 µg · day/ml (225-mg dose) to 91,493 µg · day/ml (5,000-mg dose), indicating linear pharmacokinetics. MEDI4893's terminal half-life was estimated to be 80 to 112 days, which is approximately 4-fold longer than the half-lives of other human immunoglobulin G antibodies. The alpha-toxin-neutralizing activity in serum correlated highly with the MEDI4893 concentrations in serum. Three adults transiently tested positive for ADA on day 151, but this did not have an impact on MEDI4893 serum concentrations or the MEDI4893 safety profile; no subjects exhibited serum ADA at the study end. These data support the continued development of MEDI4893 for the prevention of S. aureus-mediated pneumonia. (This study has been registered at ClinicalTrials.gov under identifier NCT02296320.).
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Staphylococcus aureus/efeitos dos fármacos , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/sangue , Anticorpos Amplamente Neutralizantes , Método Duplo-Cego , Feminino , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/tratamento farmacológico , Adulto JovemRESUMO
The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antígenos de Histocompatibilidade Classe I/fisiologia , Imunoglobulina G/fisiologia , Engenharia de Proteínas , Receptores Fc/fisiologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Bacteriófagos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Ressonância de Plasmônio de Superfície , Distribuição TecidualRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) causes large-scale epidemics of acute bacterial skin and skin structure infections (ABSSSI) within communities across the United States. Animal models that reproduce ABSSSI as they occur in humans are urgently needed to test new therapeutic strategies. Alpha-toxin plays a critical role in a variety of staphylococcal infection models in mice, but its role in the pathogenesis of ABSSSI remains to be elucidated in rabbits, which are similar to humans in their susceptibility to S. aureus superantigens and certain bicomponent pore-forming leukocidins. We report here a new rabbit model of ABSSSI and show that those infected with a mutant deficient in expression of alpha-toxin (Δhla) developed a small dermonecrotic lesion, whereas those infected with isogenic USA300 MRSA wild-type or complemented Δhla strains developed ABSSSI that mimic the severe infections that occur in humans, including the large central dermonecrotic core surrounded by erythema, induration, and marked subcutaneous hemorrhage. More importantly, immunoprophylaxis with MEDI4893*, an anti-alpha-toxin human monoclonal antibody, significantly reduced the severity of disease caused by a USA300 wild-type strain to that caused by the Δhla mutant, indicating that this toxin could be completely neutralized during infection. Thus, this study illustrates a potential high standard for the development of new immunotherapeutic agents in which a toxin-neutralizing antibody provides protection to the same degree achieved with a toxin gene knockout. When MEDI4893* was administered as adjunctive therapy with a subtherapeutic dose of linezolid, the combination was significantly more efficacious than either agent alone in reducing the severity of ABSSSI.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Dermatopatias Bacterianas/microbiologia , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados , Toxinas Bacterianas/genética , Anticorpos Amplamente Neutralizantes , Modelos Animais de Doenças , Proteínas Hemolisinas/genética , Humanos , Linezolida/sangue , Linezolida/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/imunologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Profilaxia Pré-Exposição/métodos , Coelhos , Dermatopatias Bacterianas/imunologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/microbiologiaRESUMO
AIMS: Sifalimumab, a human immunoglobulin (Ig) G1 monoclonal antibody against INF-alpha, is being studied as a treatment for systemic lupus erythematosus (SLE). This analysis characterized population pharmacokinetics (PK) of sifalimumab following repeat fixed dose and evaluated the utility of fixed dosing vs. body weight normalized dosing in SLE patients. METHODS: PK data were collected in a phase IIb study where 298 patients received multiple intravenous doses (200-1200 mg) of sifalimumab every 4 weeks for 52 weeks. A population pharmacokinetic model was developed using 3961 quantifiable serum concentrations and the impact of patient demographics, clinical indices and biomarkers on pharmacokinetic parameters was evaluated. The appropriateness of the final model was evaluated using visual predictive check and bootstrap. RESULTS: A two compartment model with first order elimination adequately described sifalimumab serum PK. The estimated typical clearance (CL) and central volume of distribution (V1 ) were 184 ml day(-1) and 2.82 l with 24% and 16% between-subject variability (BSV), respectively. Body weight, dose, 21 INF gene signature baseline and concomitant steroid use were identified as statistically significant covariates for CL and V1 and accounted for <10% of PK variability in the final model. Typical values and BSV of PK parameters from the current analysis with fixed dosing were similar to previous population PK results with body weight normalized dosing. CONCLUSIONS: The transition from body weight normalized dosing to fixed dosing did not impact sifalimumab PK. These findings support the use of fixed dosing for sifalimumab in future clinical studies evaluating it as a potential treatment for SLE.
