RESUMO
The immunogenicity of biotherapeutics presents a major challenge during the clinical development of new protein drugs including monoclonal antibodies. To address this, multiple humanization and de-immunization techniques that employ in silico algorithms and in vitro test systems have been proposed and implemented. However, the success of these approaches has been variable and to date, the ability of these techniques to predict immunogenicity has not been systematically tested in humans or other primates. This study tested whether antibody humanization and de-immunization strategies reduce the risk of anti-drug antibody (ADA) development using cynomolgus macaque as a surrogate for human. First human-cyno chimeric antibodies were constructed by grafting the variable domains of the adalimumab and golimumab monoclonal antibodies onto cynomolgus macaque IgG1 and Igκ constant domains followed by framework germlining to cyno to reduce the xenogenic content. Next, B and T cell epitopes and aggregation-prone regions were identified using common in silico methods to select domains with an ADA risk for additional modification. The resultant engineered antibodies had a comparable affinity for TNFα, demonstrated similar biophysical properties, and exhibited significantly reduced ADA levels in cynomolgus macaque compared with the parental antibodies, with a corresponding improvement in the pharmacokinetic profile. Notably, plasma concentrations of the engineered antibodies were quantifiable through 504 hours (chimeric) and 840 hours (germlined/de-immunized), compared with only 336 hours (adalimumab) or 336-672 hours (golimumab). The results point to the significant value in the investment in these engineering strategies as an important guide for monoclonal antibody optimization that can contribute to improved clinical outcomes.
Assuntos
Adalimumab/imunologia , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Animais , Feminino , Humanos , Imunização , Macaca fascicularis , Masculino , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Immunogenicity of biomolecules is one of the largest concerns in biological therapeutic drug development. Adverse immune responses as a result of immunogenicity to biotherapeutics range from mild hypersensitivity reactions to potentially life-threatening anaphylactic reactions and can negatively impact human health and drug efficacy. Numerous confounding patient-, product- or treatment-related factors can influence the development of an immune reaction against therapeutic proteins. The goal of this study was to investigate the relationship between pre-existing drug reactivity (PE-ADA), individual immunogenetics (MHC class II haplotypes), and development of treatment-induced antidrug antibodies (TE-ADA) in cynomolgus macaque. PE-ADA refers to the presence of antibodies immunoreactive against the biotherapeutic in treatment-naïve individuals. We observed that PE-ADA frequency against four different bispecific antibodies in naïve cynomolgus macaque is similar to that reported in humans. Additionally, we report a trend towards an increased incidence of TE-ADA development in macaques with high PE-ADA levels. In order to explore the relationship between MHC class II alleles and risk of ADA development, we obtained full-length MHC class II sequences from 60 cynomolgus macaques in our colony. We identified a total of 248 DR, DP, and DQ alleles and 236 unique haplotypes in our cohort indicating a genetically complex set of animals potentially reflective of the human population. Based on our observations, we propose the evaluation of the magnitude/frequency of pre-existing reactivity and consideration of MHC class II genetics as additional useful tools to understand the immunogenic potential of biotherapeutics.
Assuntos
Anticorpos Biespecíficos/imunologia , Hipersensibilidade a Drogas/imunologia , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Imunogenética , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/sangue , Hipersensibilidade a Drogas/genética , Frequência do Gene , Haplótipos , Macaca fascicularis , Masculino , FilogeniaRESUMO
We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.
Assuntos
Anticorpos Biespecíficos , Doenças Autoimunes/tratamento farmacológico , Fator Ativador de Células B/antagonistas & inibidores , Interleucina-17/antagonistas & inibidores , Anticorpos de Cadeia Única , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Fator Ativador de Células B/imunologia , Feminino , Células HEK293 , Células HT29 , Humanos , Interleucina-17/imunologia , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
OBJECTIVE: The objective of this investigation was to examine the distribution of galcanezumab and a control immunoglobulin 4 antibody containing the same constant regions as galcanezumab, into peripheral and central tissues. METHODS: Galcanezumab and a control immunoglobulin 4 antibody were radioiodinated with Iodine-125 to specific activities of 0.11 mCi/mg and 0.16 mCi/mg, respectively. At 24, 72, and 168 hours following subcutaneous injection of either antibody (4 mg/kg), cerebrospinal fluid and plasma were obtained followed by saline perfusion to remove residual blood and collection of selected tissues for determination of Iodine-125 content by gamma counting. RESULTS: The peak plasma levels of Iodine-125 galcanezumab and Iodine-125 control immunoglobulin 4 were observed at 72 hours and remained high at 168 hours post-dose. The rank order of tissue levels was dura mater = spleen > trigeminal ganglia â«hypothalamus = spinal cord = prefrontal cortex = cerebellum. Iodine-125 galcanezumab levels in peripheral tissue (dura mater, spleen, and trigeminal ganglia) averaged 5% to 11% of plasma, whereas all of the central nervous system (CNS) tissue levels and the cerebrospinal fluid levels were < 0.4% of plasma. Distribution of the antibodies into the dura mater and the trigeminal ganglia was similar to that observed in the spleen and significantly greater than exposure in the brain or spinal cord. CONCLUSIONS: The central levels of galcanezumab were relatively low, which would favor the dura mater and trigeminal ganglia as sites of action for its observed clinical efficacy. However, a central site of action cannot be excluded.
Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Encéfalo/metabolismo , Medula Espinal/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Dura-Máter/metabolismo , Radioisótopos do Iodo , Masculino , Ratos , Ratos Sprague-Dawley , Baço/metabolismo , Distribuição Tecidual , Gânglio Trigeminal/metabolismoRESUMO
Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy.
RESUMO
A recent report described a novel mechanism of action for an anti-proprotein convertase subtilisin-kexin type 9 (PCSK9) monoclonal antibody (LY3015014, or LY), wherein the antibody has improved potency and duration of action due to the PCSK9 epitope for LY binding. Unlike other antibodies, proteolysis of PCSK9 can occur when LY is bound to PCSK9. We hypothesized that this allowance of PCSK9 cleavage potentially improves LY efficiency through two pathways, namely lack of accumulation of intact PCSK9 and reduced clearance of LY. A quantitative modeling approach is necessary to further understand this novel mechanism of action. We developed a mechanism-based model to characterize the relationship between antibody pharmacokinetics, PCSK9 and LDL cholesterol levels in animals, and used the model to better understand the underlying drivers for the improved efficiency of LY. Simulations suggested that the allowance of cleavage of PCSK9 resulting in a lack of accumulation of intact PCSK9 is the major driver of the improved potency and durability of LY. The modeling reveals that this novel 'proteolysis-permitting' mechanism of LY is a means by which an efficient antibody can be developed with a total antibody dosing rate that is lower than the target production rate. We expect this engineering approach may be applicable to other targets and that the mathematical models presented herein will be useful in evaluating similar approaches.
Assuntos
Anticorpos Monoclonais/farmacocinética , Modelos Teóricos , Inibidores de PCSK9 , Animais , LDL-Colesterol/metabolismo , Humanos , Macaca fascicularis , Camundongos , Proteólise/efeitos dos fármacosRESUMO
Interleukin (IL)-17A exists as a homodimer (A/A) or as a heterodimer (A/F) with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis.
RESUMO
Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgG-scFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.
Assuntos
Anticorpos Biespecíficos/farmacocinética , Capilares/metabolismo , Fígado/metabolismo , Animais , Meia-Vida , Antígenos de Histocompatibilidade Classe I , Humanos , Macaca fascicularis , Taxa de Depuração Metabólica , Receptores FcRESUMO
Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts.
Assuntos
Reações Antígeno-Anticorpo , Modelos Imunológicos , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/metabolismo , Afinidade de Anticorpos , Antígenos de Superfície/química , Antígenos de Superfície/metabolismo , Linhagem Celular , Receptores ErbB/química , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Cinética , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/imunologia , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a significant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused significant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action. PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen to accumulate.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticolesterolemiantes/farmacologia , Pró-Proteína Convertases/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticolesterolemiantes/química , Anticolesterolemiantes/farmacocinética , LDL-Colesterol/sangue , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Furina/química , Meia-Vida , Humanos , Hipercolesterolemia/tratamento farmacológico , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/imunologia , Ligação Proteica , Proteólise , Serina Endopeptidases/imunologia , Resultado do TratamentoRESUMO
The application of protein engineering technologies toward successfully improving antibody pharmacokinetics has been challenging due to the multiplicity of biochemical factors that influence monoclonal antibody (mAb) disposition in vivo. Physiological factors including interactions with the neonatal Fc receptor (FcRn) and specific antigen binding properties of mAbs, along with biophysical properties of the mAbs themselves play a critical role. It has become evident that applying an integrated approach to understand the relative contribution of these factors is critical to rationally guide and apply engineering strategies to optimize mAb pharmacokinetics. The study presented here evaluated the influence of unintended non-specific interactions on the disposition of mAbs whose clearance rates are governed predominantly by either non-specific (FcRn) or target-mediated processes. The pharmacokinetics of 8 mAbs representing a diverse range of these properties was evaluated in cynomolgus monkeys. Results revealed complementarity-determining region (CDR) charge patch engineering to decrease charge-related non-specific binding can have a significant impact on improving the clearance. In contrast, the influence of enhanced in vitro FcRn binding was mixed, and related to both the strength of charge interaction and the general mechanism predominant in governing the clearance of the particular mAb. Overall, improved pharmacokinetics through enhanced FcRn interactions were apparent for a CDR charge-patch normalized mAb which was affected by non-specific clearance. The findings in this report are an important demonstration that mAb pharmacokinetics requires optimization on a case-by-case basis to improve the design of molecules with increased therapeutic application.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Regiões Determinantes de Complementaridade/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Engenharia de Proteínas/métodos , Receptores Fc/metabolismo , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/farmacocinética , Afinidade de Anticorpos/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Macaca fascicularis , Taxa de Depuração Metabólica , Camundongos , Ligação Proteica/imunologia , Receptores Fc/imunologiaRESUMO
