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1.
Mol Pharm ; 13(3): 915-23, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26849155

RESUMO

Antibodies at high concentrations often reveal unanticipated biophysical properties suboptimal for therapeutic development. The purpose of this work was to explore the use of point mutations based on crystal structure information to improve antibody physical properties such as viscosity and phase separation (LLPS) at high concentrations. An IgG4 monoclonal antibody (Mab4) that exhibited high viscosity and phase separation at high concentration was used as a model system. Guided by the crystal structure, four CDR point mutants were made to evaluate the role of hydrophobic and charge interactions on solution behavior. Surprisingly and unpredictably, two of the charge mutants, R33G and N35E, showed a reduction in viscosity and a lower propensity to form LLPS at high concentration compared to the wild-type (WT), while a third charge mutant S28K showed an increased propensity to form LLPS compared to the WT. A fourth mutant, F102H, had reduced hydrophobicity, but unchanged viscosity and phase separation behavior. We further evaluated the correlation of various biophysical measurements including second virial coefficient (A2), interaction parameter (kD), weight-average molecular weight (WAMW), and hydrodynamic diameters (DH), at relatively low protein concentration (4 to 15 mg/mL) to physical properties, such as viscosity and liquid-liquid phase separation (LLPS), at high concentration. Surprisingly, kD measured using dynamic light scattering (DLS) at low antibody concentration correlated better with viscosity and phase separation than did A2 for Mab4. Our results suggest that the high viscosity and phase separation observed at high concentration for Mab4 are mainly driven by charge and not hydrophobicity.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/imunologia , Extração Líquido-Líquido/métodos , Engenharia de Proteínas/métodos , Viscosidade , Substituição de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Cristalização , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Soluções Farmacêuticas , Conformação Proteica
2.
Histopathology ; 65(6): 879-96, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25039923

RESUMO

AIMS: Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method. METHODS AND RESULTS: Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P < 0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R(2)  > 0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21. CONCLUSIONS: The anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.


Assuntos
Anticorpos Monoclonais , Neoplasias/genética , Proteínas Proto-Oncogênicas c-met/análise , Adulto , Idoso , Animais , Especificidade de Anticorpos , Western Blotting , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Camundongos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Análise Serial de Tecidos
3.
Mol Pharm ; 10(4): 1322-31, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23383873

RESUMO

A key challenge in developing therapeutic antibodies is their highly variable propensities to self-associate at high antibody concentrations (>50 mg/mL) required for subcutaneous delivery. Identification of monoclonal antibodies (mAbs) in the initial discovery process that not only have high binding affinity but also have high solubility and low viscosity would simplify the development of safe and effective antibody therapeutics. Unfortunately, the low purities, small quantities and large numbers of antibody candidates during the early discovery process are incompatible with current methods of measuring antibody self-association. We report a method (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) capable of identifying mAbs with low self-association propensity that is robust even at low mAb concentrations (5-50 µg/mL) and in the presence of cell culture media. Gold nanoparticles are coated with polyclonal antibodies specific for human antibodies, and then human mAbs are captured from dilute antibody solutions. We find that the wavelength of maximum absorbance (plasmon wavelength) of antibody-gold conjugates--which red-shifts as the distance between particles is reduced due to attractive mAb self-interactions--is well correlated with light scattering measurements conducted at several orders of magnitude higher antibody concentrations. The generality of AC-SINS makes it well suited for use in diverse settings ranging from antibody discovery to formulation development.


Assuntos
Anticorpos Monoclonais/química , Imuno-Histoquímica , Adsorção , Química Farmacêutica/métodos , Meios de Cultura/química , Difusão , Desenho de Fármacos , Humanos , Cinética , Luz , Nanopartículas/química , Nanotecnologia/métodos , Espalhamento de Radiação , Solubilidade , Viscosidade
4.
Drug Metab Dispos ; 40(8): 1545-55, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22584253