Assuntos
Anticorpos Monoclonais/farmacocinética , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Administração Intravenosa , Adolescente , Adulto , Fatores Etários , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Peso Corporal , Relação Dose-Resposta a Droga , Cálculos da Dosagem de Medicamento , Feminino , Humanos , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Modelos Biológicos , Fatores Sexuais , Adulto JovemRESUMO
Recent guidelines in British Columbia, Canada have suggested that the use of a maximum of 3 monthly doses of palivizumab 15 mg/kg intramuscularly for RSV immunoprophylaxis of high risk infants born prior to the RSV season is adequate to provide protection against severe RSV disease for a 5-month RSV season. Efficacy was established, however, with 2 large, randomized controlled clinical studies using 5 monthly doses of immunoprophylaxis. To evaluate the differences in expected palivizumab exposures between the 2 dosing regimens (3 vs 5 monthly doses across a 5-month period), we used a population pharmacokinetic (PK) model that was developed using palivizumab PK data collected from 22 clinical studies with a total of 1800 subjects. This model adequately described observed palivizumab concentrations from the different pediatric studies and was subsequently used to simulate expected palivizumab serum concentrations for 3 monthly doses compared with 5 monthly doses in children younger than 24 months with chronic lung disease of prematurity and infants younger than 6 months postnatal age who were born at ≤ 35 weeks gestational age. Results from the population PK model indicated lower serum concentrations of palivizumab during the fourth and fifth months, after an abbreviated 3-monthly-dose regimen when compared with the mean trough concentrations seen with the 5-monthly-dose regimen studied in the pivotal clinical trials in premature infants. Specifically, during the fourth and fifth months, 52% and 85%, respectively, would have levels below the lowest concentration (fifth percentile) in those receiving the 5-monthly-dose regimen. Simulations using this model did not support a 3-monthly-dose regimen to protect against severe RSV disease during the typical 5-month season.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antivirais/administração & dosagem , Modelos Biológicos , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Fatores Etários , Anticorpos Monoclonais Humanizados/farmacocinética , Antivirais/farmacocinética , Colúmbia Britânica , Ensaios Clínicos como Assunto , Esquema de Medicação , Humanos , Lactente , Injeções Intramusculares , Palivizumab , Guias de Prática Clínica como Assunto , Fatores de Risco , Fatores de TempoRESUMO
The rat has been the preferred rodent toxicology species since before regulatory requirements have been in place, and there exists in the pharmaceutical industry and the regulatory agencies a significant amount of historical data for the rat. The resulting experience base with the rat makes the possibility of replacing it with the mouse for regulated toxicology studies untenable for all but the most extreme circumstances. However, toxicologists are very familiar with the mouse as a model for chronic carcinogenicity studies, and there exist multiple preclinical mouse models of disease. The authors evaluated the use of the mouse for early in vivo toxicology signal generation and prioritization of small molecule lead compounds prior to nomination of a development candidate. In five-day oral gavage studies with three test agents in the mouse, the authors were able to identify the same dose-limiting toxicities as those identified in the rat, including examples of compound-mediated hemolysis as well as microscopic lesions in the alimentary canal, kidney, and pancreas. Performing early signal generation studies in the mouse allows for earlier assessment of the safety liabilities of small molecules, requires significantly less compound, and allows evaluation of more compounds earlier in the project's life cycle.
Assuntos
Modelos Animais de Doenças , Camundongos , Testes de Toxicidade/métodos , Animais , Antineoplásicos/toxicidade , Inibidores Enzimáticos/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
Tryptophan hydroxylase (TPH) is a key enzyme in the synthesis of serotonin. As a neurotransmitter, serotonin plays important physiological roles both peripherally and centrally. Here we describe the discovery of substituted triazines as a novel class of tryptophan hydroxylase inhibitors. This class of TPH inhibitors can selectively reduce serotonin levels in murine intestine after oral administration without affecting levels in the brain. These TPH inhibitors may provide novel treatments for gastrointestinal disorders associated with dysregulation of the serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.