Basal insulin peglispro (BIL) comprises insulin lispro covalently bound to a 20-kDa polyethylene glycol (PEG) at lysine B28. The biologic fate of BIL and unconjugated PEG were examined in rats given a single 0.5-mg/kg i.v. or 2-mg/kg s.c. dose of BIL with (14)C label in 20-kDa PEG or (125)I label in lispro. Unconjugated (14)C-labeled 20-kDa PEG was dosed at 10 mg/kg i.v. or s.c. Blood, urine, and feces were collected up to 336 hours after dosing. Radioactivity was measured by scintillation spectrometry, and BIL was quantitated by enzyme-linked immunosorbent assay. Area under the curve and half-life for immunoreactive BIL were lower than those for both (14)C and (125)I after subcutaneous and intravenous administration. The half-lives of (14)C after BIL and PEG dosing were similar. The clearance of immunoreactive BIL was 2.4-fold faster than that of (14)C and 1.6-fold faster than (125)I. After a subcutaneous dose of BIL, immunoreactive BIL accounted for 31% of the circulating (125)I and 16% of the circulating (14)C, indicating extensive catabolism of BIL. Subcutaneous bioavailability of BIL was 23%-29%; bioavailability for unconjugated PEG was 78%. For unconjugated PEG, most of the (14)C dose was recovered in urine. For BIL, ≥86% of (125)I was eliminated in urine and (14)C was eliminated about equally in urine and feces. The major (14)C-labeled catabolism product of BIL in urine was 20-kDa PEG with lysine attached. The attachment of 20-kDa PEG to lispro in BIL led to a different elimination pathway for PEG compared with unconjugated 20-kDa PEG.
Assuntos
Insulina/análogos & derivados , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Animais , Disponibilidade Biológica , Injeções Intravenosas , Injeções Subcutâneas , Insulina/administração & dosagem , Insulina/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
LY2951742, a monoclonal antibody targeting calcitonin gene-related peptide (CGRP), is being developed for migraine prevention and osteoarthritis pain. To support the clinical development of LY2951742, capsaicin-induced dermal blood flow (DBF) was used as a target engagement biomarker to assess CGRP activity in nonhuman primates and healthy volunteers. Inhibition of capsaicin-induced DBF in nonhuman primates, measured with laser Doppler imaging, was dose dependent and sustained for at least 29 days after a single intravenous injection of the CGRP antibody. This information was used to generate a pharmacokinetic/pharmacodynamic model, which correctly predicted inhibition of capsaicin-induced DBF in humans starting at a single subcutaneous 5-mg dose. As expected, the degree of inhibition in capsaicin-induced DBF increased with higher LY2951742 plasma concentrations. Utilization of this pharmacodynamic biomarker with pharmacokinetic data collected in phase I studies provided the dose-response relationship that assisted in dose selection for the phase II clinical development of LY2951742.
Assuntos
Anticorpos Monoclonais/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/farmacologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Administração Cutânea , Adolescente , Adulto , Animais , Biomarcadores/metabolismo , Método Duplo-Cego , Humanos , Fluxometria por Laser-Doppler/métodos , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Dor/tratamento farmacológico , Adulto JovemRESUMO
Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Neutralizantes/farmacologia , Receptores de Somatomedina/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Nus , Microscopia Confocal , Neoplasias Experimentais/tratamento farmacológico , Estabilidade Proteica , Receptor IGF Tipo 1 , Ressonância de Plasmônio de Superfície , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non-tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass- and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non-tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting.
Assuntos
Anticorpos Neutralizantes/farmacologia , Músculo Esquelético/efeitos dos fármacos , Miostatina/imunologia , Neoplasias/tratamento farmacológico , Síndrome de Emaciação/tratamento farmacológico , Animais , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos/imunologia , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos SCID , Força Muscular/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Miofibrilas/efeitos dos fármacos , Neoplasias/complicações , Neoplasias Experimentais/complicações , Neoplasias Experimentais/tratamento farmacológico , Transplante Heterólogo , Resultado do Tratamento , Síndrome de Emaciação/etiologiaRESUMO
Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an â¼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.