RESUMO

The pH-dependent binding of IgGs to the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. Enhancing interactions between Fc and FcRn via protein engineering has been successfully used as an approach for improving the pharmacokinetics of monoclonal antibodies (mAbs). Although the quantitative translatability of the in vitro FcRn affinity enhancement to an in vivo pharmacokinetic benefit has been supported by several studies, there are also published reports indicating a disconnect in this relation. The body of literature suggests there are likely additional biochemical and biophysical properties of the mAbs along with their FcRn affinity that influence the in vivo pharmacokinetics. Herein, we more broadly evaluate the in vitro Fc-FcRn interactions and biochemical properties of five humanized IgG4 antibodies each with two Fc variant sequences (T250Q/M428L and V308P) and their corresponding pharmacokinetics in cynomolgus monkeys. Our findings indicate that the FcRn affinity-pharmacokinetic relationship does not show a direct correlation either across different IgGs or between the two variant sequences within a platform. Other parameters that have been suggested to contribute to mAb pharmacokinetic properties, such as the pH-dependent dissociation of the FcRn-IgG complexes, mAb biophysical properties, and nonspecific/charge binding characteristics of the mAbs, also did not independently explain the differing pharmacokinetic behaviors. Our results suggest that there is likely not a single in vitro parameter that readily predicts in vivo pharmacokinetics, but that the relative contribution and interplay of several factors along with the FcRn binding affinity are important determinants of mAb pharmacokinetic properties.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Animais , Linhagem Celular , Humanos , Imunoglobulina G/química , Técnicas In Vitro , Macaca fascicularis , Ligação Proteica
5.
BMC Bioinformatics ; 11: 241, 2010 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-20459827

RESUMO

BACKGROUND: Feature gene extraction is a fundamental issue in microarray-based biomarker discovery. It is normally treated as an optimization problem of finding the best predictive feature genes that can effectively and stably discriminate distinct types of disease conditions, e.g. tumors and normals. Since gene microarray data normally involves thousands of genes at, tens or hundreds of samples, the gene extraction process may fall into local optimums if the gene set is optimized according to the maximization of classification accuracy of the classifier built from it. RESULTS: In this paper, we propose a novel gene extraction method of error margin analysis to optimize the feature genes. The proposed algorithm has been tested upon one synthetic dataset and two real microarray datasets. Meanwhile, it has been compared with five existing gene extraction algorithms on each dataset. On the synthetic dataset, the results show that the feature set extracted by our algorithm is the closest to the actual gene set. For the two real datasets, our algorithm is superior in terms of balancing the size and the validation accuracy of the resultant gene set when comparing to other algorithms. CONCLUSION: Because of its distinct features, error margin analysis method can stably extract the relevant feature genes from microarray data for high-performance classification.


Assuntos
Perfilação da Expressão Gênica/métodos , Algoritmos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reconhecimento Automatizado de Padrão/métodos
6.
MAbs ; 11(6): 1175-1190, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31181988

RESUMO

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/farmacologia
7.
J Inflamm Res ; 9: 39-50, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27143947

RESUMO

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.

8.
Biotechnol Prog ; 20(2): 449-56, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15058989

RESUMO

The Saccharomyces cerevisiae cell wall provides a semipermeable barrier that can retain intracellular proteins but still permits small molecules to pass through. When S. cerevisiae cells expressing E. coli lacZ are treated with detergent to extract the cell membrane, beta-galactosidase activity in the permeabilized cells is approximately 40% of the activity of the protein in cell extract. However, the permeabilized cells can easily be collected and reused over 15 times without appreciable loss in activity. Cell wall composition and thickness can be modified using different cell strains for enzyme expression or by mutating genes involved in cell wall biosynthesis or degradation. The Sigma1278b strain cell wall is less permeable than the walls of BY4742 and W303 cells, and deleting EXG1, which encodes a 1,3-beta-glucanase, can further reduce permeability. A short Zymolyase treatment can increase cell wall permeability without rupturing the cells. Encapsulating multiple enzymes in permeabilized cells can offer kinetic advantages over the same enzymes in solution. Regeneration of ATP from AMP by adenylate kinase and pyruvate kinase encapsulated in the same cell proceeded more rapidly than regeneration using a cell extract. Combining permeabilized cells containing adenylate kinase with permeabilized cells containing pyruvate kinase can also regenerate ATP from AMP, but the kinetics of this reaction are slower than regeneration using cell extract or permeabilized cells expressing both enzymes.


Assuntos
Técnicas de Cultura de Células/métodos , Permeabilidade da Membrana Celular/fisiologia , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , beta-Galactosidase/biossíntese , beta-Galactosidase/genética , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Clin Cancer Res ; 20(23): 6059-70, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25231402

RESUMO

PURPOSE: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. EXPERIMENTAL DESIGN/RESULTS: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. CONCLUSIONS: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação , Transporte Proteico , Proteólise , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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