Assuntos
Inibidores Enzimáticos/química , Pirazinas/química , Triptofano Hidroxilase/antagonistas & inibidores , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Conformação Molecular , Pirazinas/síntese química , Pirazinas/farmacologia , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Serotonina/biossíntese , Relação Estrutura-Atividade , Triptofano Hidroxilase/metabolismoRESUMO
5-Hydroxytryptamine (serotonin) (5-HT) is a neurotransmitter with both central and peripheral functions, including the modulation of mood, appetite, hemodynamics, gastrointestinal (GI) sensation, secretion, and motility. Its synthesis is initiated by the enzyme tryptophan hydroxylase (TPH). Two isoforms of TPH have been discovered: TPH1, primarily expressed in the enterochromaffin cells of the gastrointestinal tract, and TPH2, expressed exclusively in neuronal cells. Mice lacking Tph1 contain little to no 5-HT in the blood and GI tract while maintaining normal levels in the brain. Because GI 5-HT is known to play important roles in normal and pathophysiology, we set out to discover and characterize novel compounds that selectively inhibit biosynthesis of GI 5-HT. Here, we describe two of a series of these inhibitors that are potent for TPH activity both in biochemical and cell-based assays. This class of compounds has unique properties with respect to pharmacokinetic and pharmacodynamic effects on GI serotonin production. Similar to the Tph1 knockout results, these TPH inhibitors have the ability to selectively reduce 5-HT levels in the murine GI tract without affecting brain 5-HT levels. In addition, administration of these compounds in a ferret model of chemotherapy-induced emesis caused modest reductions of intestinal serotonin levels and a decreased emetic response. These findings suggest that GI-specific TPH inhibitors may provide novel treatments for various gastrointestinal disorders associated with dysregulation of the GI serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.
Assuntos
Trato Gastrointestinal/metabolismo , Serotonina/biossíntese , Triptofano Hidroxilase/antagonistas & inibidores , Animais , Química Encefálica , Furões , Trato Gastrointestinal/efeitos dos fármacos , Síndrome do Intestino Irritável/tratamento farmacológico , Camundongos , Camundongos Knockout , Triptofano Hidroxilase/deficiência , Vômito/tratamento farmacológicoRESUMO
Objectives: MEDI4893 is a novel, long-acting human monoclonal antibody targeting Staphylococcus aureus (SA) alpha toxin (AT). This report presents the results of the exploratory analyses from a randomised phase 1 dose-escalation study in healthy human subjects receiving single intravenous MEDI4893 doses or placebo. Methods: Anti-AT antibodies and AT expression were measured as described previously. Nasal swabs were analysed by culture and PCR. Data were summarised by treatment groups and visits by using SAS System Version 9.3. Results: Subjects receiving 2250 or 5000 mg of MEDI4893 had the highest serum anti-AT neutralising antibody (NAb) levels: approximately 180- to 240-, 70- to 100- and sevenfold to 10-fold higher than respective baseline levels at peak, 30 and 360 days, respectively. In these subjects, levels of serum anti-AT NAbs were >3.2 International Units (IU) mL-1 for at least 211 days. In the upper respiratory tract, anti-AT NAb levels increased with MEDI4893 dose. No apparent effect of MEDI4893 on SA nasal colonisation, hla gene sequence or AT expression was observed. Five AT variants were detected, their lytic activity was fully neutralised by MEDI4893. Discussion: Our results indicate that (1) MEDI4893 administration at 2250 and 5000 mg would provide effective immunoprophylaxis against systemic SA disease; (2) MEDI4983 distributes to the upper respiratory tract and retains neutralising activity against AT; and (3) potential for emergence of MEDI4893 resistance is low. Conclusion: Intravenous administration of MEDI4893 maintained levels of anti-AT NAbs in serum and nasal mucosa that may provide effective immunoprophylaxis against SA disease and support continued clinical development of MEDI4893.