Assuntos
Anticorpos Monoclonais Humanizados , Especificidade de Anticorpos/genética , Regiões Determinantes de Complementaridade , Imunoglobulina G , Modelos Moleculares , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais Humanizados/farmacologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Regiões Determinantes de Complementaridade/farmacologia , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Ponto Isoelétrico , CamundongosRESUMO
Monoclonal antibodies (mAbs) represent an important class of therapeutic modalities. To optimize their pharmaceutical properties, studies have focused on improving mAb pharmacokinetic/pharmacodynamic profiles by modulating their interactions with the neonatal Fc receptor (FcRn). The influence of both the chemical and physical properties of IgGs has been examined in the context of FcRn interactions. In this regard, a variety of FcRn binding assays and tools have been developed and used to characterize the interaction with IgGs. However, a predictive relationship between the FcRn binding interaction of IgGs in vitro and their pharmacokinetics in vivo broadly across mAbs remains elusive. Many studies have increasingly suggested that the interplay between the characteristics of the mAb and the nature of its target can influence disposition and elimination. Thus, it is becoming increasingly evident that along with FcRn interactions, consideration of the non-FcRn-based biologic processes active in mAb disposition should be integrated into mAb development and optimization. Herein, we describe how the pharmacokinetics of mAbs can be modulated through FcRn interactions and provide perspectives on interpreting the receptor binding parameters in relation to other mechanisms involved in antibody disposition to aid in guiding mAb development.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/metabolismo , Animais , Humanos , Farmacocinética , Ligação ProteicaRESUMO
Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator that represents a promising target for the treatment of several metabolic diseases. Administration of recombinant wild type FGF21 to diabetic animals leads to a dramatic improvement in glycaemia and ameliorates other systemic measures of metabolic health. Here we report the pharmacologic outcomes observed in non-human primates upon administration of a recently described FGF21 analogue, LY2405319 (LY). Diabetic rhesus monkeys were treated subcutaneously with LY once daily for a period of seven weeks. The doses of LY used were 3, 9 and 50 mg/kg each delivered in an escalating fashion with washout measurements taken at 2, 4, 6 and 8 weeks following the final LY dose. LY therapy led to a dramatic and rapid lowering of several important metabolic parameters including glucose, body weight, insulin, cholesterol and triglyceride levels at all doses tested. In addition, we observed favorable changes in circulating profiles of adipokines, with increased adiponectin and reduced leptin indicative of direct FGF21 action on adipose tissue. Importantly, and for the first time we show that FGF21 based therapy has metabolic efficacy in an animal with late stage diabetes. While the glycemic efficacy of LY in this animal was partially attenuated its lipid lowering effect was fully preserved suggesting that FGF21 may be a viable treatment option even in patients with advanced disease progression. These findings support continued exploration of the FGF21 pathway for the treatment of metabolic disease.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Adipocinas/sangue , Animais , Glicemia/análise , Progressão da Doença , Relação Dose-Resposta a Droga , Ingestão de Energia/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacocinética , Insulina/sangue , Macaca mulatta , Redução de Peso/efeitos dos fármacosRESUMO
Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7-14 days resulted in a 25-50% lowering of plasma glucose coupled with a 10-30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.
Assuntos
Fatores de Crescimento de Fibroblastos/farmacologia , Proteínas Recombinantes , Células 3T3 , Substituição de Aminoácidos , Animais , Linhagem Celular , Desenho de Fármacos , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/genética , Expressão Gênica , Variação Genética , Células Hep G2 , Humanos , Proteínas Klotho , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Pichia/genética , Pichia/metabolismo , Conformação Proteica , Estabilidade Proteica , TemperaturaRESUMO
The aim of this work was to develop and characterize an ELISA to measure free ligand concentrations in rat serum in the presence of a Fab to the same ligand. A variety of experiments were conducted to understand optimal assay conditions and to verify that only free ligand was detected. The parameters explored included sample incubation time on plate, the initial concentrations of Fab and ligand, and the pre-incubation time required for the Fab-ligand complex concentrations to reach equilibrium. We found the optimal experimental conditions to include a 10-minute on-plate incubation of ligand-containing samples, with a 24-hour pre-incubation time for test samples of Fab and ligand to reach equilibrium. An alternative approach, involving removal of Fab-ligand complexes from the solution prior to measuring concentrations of the ligand, was also used to verify that the assay only measured free ligand. Rats were dosed subcutaneously with Fab and the assay was used to demonstrate dose-dependent suppression of endogenous free ligand levels in vivo.