RESUMO
By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/biossíntese , Imunoglobulina G/farmacologia , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/farmacologia , Angiopoietina-2/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/biossíntese , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Humanos , Imunoglobulina G/biossíntese , Camundongos , Anticorpos de Cadeia Única/biossíntese , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibodies carry out a plethora of functions through their crystallizable fragment (Fc) regions, which can be naturally tuned by the adoption of several isotypes and post-translational modifications. Protein engineering enables further Fc function modulations through modifications of the interactions between the Fc and its functional partners, including FcγR, FcRn, complement complex, and additions of auxiliary functional units. Due to the many functions embedded within the confinement of an Fc, a suitable balance must be maintained for a therapeutic antibody to be effective and safe. The outcome of any Fc engineering depends on the interplay among all the effector molecules involved. In this report, we assessed the effects of Fc multiplication (or tandem Fc) on antibody functions. Using IgG1 as a test case, we found that, depending on the specifically designed linker, Fc multiplication led to differentially folded, stable molecules with unique pharmacokinetic profiles. Interestingly, the variants with 3 copies of Fc improved in vitro opsonophagocytic killing activity and displayed significantly improved protective efficacies in a Klebsiella pneumoniae mouse therapeutic model despite faster clearance compared with its IgG1 counterpart. There was no adverse effect observed or pro-inflammatory cytokine release when the Fc variants were administered to animals. We further elucidated that enhanced binding to various effector molecules by IgG-3Fc created a "sink" leading to the rapid clearance of the 3Fc variants, and identified the increased FcRn binding as one strategy to facilitate "sink" escape. These findings reveal new opportunities for novel Fc engineering to further expand our abilities to manipulate and improve antibody therapeutics.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Engenharia de Proteínas/métodos , Animais , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/química , Imunoglobulina G/farmacologia , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Purpose: To use preclinical models to identify a dosing schedule that improves tolerability of highly potent pyrrolobenzodiazepine dimers (PBDs) antibody drug conjugates (ADCs) without compromising antitumor activity.Experimental Design: A series of dose-fractionation studies were conducted to investigate the pharmacokinetic drivers of safety and efficacy of PBD ADCs in animal models. The exposure-activity relationship was investigated in mouse xenograft models of human prostate cancer, breast cancer, and gastric cancer by comparing antitumor activity after single and fractionated dosing with tumor-targeting ADCs conjugated to SG3249, a potent PBD dimer. The exposure-tolerability relationship was similarly investigated in rat and monkey toxicology studies by comparing tolerability, as assessed by survival, body weight, and organ-specific toxicities, after single and fractionated dosing with ADCs conjugated to SG3249 (rats) or SG3400, a structurally related PBD (monkeys).Results: Observations of similar antitumor activity in mice treated with single or fractionated dosing suggests that antitumor activity of PBD ADCs is more closely related to total exposure (AUC) than peak drug concentrations (Cmax). In contrast, improved survival and reduced toxicity in rats and monkeys treated with a fractionated dosing schedule suggests that tolerability of PBD ADCs is more closely associated with Cmax than AUC.Conclusions: We provide the first evidence that fractionated dosing can improve preclinical tolerability of at least some PBD ADCs without compromising efficacy. These findings suggest that preclinical exploration of dosing schedule could be an important clinical strategy to improve the therapeutic window of highly potent ADCs and should be investigated further. Clin Cancer Res; 23(19); 5858-68. ©2017 AACR.
Assuntos
Benzodiazepinas/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Pirróis/administração & dosagem , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Benzodiazepinas/química , Benzodiazepinas/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Haplorrinos , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Masculino , Camundongos , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Pirróis/química , Pirróis/imunologia , Ratos , Índice Terapêutico , Trastuzumab/administração & dosagem , Trastuzumab/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with in vitro evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS) to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW) reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.
Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Animais , Cromatografia em Gel , Cristalografia por Raios X , Difusão Dinâmica da Luz , Ensaio de Imunoadsorção Enzimática , Meia-Vida , Humanos , Fragmentos Fc das Imunoglobulinas/sangue , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/genética , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Biblioteca de Peptídeos , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , UltracentrifugaçãoRESUMO
Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Animais , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Cisteína/química , Desenho de Fármacos , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacocinética , Camundongos Nus , Terapia de Alvo Molecular , Estabilidade Proteica , Receptores Fc/químicaRESUMO
Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, "DuetMab," for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.
Assuntos
Anticorpos Biespecíficos , Afinidade de Anticorpos/genética , Cetuximab , Imunoglobulina G , Trastuzumab , Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Cetuximab/biossíntese , Cetuximab/química , Cetuximab/genética , Células HEK293 , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Trastuzumab/biossíntese , Trastuzumab/química , Trastuzumab/genéticaRESUMO
PURPOSE: This phase I, multicenter, open-label, single-arm, dose-escalation, and dose-expansion study evaluated the safety, tolerability, and antitumor activity of MEDI-573 in adults with advanced solid tumors refractory to standard therapy or for which no standard therapy exists. EXPERIMENTAL DESIGN: Patients received MEDI-573 in 1 of 5 cohorts (0.5, 1.5, 5, 10, or 15 mg/kg) dosed weekly or 1 of 2 cohorts (30 or 45 mg/kg) dosed every 3 weeks. Primary end points included the MEDI-573 safety profile, maximum tolerated dose (MTD), and optimal biologic dose (OBD). Secondary end points included MEDI-573 pharmacokinetics (PK), pharmacodynamics, immunogenicity, and antitumor activity. RESULTS: In total, 43 patients (20 with urothelial cancer) received MEDI-573. No dose-limiting toxicities were identified, and only 1 patient experienced hyperglycemia related to treatment. Elevations in levels of insulin and/or growth hormone were not observed. Adverse events observed in >10% of patients included fatigue, anorexia, nausea, diarrhea, and anemia. PK evaluation demonstrated that levels of MEDI-573 increased with dose at all dose levels tested. At doses >5 mg/kg, circulating levels of insulin-like growth factor (IGF)-I and IGFII were fully suppressed. Of 39 patients evaluable for response, none experienced partial or complete response and 13 had stable disease as best response. CONCLUSIONS: The MTD of MEDI-573 was not reached. The OBD was 5 mg/kg weekly or 30 or 45 mg/kg every 3 weeks. MEDI-573 showed preliminary antitumor activity in a heavily pretreated population and had a favorable tolerability profile, with no notable perturbations in metabolic homeostasis.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias/tratamento farmacológico , Idoso , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/farmacocinética , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like II/antagonistas & inibidores , Masculino , Dose Máxima Tolerável , Pessoa de Meia-IdadeRESUMO
Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4αPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.
Assuntos
Anticorpos Antibacterianos/uso terapêutico , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/imunologia , Animais , Antibacterianos/farmacologia , Anticorpos Antibacterianos/química , Anticorpos Biespecíficos/química , Anticorpos Monoclonais/química , Antígenos de Bactérias/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Humanos , Camundongos , Conformação Molecular , Fagocitose , Infecções por Pseudomonas/imunologiaRESUMO
PURPOSE: Goals of this study were to determine if pharmacological or genetic inhibition of Rho-associated coiled coil containing protein kinases (known as ROCK1 and ROCK2) alters intraocular pressure (IOP) in mice. METHODS: Micro-cannulation of the anterior chamber was used to measure IOP in wild-type B6.129 hybrid mice following treatment with ROCK inhibitors Y-27632 or Y-39983. For comparative purposes, wild-type mice were also treated with timolol, acetazolamide, pilocarpine, or latanoprost. Mice deficient in either Rock1 or Rock2 were generated by homologous recombination or gene trapping, respectively, and their IOP was determined using identical methods employed in the pharmacology studies. RESULTS: Treatment of wild-type B6.129 hybrid mice with ROCK inhibitors (Y-27632 and Y-39983) resulted in significant reductions in IOP. The magnitude of IOP reduction observed with topical Y-39983 was comparable to timolol, and exceeded the IOP effects of latanoprost in this study. Pilocarpine had no discernible effect on IOP in mice. Moreover, mice deficient in either Rock1 or Rock2 exhibited a significant decrease in IOP compared to their B6.129 wild-type littermates. CONCLUSIONS: Pharmacological or genetic inhibition of ROCKs results in decreased IOP in mice. The magnitude of IOP reduction is significant as demonstrated with comparative pharmacology using agents that lower IOP in humans. These studies support the ROCK pathway as a therapeutic target for treating ocular hypertension.
Assuntos
Amidas/farmacologia , Pressão Intraocular/efeitos dos fármacos , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Acetazolamida/administração & dosagem , Acetazolamida/farmacologia , Administração Tópica , Antagonistas Adrenérgicos beta/administração & dosagem , Antagonistas Adrenérgicos beta/farmacologia , Amidas/administração & dosagem , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/farmacologia , Relação Dose-Resposta a Droga , Latanoprosta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agonistas Muscarínicos/administração & dosagem , Agonistas Muscarínicos/farmacologia , Pilocarpina/administração & dosagem , Pilocarpina/farmacologia , Prostaglandinas F Sintéticas/administração & dosagem , Prostaglandinas F Sintéticas/farmacologia , Piridinas/administração & dosagem , Timolol/administração & dosagem , Timolol/farmacologiaRESUMO
The discovery of a novel class of peripheral tryptophan hydroxylase (TPH) inhibitors is described. This class of TPH inhibitors exhibits excellent potency in in vitro biochemical and cell-based assays, and it selectively reduces serotonin levels in the murine intestine after oral administration without affecting levels in the brain. These TPH1 inhibitors may provide novel treatments for gastrointestinal disorders associated with dysregulation of the serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.